The following alphabetical list represents papers published in 2014 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.
P.S. The journal links only work if you have a license to those respective online journals.
2014 Titles :
Abdi, K., Singh, N.J., Spooner, E., Kessler, B.M., Radaev, S., Lantz, L., Xiao, T.S., Matzinger, P., Sun, P.D., and Ploegh, H.L. (2014). Free IL-12p40 Monomer Is a Polyfunctional Adaptor for Generating Novel IL-12-like Heterodimers Extracellularly. J Immunol 192, 6028-6036.IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion. PDF
Alpatov, R., Lesch, B.J., Nakamoto-Kinoshita, M., Blanco, A., Chen, S., Stutzer, A., Armache, K.J., Simon, M.D., Xu, C., Ali, M., David C Page, et al.. (2014). A Chromatin-Dependent Role of the Fragile X Mental Retardation Protein FMRP in the DNA Damage Response. Cell 157, 869-881.Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome .Full Text.
AlvarezDominguez2, J.R., Hu, W.Q., Yuan, B.B., Shi, J.H., Park, S.S., Gromatzky, A.A., van Oudenaarden, A., and Lodish, H.F. (2014). Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation. Blood 123, 570-581.Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis. Full Text.
AlvarezDominguez, J.R., Hu, W., Gromatzky, A.A., and Lodish, H.F. (2014). Long noncoding RNAs during normal and malignant hematopoiesis. International Journal of Hematology Mar 9. [Epub ahead of print] .Long noncoding RNAs (lncRNAs) are increasingly recognized to contribute to cellular development via diverse mechanisms during both health and disease. Here, we highlight recent progress on the study of lncRNAs that function in the development of blood cells. We emphasize lncRNAs that regulate blood cell fates through epigenetic control of gene expression, an emerging theme among functional lncRNAs. Many of these noncoding genes and their targets become dysregulated during malignant hematopoiesis, directly implicating lncRNAs in blood cancers such as leukemia. In a few cases, dysregulation of an lncRNA alone leads to malignant hematopoiesis in a mouse model. Thus, lncRNAs may be not only useful as markers for the diagnosis and prognosis of cancers of the blood, but also as potential targets for novel therapies. Full Text
Anders, L., Guenther, M.G., Qi, J., Fan, Z.P., Marineau, J.J., Rahl, P.B., Loven, J., Sigova, A.A., Smith, W.B., Lee, T.I., James E Bradner & Richard A Young (2014). Genome-wide localization of small molecules. Nature Biotechnology 32, 92-+.A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins. Full Text
Armond, J.W., Saha, K., Rana, A.A., Oates, C.J., Jaenisch, R., Nicodemi, M., and Mukherjee, S. (2014). A stochastic model dissects cell states in biological transition processes. Sci Rep 4, 3692.Many biological processes, including differentiation, reprogramming, and disease transformations, involve transitions of cells through distinct states. Direct, unbiased investigation of cell states and their transitions is challenging due to several factors, including limitations of single-cell assays. Here we present a stochastic model of cellular transitions that allows underlying single-cell information, including cell-state-specific parameters and rates governing transitions between states, to be estimated from genome-wide, population-averaged time-course data. The key novelty of our approach lies in specifying latent stochastic models at the single-cell level, and then aggregating these models to give a likelihood that links parameters at the single-cell level to observables at the population level. We apply our approach in the context of reprogramming to pluripotency. This yields new insights, including profiles of two intermediate cell states, that are supported by independent single-cell studies. Our model provides a general conceptual framework for the study of cell transitions, including epigenetic transformations. Full Text
Ashour, J., Schmidt, F., Hanke, L., Cragnolini, J., Cavallari, M., Altenburg, A., Brewer, R., Ingram, J., Shoemaker, C., and Ploegh, H. (2014). Intracellular expression of camelid single domain antibodies specific for influenza virus nucleoprotein (NP) uncovers distinct features of its nuclear localization. Journal of Virology (online ahead of print ). Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of flu-encoded proteins. We generated a panel of camelid-derived single domain antibody fragments (VHHs) against influenza nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (alphaNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the vRNP complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. IMPORTANCE: Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function, and how to disrupt said function through pharmaceutical intervention, is difficult. We present a novel method based on single domain antibody technology that permits specific targeting and disruption of an essential flu protein in the absence of genetic manipulation of the flu virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens. Full Text.
Avalos3, A.M., Meyer-Wentrup, F., and Ploegh, H.L. (2014). B-cell receptor signaling in lymphoid malignancies and autoimmunity. Adv Immunol 123, 1-49.The B-cell receptor (BCR) for antigen is a key sensor required for B-cell development, survival, and activation. Rigorous selection checkpoints ensure that the mature B-cell compartment in the periphery is largely purged of self-reactive B cells. However, autoreactive B cells escape selection and persist in the periphery as anergic or clonally ignorant B cells. Under the influence of genetic or environmental factors, which are not completely understood, autoreactive B cells may be activated. Similar activation can also occur at different stages of B-cell maturation in the bone marrow or in peripheral lymphoid organs and give rise to malignant B cells. The pathology that typifies neoplastic lymphocytes and autoreactive B cells differs: malignant B cells proliferate and occupy niches otherwise taken up by healthy leukocytes or erythrocytes, while autoreactive B cells produce pathogenic antibodies or present self-antigen to T cells. However, both malignant and autoreactive B cells share the commonality of deregulated BCR pathways as principal contributors to pathogenicity. We first summarize current views of BCR activation. We then explore how anomalous BCR pathways correlate with malignancies and autoimmunity. We also elaborate on the activation of TLR pathways in abnormal B cells and how they contribute to maintenance of pathology. Finally, we outline the benefits and emergence of mouse models generated by somatic cell nuclear transfer to study B-cell function in manners for which current transgenic models may be less well suited.
Avalos2, A.M., and Ploegh, H.L. (2014). Early BCR Events and Antigen Capture, Processing, and Loading on MHC Class II on B Cells. Frontiersin Immunoogyl 5, 92.B cells are efficient antigen-presenting cells (APCs), relying on antigen uptake through the B cell receptor (BCR). The mechanism of antigen recognition remains a topic of debate; while the prevalent view holds that antigens need to be multivalent for BCR activation, monovalent antigens can also initiate B cell responses. In this review, we describe the steps required for antigen uptake, processing, and loading of peptides onto MHC Class II compartments in B cells for efficient presentation to CD4 T cells, with a special focus in the initial steps of BCR recognition of antigen. Full Text
Avalos, A.M., Bilate, A.M., Witte, M.D., Tai, A.K., He, J., Frushicheva, M.P., Thill, P.D., Meyer-Wentrup, F., Theile, C.S., Chakraborty, A.K,. Xiaowei Zhuang and Hidde L. Ploegh (2014). Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells. JEM vol. 211 no. 2 365-379 Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition. Full Text
BarPeled, L., and Sabatini, D.M. (2014). Regulation of mTORC1 by amino acids. Trends in Cell Bioogyl.Available online 31 March 2014. The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of cellular and organismal growth, and hyperactivation of this pathway is implicated in the pathogenesis of many human diseases including cancer and diabetes. mTORC1 promotes growth in response to the availability of nutrients, such as amino acids, which drive mTORC1 to the lysosomal surface, its site of activation. How amino acid levels are communicated to mTORC1 is only recently coming to light by the discovery of a lysosome-based signaling system composed of Rags (Ras-related GTPases) and Ragulator v-ATPase, GATOR (GAP activity towards Rags), and folliculin (FLCN) complexes. Increased understanding of this pathway will not only provide insight into growth control but also into the human pathologies triggered by its deregulation. Full Text
Bell2, E.L., Nagamori, I., Williams, E.O., Del Rosario, A.M., Bryson, B.D., Watson, N., White, F.M., Sassone-Corsi, P., and Guarente, L. (2014). SirT1 is required in the male germ cell for differentiation and fecundity in mice. Development 141, 3495-3504.Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis. Full Text.
Bell, G.W., and Lewitter, F. (2014). Resources for Small Regulatory RNAs. Curr Protoc Mol Biol 107, 19 18 11- 14.Over the past twenty years, new classes of regulatory RNAs have been discovered, previously hidden in the transcriptome mostly due to their small size. These small regulatory RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Numerous databases have been developed to store information about these small regulatory RNAs, and many tools have been developed to work with the data. This overview introduces the reader to the many resources available for working with small regulatory RNAs. Curr. Protoc. Mol. Biol. 107:19.8.1-19.8.14. .Full Text.
Bellott, D.W., Hughes, J.F., Skaletsky, H., Brown, L.G., Pyntikova, T., Cho, T.J., Koutseva, N., Zaghlul, S. ,Steve Rozen & David C. Page, et al. (2014). Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators. Nature 508, 494-499.The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three per cent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was nonrandom, and in two cases, convergent across placental and marsupial mammals. We conclude that the gene content of the Y chromosome became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and has unappreciated roles in Turner's syndrome and in phenotypic differences between the sexes in health and disease. Full Text
Bhattacharya, S., Iyer, E.P., Iyer, S.C., and Cox, D.N. (2014). Cell-type specific transcriptomic profiling to dissect mechanisms of differential dendritogenesis. Genomics Data 2, 378-381.The establishment, maintenance and modulation of cell-type specific neural architectures is critically important to the formation of functional neural networks. At the neuroanatomical level, differential patterns of dendritic arborization directly impact neural function and connectivity, however the molecular mechanisms underlying the specification of distinct dendrite morphologies remains incompletely understood. To address this question, we analyzed global gene expression from purified populations of wild-type class I and class IV Drosophila melanogaster dendritic arborization (da) sensory neurons compared to wild-type whole larval RNA using oligo DNA microarray expression profiling. Herein we present detailed experimental methods and bioinformatic analyses to correspond with our data reported in the Gene Expression Omnibus under accession number GSE46154. We further provide R code to facilitate data accession, perform quality controls, and conduct bioinformatic analyses relevant to this dataset. Our cell-type specific gene expression datasets provide a valuable resource for guiding further investigations designed to explore the molecular mechanisms underlying differential patterns of neuronal patterning. Full Text.
Birsoy, K., Possemato, R., Lorbeer, F.K., Bayraktar, E.C., Thiru, P., Yucel, B., Wang, T., Chen, W.W., Clish, C.B., and Sabatini, D.M. (2014). Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides. Nature.Published online 16 March 2014. As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors. Full Text
Blanco, F.F., Sanduja, S., Deane, N.G., Blackshear, P.J., and Dixon, D.A. (2014). Transforming Growth Factor beta Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin. Molecular and Cellular Biology 34, 180-195.Transforming growth factor beta (TGF-beta) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3= untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-beta inhibited ARE-mRNA expression. This effect of TGF-beta was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-beta /Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-beta induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-beta -dependent P-body formation and promoted growth inhibition by TGF-beta. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-beta /Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-beta's antiproliferative properties and identify TGF-beta as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation. Full Text
Breker, M., Gymrek, M., Moldavski, O., and Schuldiner, M. (2014). LoQAtE-Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast. Nucleic Acids Research 42, D726-D730.Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called 'LoQAtE' (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H2O2 and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations. Full Text
Buganim, Y., Markoulaki, S., van Wietmarschen, N., Hoke, H., Wu, T., Ganz, K., Akhtar-Zaidi, B., He, Y., Abraham, B.J., Porubsky, D, Kulenkampff E, Faddah DA, Shi L, Gao Q, Sarkar S, Cohen M, Goldmann J, Nery JR, Schultz MD, Ecker JR, Xiao A, Young RA, Lansdorp PM, and Jaenisch R. (2014). The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection. Cell Stem Cell 15, 295-309.Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human. Full Text.
Caraveo, G., Auluck, P.K., Whitesell, L., Chung, C.Y., Baru, V., Mosharov, E.V., Yan, X., Ben-Johny, M., Soste, M., Picotti, P., et al and Susan Lindquist . (2014). Calcineurin determines toxic versus beneficial responses to alpha-synuclein. Proc Natl Acad Sci U S A Early Edition Calcineurin (CN) is a highly conserved Ca2+-calmodulin (CaM)-dependent phosphatase that senses Ca2+ concentrations and transduces that information into cellular responses. Ca2+ homeostasis is disrupted by alpha-synuclein (alpha-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that alpha-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca2+, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies. Full Text.
Cassady, J.P., D'Alessio, A.C., Sarkar, S., Dani, V.S., Fan, Z.P., Ganz, K., Roessler, R., Sur, M., Young, R.A., and Jaenisch, R. (2014). Direct Lineage Conversion of Adult Mouse Liver Cells and B Lymphocytes to Neural Stem Cells. Stem Cell Reports. Available online 6 November 2014 Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors. Full Text.
Chantranupong, L., Wolfson, R.L., Orozco, J.M., Saxton, R.A., Scaria, S.M., Bar-Peled, L., Spooner, E., Isasa, M., Gygi, S.P., and Sabatini, D.M. (2014). The Sestrins Interact with GATOR2 to Negatively Regulate the Amino-Acid-Sensing Pathway Upstream of mTORC1. Cell Reports Sep 24. [Epub ahead of print] The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway. Full Text.
Cheeseman, I.M. (2014). The Kinetochore. Cold Spring Harb Perspect Biol 6(7). pii: a015826. Critical requirement for mitosis is the distribution of genetic material to the two daughter cells. The central player in this process is the macromolecular kinetochore structure, which binds to both chromosomal DNA and spindle microtubule polymers to direct chromosome alignment and segregation. This review will discuss the key kinetochore activities required for mitotic chromosome segregation, including the recognition of a specific site on each chromosome, kinetochore assembly and the formation of kinetochore-microtubule connections, the generation of force to drive chromosome segregation, and the regulation of kinetochore function to ensure that chromosome segregation occurs with high fidelity. Full Text.
Chen2, C., and Lodish, H.F. (2014). Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis. Experimental Hematology Mar 6. [Epub ahead of print] .Key transcriptional regulators of terminal erythropoiesis, such as GATA1 and TAL1, have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation and whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor Tfdp2 were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression, and knockdown of Tfdp2 results in significantly reduced rates of proliferation, as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis. Full Text
Chen, D., Sun, Y., Yuan, Y., Han, Z., Zhang, P., Zhang, J., You, M.J., Teruya-Feldstein, J., Wang, M., Gupta, S., et al. (2014). miR-100 Induces Epithelial-Mesenchymal Transition but Suppresses Tumorigenesis, Migration and Invasion. PLoS Genet 10, e1004177.Whether epithelial-mesenchymal transition (EMT) is always linked to increased tumorigenicity is controversial. Through microRNA (miRNA) expression profiling of mammary epithelial cells overexpressing Twist, Snail or ZEB1, we identified miR-100 as a novel EMT inducer. Surprisingly, miR-100 inhibits the tumorigenicity, motility and invasiveness of mammary tumor cells, and is commonly downregulated in human breast cancer due to hypermethylation of its host gene MIR100HG. The EMT-inducing and tumor-suppressing effects of miR-100 are mediated by distinct targets. While miR-100 downregulates E-cadherin by targeting SMARCA5, a regulator of CDH1 promoter methylation, this miRNA suppresses tumorigenesis, cell movement and invasion in vitro and in vivo through direct targeting of HOXA1, a gene that is both oncogenic and pro-invasive, leading to repression of multiple HOXA1 downstream targets involved in oncogenesis and invasiveness. These findings provide a proof-of-principle that EMT and tumorigenicity are not always associated and that certain EMT inducers can inhibit tumorigenesis, migration and invasion. Full Text
Chen3, X., Iliopoulos, D., Zhang, Q., Tang, Q., Greenblatt, M.B., Hatziapostolou, M., Lim, E., Tam, W.L., Ni, M., Chen, Y., et al. (2014). XBP1 promotes triple-negative breast cancer by controlling the HIF1alpha pathway. Nature. Apr 3 508(7494):103-7. Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)-a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)-is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1alpha (HIF1alpha) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1alpha that regulates the expression of HIF1alpha targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1alpha and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer. Full Text
Chen4, W.W., Birsoy, K., Mihaylova, M.M., Snitkin, H., Stasinski, I., Yucel, B., Bayraktar, E.C., Carette, J.E., Clish, C.B., Brummelkamp, T.R , David D. Sabatini, David M. Sabatini. (2014). Inhibition of ATPIF1 Ameliorates Severe Mitochondrial Respiratory Chain Dysfunction in Mammalian Cells. Cell Reports Volume 7, Issue 1, p27–34. Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human rho degrees cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology. Full Text
Cheng, A.W., Shi, J.H., Wong, P., Luo, K.L., Trepman, P., Wang, E.T., Choi, H., Burge, C.B., and Lodish, H.F. (2014). Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis. Blood 124, 598-610.The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation. Full Text.
Chipumuro, E., Marco, E., Christensen, C.L., Kwiatkowski, N., Zhang, T., Hatheway, C.M., Abraham, B.J., Sharma, B., Yeung, C., Altabef, A., Richard A. Young, et al. (2014). CDK7 Inhibition Suppresses Super-Enhancer-Linked Oncogenic Transcription in MYCN-Driven Cancer. Cell 159, 1126-1139.The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins. Full Text.
Choi3, S.H., Kim, Y.H., Hebisch, M., Sliwinski, C., Lee, S., D'Avanzo, C., Chen, H., Hooli, B., Asselin, C., Muffat, J., et al. (2014). A three-dimensional human neural cell culture model of Alzheimer's disease. Nature Published online 12 October . Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-beta plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-beta peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-beta-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-beta species and phosphorylated tau but did not demonstrate amyloid-beta plaques or neurofibrillary tangles. Here we report that FAD mutations in beta-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-beta, including amyloid-beta plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-beta generation with beta- or gamma-secretase inhibitors not only decreased amyloid-beta pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-beta-mediated tau phosphorylation. We have successfully recapitulated amyloid-beta and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders. Full Text.
Choi2, Y.J., Saez, B., Anders, L., Hydbring, P., Stefano, J., Bacon, N.A., Cook, C., Kalaszczynska, I., Signoretti, S., Young, R.A., et al. (2014). D-Cyclins Repress Apoptosis in Hematopoietic Cells by Controlling Death Receptor Fas and Its Ligand FasL. Developmental Cell Epub 2014 Jul 31. D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis. Full Text.
Choi, Y.J., and Anders, L. (2014). Signaling through cyclin D-dependent kinases. Oncogene 33, 1890-1903.Research over the past quarter century has identified cyclin D-dependent kinases, CDK4 and CDK6, as the major oncogenic drivers among members of the CDK superfamily. CDK4/6 are rendered hyperactive in the majority of human cancers through a multitude of genomic alterations. Sustained activation of these protein kinases provides cancer cells with the power to enter the cell cycle continuously by triggering G1-S-phase transitions and dramatically shortening the duration of the G1 phase. It has also become clear, however, that CDK4/6 effectively counter cancer cell-intrinsic tumor suppression mechanisms, senescence and apoptosis, which must be overcome during cell transformation and kept at bay throughout all stages of tumorigenesis. As a central 'node' in cellular signaling networks, cyclin D-dependent kinases sense a plethora of mitogenic signals to orchestrate specific transcriptional programs. As the complexity of the cellular signaling network regulated by these oncogenic kinases unfolds, much remains to be learned about its architecture, its dynamics and the consequences of its perturbation. Full Text.
Christensen, C.L., Kwiatkowski, N., Abraham, B.J., Carretero, J., Al-Shahrour, F., Zhang, T., Chipumuro, E., Herter-Sprie, G.S., Akbay, E.A., Altabef, A, Richard A. Young, et al. (2014). Targeting Transcriptional Addictions in Small Cell Lung Cancer with a Covalent CDK7 Inhibitor. Cancer Cell 26, 909-922.Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy. Full Text.
Chudnovsky, Y., Kim, D., Zheng, S., Whyte, W.A., Bansal, M., Bray, M.A., Gopal, S., Theisen, M.A., Bilodeau, S., Thiru, P., Julien Muffat, Omer H. Yilmaz, Maya Mitalipova, Richard A. Young, David M. Sabatini, et al(2014). ZFHX4 Interacts with the NuRD Core Member CHD4 and Regulates the Glioblastoma Tumor-Initiating Cell State. Cell Reports, 16 January 2014 .Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state. Full Text
Claessen, J.H., Sanyal, S., and Ploegh, H.L. (2014). The Chaperone BAG6 Captures Dislocated Glycoproteins in the Cytosol. PLoS One 9, e90204.Secretory and membrane (glyco)proteins are subject to quality control in the endoplasmic reticulum (ER) to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle. Full Text
Claw, K.G., George, R.D., and Swanson, W.J. (2014). Detecting Coevolution in Mammalian Sperm-Egg Fusion Proteins. Molecular Reproduction and Development 81, 531-538.Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs.
Corpas, M., Jimenez, R.C., Bongcam-Rudloff, E., Budd, A., Brazas, M.D., Fernandes, P.L., Gaeta, B., van Gelder, C., Korpelainen, E., Lewitter, F., et al. (2014). The GOBLET Training Portal: A Global Repository of Bioinformatics Training Materials, Courses and Trainers. Bioinformatics published online: September 4, 2014. Rapid technological advances have led to an explosion of biomedical data in recent years. The pace of change has inspired new, collaborative approaches for sharing materials and resources to help train life scientists both in the use of cutting-edge bioinformatics tools and databases, and in how to analyse and interpret large datasets. A prototype platform for sharing such training resources was recently created by the Bioinformatics Training Network (BTN). Building on this work, we have created a centralised portal for sharing training materials and courses, including a catalogue of trainers and course organisers, and an announcement service for training events. For course organisers, the portal provides opportunities to promote their training events; for trainers, the portal offers an environment for sharing materials, for gaining visibility for their work and promoting their skills; for trainees, it offers a convenient one-stop shop for finding suitable training resources and identifying relevant training events and activities locally and world-wide. Availability: http://mygoblet.org/training-portal CONTACT: manuel.corpas@tgac.ac.uk .Full Text.
Cuiffo, B.G., Campagne, A., Bell, G.W., Lembo, A., Orso, F., Lien, E.C., Bhasin, M.K., Raimo, M., Hanson, S.E., Marusyk, A., et al. (2014). MSC-Regulated MicroRNAs Converge on the Transcription Factor FOXP2 and Promote Breast Cancer Metastasis. Cell Stem Cell 15, 762-774.Mesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer. Full Text.
Damon, J.R., Pincus, D., and Ploegh, H.L. (2014). tRNA Thiolation Links Translation to Stress Responses in Saccharomyces cerevisiae. Molecular Biologyof theCell [Epub ahead of print] Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. While tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGluUUC, tGlnUUG and tLysUUU in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable, and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need. Full Text.
Dawlaty, M.M., Breiling, A., Le, T., Barrasa, M.I., Raddatz, G., Gao, Q., Powell, B.E., Cheng, A.W., Faull, K.F., Lyko, F., and Rudolph Jaenisch. (2014). Loss of tet enzymes compromises proper differentiation of embryonic stem cells. Dev Cell 29, 102-111.Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development. Full Text
DelVecchio, C.A., Feng, Y., Sokol, E.S., Tillman, E.J., Sanduja, S., Reinhardt, F., and Gupta, P.B. (2014). De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling. PLoS Biol 12, e1001945.Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. Full Text.
Denzler, R., Agarwal, V., Stefano, J., Bartel, D.P., and Stoffel, M. (2014). Assessing the ceRNA Hypothesis with Quantitative Measurements of miRNA and Target Abundance. Mol Cel Available online 1 May 201l Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (>/=1.5 x 105 added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effec t.Full Text.
Deuse, T., Wang, D., Stubbendorff, M., Itagaki, R., Grabosch, A., Greaves, L.C., Alawi, M., Grunewald, A., Hu, X., Hua, X. , Rudolf Jaenisch, et al. (2014). SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts. Cell Stem Cell Nov 20. [Epub ahead of print] The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies. Full Text.
Dowen2, J.M., Fan, Z.P., Hnisz, D., Ren, G., Abraham, B.J., Zhang, L.N., Weintraub, A.S., Schuijers, J., Lee, T.I., Zhao, K.and Richard A. Young (2014). Control of cell identity genes occurs in insulated neighborhoods in Mammalian chromosomes. Cell 159, 374-387.The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure. Full Text.
Dowen, J.M., and Young, R.A. (2014). SMC complexes link gene expression and genome architecture. Curr Opin Genet Dev 25C, 131-137.The structural maintenance of chromosomes (SMC) complexes are associated with transcriptional enhancers, promoters and insulators, where they contribute to the control of gene expression and genome structure. We review here recent insights into the interlinked roles of SMC complexes in gene expression and genome architecture. Among these, we note evidence that SMC complexes play important roles in the regulation of genes that control cell identity. We conclude by reviewing diseases associated with SMC mutations. Full Text.
Edwards, M.D., Symbor-Nagrabska, A., Dollard, L., Gifford, D.K., and Fink, G.R. (2014). Interactions between chromosomal and nonchromosomal elements reveal missing heritability. Proc Natl Acad Sci U S A 111(21):7719-7722.The measurement of any nonchromosomal genetic contribution to the heritability of a trait is often confounded by the inability to control both the chromosomal and nonchromosomal information in a population. We have designed a unique system in yeast where we can control both sources of information so that the phenotype of a single chromosomal polymorphism can be measured in the presence of different cytoplasmic elements. With this system, we have shown that both the source of the mitochondrial genome and the presence or absence of a dsRNA virus influence the phenotype of chromosomal variants that affect the growth of yeast. Moreover, by considering this nonchromosomal information that is passed from parent to offspring and by allowing chromosomal and nonchromosomal information to exhibit nonadditive interactions, we are able to account for much of the heritability of growth traits. Taken together, our results highlight the importance of including all sources of heritable information in genetic studies and suggest a possible avenue of attack for finding additional missing heritability. Full Text.
Efeyan A., Schweitzer, L.D., Bilate, A.M., Chang, S., Kirak, O., Lamming, D.W., and Sabatini, D.M. (2014). RagA, but Not RagB, Is Essential for Embryonic Development and Adult Mice. Developmental Cell Apr 22;. [Epub ahead of print].The mechanistic target of rapamycin complex 1 (mTORC1) integrates cues from growth factors and nutrients to control metabolism. In contrast to the growth factor input, genetic disruption of nutrient-dependent activation of mTORC1 in mammals remains unexplored. We engineered mice lacking RagA and RagB genes, which encode the GTPases responsible for mTORC1 activation by nutrients. RagB has limited expression, and its loss shows no effects on mammalian physiology. RagA deficiency leads to E10.5 embryonic death, loss of mTORC1 activity, and severe growth defects. Primary cells derived from these mice exhibit no regulation of mTORC1 by nutrients and maintain high sensitivity to growth factors. Deletion of RagA in adult mice is lethal. Upon RagA loss, a myeloid population expands in peripheral tissues. RagA-specific deletion in liver increases cellular responses to growth factors. These results show the essentiality of nutrient sensing for mTORC1 activity in mice and its suppression of PI3K/Akt signaling. Full Text
Eichhorn, S.W., Guo, H., McGeary, S.E., Rodriguez-Mias, R.A., Shin, C., Baek, D., Hsu, S.H., Ghoshal, K., Villen, J., and Bartel, D.P. (2014). mRNA Destabilization Is the Dominant Effect of Mammalian MicroRNAs by the Time Substantial Repression Ensues. Mol Cell Sep 24. [Epub ahead of print] MicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined globally. Here, we extend the global steady-state measurements to additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition, or translational state, mRNA destabilization explains most (66%->90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results imply that consequential miRNA-mediated repression is largely irreversible and provide other insights into the nature of miRNA-mediated regulation. They also simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects. Full Text.
Emlet, D.R., Gupta, P., Holgado-Madruga, M., Del Vecchio, C.A., Mitra, S.S., Han, S.Y., Li, G., Jensen, K.C., Vogel, H., Xu, L.W., et al. (2014). Targeting a Glioblastoma Cancer Stem-Cell Population Defined by EGF Receptor Variant III. Cancer Research 74, 1238-1249.The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII(+)/CD133(+) defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII+ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII(-) cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII(+)/CD133(+) coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII(+)/CD133(+) population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Full Text
Erdmann, R.M., Souza, A.L., Clish, C.B., and Gehring, M. (2014). 5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA. G3 Nov 6. [Epub ahead of print] (Bethesda) 5-hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. Here we systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we employed, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA. Full Text.
Erlich2, Y., Williams, J.B., Glazer, D., Yocum, K., Farahany, N., Olson, M., Narayanan, A., Stein, L.D., Witkowski, J.A., and Kain, R.C. (2014). Redefining genomic privacy: trust and empowerment. PLoS Biol 12, e1001983.Fulfilling the promise of the genetic revolution requires the analysis of large datasets containing information from thousands to millions of participants. However, sharing human genomic data requires protecting subjects from potential harm. Current models rely on de-identification techniques in which privacy versus data utility becomes a zero-sum game. Instead, we propose the use of trust-enabling techniques to create a solution in which researchers and participants both win. To do so we introduce three principles that facilitate trust in genetic research and outline one possible framework built upon those principles. Our hope is that such trust-centric frameworks provide a sustainable solution that reconciles genetic privacy with data sharing and facilitates genetic research. Full Text.
Erlich, Y., and Narayanan, A. (2014). Routes for breaching and protecting genetic privacy. Nature Rev Genetics Published online 08 May 2014. We are entering an era of ubiquitous genetic information for research, clinical care and personal curiosity. Sharing these data sets is vital for progress in biomedical research. However, a growing concern is the ability to protect the genetic privacy of the data originators. Here, we present an overview of genetic privacy breaching strategies. We outline the principles of each technique, indicate the underlying assumptions, and assess their technological complexity and maturation. We then review potential mitigation methods for privacy-preserving dissemination of sensitive data and highlight different cases that are relevant to genetic applications. Full Text.
Fallon, T.R., and Weng, J.K. (2014). A molecular gauge for nitrogen economy in plants. Cell 159, 977-978.Understanding nitrogen metabolism in plants holds promise for future agricultural improvements. Chellamuthu et al. now identify a feedback regulation in plant nitrogen metabolism through glutamine sensing. This mechanism appears to be conserved from algae to flowering plants with a few surprising exceptions. Full Text.
Feng, Y., Sokol, E.S., Del Vecchio, C.A., Sanduja, S., Claessen, J.H., Proia, T., Jin, D.X., Reinhardt, F., Ploegh, H.L., Wang, ,and Piyush B. Gupta. (2014). Epithelial-to-mesenchymal transition activates PERK-eIF2a and sensitizes cells to endoplasmic reticulum stress. Cancer Discovery Published OnlineFirst April 4, 2014. Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and drug resistance, yet few vulnerabilities of this state have been identified. Using selective small molecules as cellular probes, we show that induction of EMT greatly sensitizes cells to agents that perturb endoplasmic reticulum (ER) function. This sensitivity to ER perturbations is caused by the synthesis and secretion of large quantities of extracellular matrix (ECM) proteins by EMT cells. Consistent with their increased secretory output, EMT cells display a branched ER morphology and constitutively activate the PERK-eIF2alpha axis of the unfolded protein response (UPR). PERK activation is also required for EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression correlates strongly with both ECM and PERK-eIF2alpha genes, but not with other branches of the UPR. Taken together, our findings identify a novel vulnerability of EMT cells, and demonstrate that the PERK branch of the UPR is required for their malignancy. Full Text
Festuccia, W.T., Pouliot, P., Bakan, I., Sabatini, D.M., and Laplante, M. (2014). Myeloid-specific rictor deletion induces m1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide. PLoS One 9, e95432.The phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) axis plays a central role in attenuating inflammation upon macrophage stimulation with toll-like receptor (TLR) ligands. The mechanistic target of rapamycin complex 2 (mTORC2) relays signal from PI3K to Akt but its role in modulating inflammation in vivo has never been investigated. To evaluate the role of mTORC2 in the regulation of inflammation in vivo, we have generated a mouse model lacking Rictor, an essential mTORC2 component, in myeloid cells. Primary macrophages isolated from myeloid-specific Rictor null mice exhibited an exaggerated response to TLRs ligands, and expressed high levels of M1 genes and lower levels of M2 markers. To determine whether the loss of Rictor similarly affected inflammation in vivo, mice were either fed a high fat diet, a situation promoting chronic but low-grade inflammation, or were injected with lipopolysaccharide (LPS), which mimics an acute, severe septic inflammatory condition. Although high fat feeding contributed to promote obesity, inflammation, macrophage infiltration in adipose tissue and systemic insulin resistance, we did not observe a significant impact of Rictor loss on these parameters. However, mice lacking Rictor exhibited a higher sensitivity to sceptic shock when injected with LPS. Altogether, these results indicate that mTORC2 is a key negative regulator of macrophages TLR signalling and that its role in modulating inflammation is particularly important in the context of severe inflammatory challenges. These observations suggest that approaches aimed at modulating mTORC2 activity may represent a possible therapeutic approach for diseases linked to excessive inflammation. PDF
Filone, C.M., Caballero, I.S., Dower, K., Mendillo, M.L., Cowley, G.S., Santagata, S., Rozelle, D.K., Yen, J., Rubins, K.H., Hacohen, N., et al. (2014). The Master Regulator of the Cellular Stress Response (HSF1) Is Critical for Orthopoxvirus Infection. PLoS Pathog 10, e1003904.The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development. Full Text
Fink, G.R., Leshner, A.I., and Turekian, V.C. (2014) Science diplomacy with Cuba .Science 344, 1065 . Over a century ago, interactions between a Cuban scientist, Carlos Finlay, and a U.S. scientist, Jesse Lazear, led to an understanding of the role of the mosquito in transmitting yellow fever and to the development of effective countermeasures. Today, new infectious diseases confront both Cuba and the United States, but a longstanding diplomatic breach between the two nations now makes such valuable joint research more complicated, if not impossible. It is time for both governments to reconsider the rules that stand in the way of scientific collaboration, before a potentially deadly outbreak spreads through both countries and beyond. In particular, the U.S. government can make a relatively straightforward change to rules that would make collaboration much easier.. Full Text.
Flierl, A., Oliveira, L.M., Falomir-Lockhart, L.J., Mak, S.K., Hesley, J., Soldner, F., Arndt-Jovin, D.J., Jaenisch, R., Langston, J.W., Jovin, T.M., et al. (2014). Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication. PLoS One 9, e112413.Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered alpha-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of alpha-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD. Full Text.
Forster, R., Chiba, K., Schaeffer, L., Regalado, S.G., Lai, C.S., Gao, Q., Kiani, S., Farin, H.F., Clevers, H., Cost, G.J., Rudolf Jaenisch, et al. (2014). Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells. Stem Cell Reports 2, 838-852.Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.Full Text.
Frederick, K.K., Debelouchina, G.T., Kayatekin, C., Dorminy, T., Jacavone, A.C., Griffin, R.G., and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology 30 January 2014 .Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance. Full Text
Gilbert, L.A., Horlbeck, M.A., Adamson, B., Villalta, J.E., Chen, Y., Whitehead, E.H., Guimaraes, C., Panning, B., Ploegh, H.L., Bassik, M.C., et al. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell (in press). While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a approximately 1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways. Full Text.
Grabiner, B.C., Nardi, V., Birsoy, K., Possemato, R., Shen, K., Sinha, S., Jordan, A., Beck, A.H., and Sabatini, D.M. (2014). A diverse array of cancer-associated mTOR mutations are hyperactivating and can predict rapamycin sensitivity. Cancer Discovery Published OnlineFirst March 14, 2014 Genes encoding components of the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene for the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor Deptor. mTORC1 signaling in cells expressing various activating mutations remains sensitive to pharmacological mTOR inhibition, but is partially resistant to nutrient deprivation. Lastly, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and as in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency. Full Text
Guo, J.U., Agarwal, V., Guo, H.L., and Bartel, D.P. (2014). Expanded identification and characterization of mammalian circular RNAs. Genome Biology 15 409. Background: The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. Results: We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type-specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their neighboring linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a gene encoding a primate-specific zinc-finger protein, ZNF91. Conclusions: Our results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs. Full Text.
Gymrek, M. (2014). PyBamView: a browser based application for viewing short read alignments. Bioinformatics Aug 21. [Epub ahead of print] SUMMARY: Current sequence alignment browsers allow visualization of large and complex next-generation sequencing datasets. However, most of these tools provide inadequate display of insertions and can be cumbersome to use on large datasets. I implemented PyBamView, a lightweight web application for visualizing short read alignments. It provides an easy-to-use web interface for viewing alignments across multiple samples, with a focus on accurate visualization of insertions. Availability and Implementation: PyBamView is available as a standard python package. The source code is freely available under the MIT license at https://mgymrek.github.io/pybamview. CONTACT: mgymrek@mit.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Full Text.
Hacisuleyman, E., Goff, L.A., Trapnell, C., Williams, A., Henao-Mejia, J., Sun, L., McClanahan, P., Hendrickson, D.G., Sauvageau, M., Kelley, D.R. , Harvey F Lodish et al. (2014). Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nature Struct Mol Biol Published online 26 January 2014 . RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes. Full Text
Hattangadi, S.M., Martinez-Morilla, S., Patterson, H.C., Shi, J., Burke, K., Avila-Figueroa, A., Venkatesan, S., Wang, J., Paulsen, K., Gorlich, D , Harvey Lodish. (2014). Histones to the cytosol: Exportin 7 is essential for normal terminal erythroid nuclear maturation. Blood Aug 4. [Epub ahead of print] Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7) is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly, many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7. Full Text.
Hu, W., Yuan, B., and Lodish, H.F. (2014). Cpeb4-Mediated Translational Regulatory Circuitry Controls Terminal Erythroid Differentiation. Developmental Cell Sep 9 [Epub ahead of print] While we have considerable understanding of the transcriptional networks controlling mammalian cell differentiation, our knowledge of posttranscriptional regulatory events is very limited. Using differentiation of primary erythroid cells as a model, we show that the sequence-specific mRNA-binding protein Cpeb4 is strongly induced by the erythroid-important transcription factors Gata1 and Tal1 and is essential for terminal erythropoiesis. By interacting with the translation initiation factor eIF3, Cpeb4 represses the translation of a large set of mRNAs, including its own mRNA. Thus, transcriptional induction and translational repression combine to form a negative feedback loop to control Cpeb4 protein levels within a specific range that is required for terminal erythropoiesis. Our study provides an example of how translational control is integrated with transcriptional regulation to precisely control gene expression during mammalian cell differentiation. Full Text.
Hua, B.L., Li, S.R., and Orr-Weaver, T.L. (2014). The Role of Transcription in the Activation of a Drosophila Amplification Origin. G3-Genes Genomes Genetics 4, 2403-2408.The mechanisms that underlie metazoan DNA replication initiation, especially the connection between transcription and replication origin activation, are not well understood. To probe the role of transcription in origin activation, we exploited a specific replication origin in Drosophila melanogaster follicle cells, ori62, which coincides with the yellow-g2 transcription unit and exhibits transcription-dependent origin firing. Within a 10-kb genomic fragment that contains ori62 and is sufficient for amplification, RNA-sequencing analysis revealed that all detected RNAs mapped solely to the yellow-g2 gene. To determine whether transcription is required in cis for ori62 firing, we generated a set of tagged yellow-g2 transgenes in which we could prevent local transcription across ori62 by deletions in the yellow-g2 promoter. Surprisingly, inhibition of yellow-g2 transcription by promoter deletions did not affect ori62 firing. Our results reveal that transcription in cis is not required for ori62 firing, raising the possibility that a trans-acting factor is required specifically for the activation of ori62. This finding illustrates that a diversity of mechanisms can be used in the regulation of metazoan DNA replication initiation. Full Text.
Huang, Y.H., Zhu, C., Kondo, Y., Anderson, A.C., Gandhi, A., Russell, A., Dougan, S.K., Petersen, B.S., Melum, E., Pertel, T, .Hidde L. Ploegh, et al. (2014). CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature Published online 26 October. T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity. Full Text.
Jackson, W.S., Krost, C., Borkowski, A.W., and Kaczmarczyk, L. (2014). Translation of the Prion Protein mRNA Is Robust in Astrocytes but Does Not Amplify during Reactive Astrocytosis in the Mouse Brain. PLoS One 9, e95958.Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP), could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp), was replaced with that for green fluorescent protein (GFP). Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments. Full Text
Jacox2, L., Sindelka, R., Chen, J., Rothman, A., Dickinson, A., and Sive, H. (2014). The Extreme Anterior Domain Is an Essential Craniofacial Organizer Acting through Kinin-Kallikrein Signaling. Cell Reports
Jul 15. [Epub ahead of print]. The extreme anterior domain (EAD) is a conserved embryonic region that includes the presumptive mouth. We show that the Kinin-Kallikrein pathway is active in the EAD and necessary for craniofacial development in Xenopus and zebrafish. The mouth failed to form and neural crest (NC) development and migration was abnormal after loss of function (LOF) in the pathway genes kng, encoding Bradykinin (xBdk), carboxypeptidase-N (cpn), which cleaves Bradykinin, and neuronal nitric oxide synthase (nNOS). Consistent with a role for nitric oxide (NO) in face formation, endogenous NO levels declined after LOF in pathway genes, but these were restored and a normal face formed after medial implantation of xBdk-beads into LOF embryos. Facial transplants demonstrated that Cpn function from within the EAD is necessary for the migration of first arch cranial NC into the face and for promoting mouth opening. The study identifies the EAD as an essential craniofacial organizer acting through Kinin-Kallikrein signaling .Full Text.
Jacox, L.A., Dickinson, A.J., and Sive, H. (2014). Facial Transplants in Xenopus laevis Embryos. J Vis Exp. 2014 Mar 26 ; (85) .Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals. Full Text
Jarosz2, D.F., Lancaster, A.K., Brown, J.C., and Lindquist, S. (2014). An Evolutionarily Conserved Prion-like Element Converts Wild Fungi from Metabolic Specialists to Generalists. Cell 158, 1072-1082.[GAR(+)] is a protein-based element of inheritance that allows yeast (Saccharomyces cerevisiae) to circumvent a hallmark of their biology: extreme metabolic specialization for glucose fermentation. When glucose is present, yeast will not use other carbon sources. [GAR(+)] allows cells to circumvent this "glucose repression." [GAR(+)] is induced in yeast by a factor secreted by bacteria inhabiting their environment. We report that de novo rates of [GAR(+)] appearance correlate with the yeast's ecological niche. Evolutionarily distant fungi possess similar epigenetic elements that are also induced by bacteria. As expected for a mechanism whose adaptive value originates from the selective pressures of life in biological communities, the ability of bacteria to induce [GAR(+)] and the ability of yeast to respond to bacterial signals have been extinguished repeatedly during the extended monoculture of domestication. Thus, [GAR(+)] is a broadly conserved adaptive strategy that links environmental and social cues to heritable changes in metabolism. Full Text.
Jarosz, D.F., Brown, J.C., Walker, G.A., Datta, M.S., Ung, W.L., Lancaster, A.K., Rotem, A., Chang, A., Newby, G.A., Weitz, D.A, .Linda F. Bisson, and Susan Lindquist (2014). Cross-kingdom chemical communication drives a heritable, mutually beneficial prion-based transformation of metabolism. Cell 158, 1083-1093.In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR(+)], a protein-based epigenetic element, allows yeast to circumvent this "glucose repression" and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR(+)]. [GAR(+)] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR(+)] presents a unique example of Lamarckian inheritance. Full Text.
Jurkin, J., Henkel, T., Nielsen, A.F., Minnich, M., Popow, J., Kaufmann, T., Heindl, K., Hoffmann, T., Busslinger, M., and Martinez, J. (2014). The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells. The EMBO journal Published online. The unfolded protein response (UPR) is a conserved stress-signaling pathway activated after accumulation of unfolded proteins within the endoplasmic reticulum (ER). Active UPR signaling leads to unconventional, enzymatic splicing of XBP1 mRNA enabling expression of the transcription factor XBP1s to control ER homeostasis. While IRE1 has been identified as the endoribonuclease required for cleavage of this mRNA, the corresponding ligase in mammalian cells has remained elusive. Here, we report that RTCB, the catalytic subunit of the tRNA ligase complex, and its co-factor archease mediate XBP1 mRNA splicing both in vitro and in vivo. Depletion of RTCB in plasma cells of Rtcbfl/fl Cd23-Cre mice prevents XBP1s expression, which normally is strongly induced during plasma cell development. RTCB-depleted plasma cells show reduced and disorganized ER structures as well as severe defects in antibody secretion. Targeting RTCB and/or archease thus represents a promising strategy for the treatment of a growing number of diseases associated with elevated expression of XBP1s. Full Text.
Kanarek, N., Grivennikov, S.I., Leshets, M., Lasry, A., Alkalay, I., Horwitz, E., Shaul, Y.D., Stachler, M., Voronov, E., Apte, R.N., et al. (2014). Critical role for IL-1 beta in DNA damage-induced mucositis. Proceedings of the National Academy of Sciences of the United States of America 111, E702-E711. beta-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-kappa B inhibitor I kappa B. To appreciate tissue-specific roles of beta-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1 beta as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1 beta is induced by DNA damage via an NF-kappa B-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-kappa B, with failure to express the endogenous IL-1 beta receptor antagonist IL-1Ra upon fourallele loss. Antibody neutralization of IL-1 beta prevents epithelial tight junction dysfunction and alleviates mucositis in beta-TrCP-deficient mice. IL-1 beta antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis. Full Text
Karreman, M.A., Mercier, L., Schieber, N.L., Shibue, T., Schwab, Y., and Goetz, J.G. (2014). Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points. PLoS One 9, e114448.Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. Full Text.
Katz, Y., Li, F., Lambert, N.J., Sokol, E.S., Tam, W.L., Cheng, A.W., Airoldi, E.M., Lengner, C.J., Gupta, P.B., Yu, Z .,Rudolph Jaenisch, et al. (2014). Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state. Elife [Epub ahead of print] The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers, and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis of genetic mouse models in neuronal and mammary cell types, we found that Msis regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT) and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited translation of genes required for EMT, including Jagged1, and repressed EMT in cell culture and in mammary gland in vivo, while knockdown in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types. Full Text.
Kayatekin, C., Matlack, K.E., Hesse, W.R., Guan, Y., Chakrabortee, S., Russ, J., Wanker, E.E., Shah, J.V., and Lindquist, S. (2014). Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1. Proc Natl Acad Sci U S A Aug 4. pii: 201412504. [Epub ahead of print] Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings. Full Text.
Keitel, U., Scheel, A., Thomale, J., Halpape, R., Kaulfuss, S., Scheel, C., and Dobbelstein, M. (2014). Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation. Oncotarget Nov 17. [Epub ahead of print] The transition from an epithelial to a mesenchymal phenotype (EMT) confers increased invasiveness and clonogenic potential to tumor cells. We used a breast epithelium-derived cell culture model to evaluate the impact of EMT on the cellular sensitivity towards chemotherapeutics and apoptotic stimuli. Cells that had passed through an EMT acquired resistance towards chemotherapeutics and death ligands. Mechanistically, we found that the levels of the apoptosis inhibitor Bcl-xL were strongly enhanced in mesenchymal versus epithelial cells, whereas the pro-apoptotic proteins Bim and Puma were diminished. Clinical samples from breast cancer showed enhanced Bcl-xL staining in cells that had dispersed into the desmoplastic stroma, as compared to cells that were part of large tumor cell aggregates, suggesting increased Bcl-xL expression when cells invade the stroma. Bcl-xL was necessary for apoptotic resistance in mesenchymal cells, and its expression was sufficient to confer such resistance to epithelial cells. To antagonize Bcl-xL, BH3-mimetics were used. They successfully interfered with the proliferation and survival of mesenchymal cells, and also inhibited the growth of xenograft tumors raised from the mesenchymal subpopulation. We conclude that enhanced Bcl-xL levels confer resistance to cells upon EMT, and that Bcl-xL represents a promising target for therapy directed against invasive cancer cells. Full Text.
Kern, D.M., Kim, T., Rigney, M., Hattersley, N., Desai, A., and Cheeseman, I.M. (2014). The outer kinetochore protein KNL-1 contains a defined oligomerization domain in nematodes. Mol Biol Cell [Epub ahead of print] The kinetochore is a large, macromolecular assembly that is essential for connecting chromosomes to microtubules during mitosis. Despite the recent identification of multiple kinetochore components, the nature and organization of the higher order kinetochore structure remain unknown. The outer kinetochore KNL-1/Mis12 complex/Ndc80 complex (KMN) network plays a key role in generating and sensing microtubule attachments. Here, we demonstrate that Caenorhabditis elegans KNL-1 exists as an oligomer and we identify a specific domain in KNL-1 responsible for this activity. An N-terminal KNL-1 domain from both C. elegans and the related nematode C. remanei oligomerizes into a decameric assembly that appears roughly circular when visualized by electron microscopy. Based on sequence and mutational analysis, we identify a small hydrophobic region as responsible for this oligomerization activity. However, mutants that precisely disrupt KNL-1 oligomerization did not alter KNL-1 localization or result in the loss of embryonic viability based on gene replacements in C. elegans. In C. elegans, KNL-1 oligomerization may coordinate with other kinetochore activities to ensure the proper organization, function, and sensory capabilities of the kinetochore-microtubule attachment. Full Text.
Kim, H.J., Cho, H., Alexander, R., Patterson, H.C., Gu, M., Lo, K.A., Xu, D., Goh, V.J., Nguyen, L.N., Chai, X.,Cher X. Huang , Harvey Lodish, et al. (2014). MicroRNAs are required for the feature maintenance and differentiation of brown adipocytes. Diabetes. Published online before print July 9, 2014. Brown adipose tissue is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs as essential regulators of brown adipocyte differentiation, but it remains unknown whether microRNAs are required for the feature maintenance of mature brown adipocytes. To address this question, we ablated Dgcr8, a key regulator of the microRNA biogenesis pathway, in mature brown as well as white adipocytes. The adipose tissue -specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat, and the mice were intolerant to cold exposure. In vitro primary brown adipocyte cultures confirmed that microRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that microRNAs are essential for the browning of subcutaneous white adipocyte both in vitro and in vivo. Using this animal model, we performed microRNA expression profiling analysis and identified a set of BAT-specific microRNAs that are up-regulated during brown adipocyte differentiation and enriched in brown fat compared to other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of microRNAs in the maintenance as well as the differentiation of brown adipocytes Full Text.
Klemm, S., Semrau, S., Wiebrands, K., Mooijman, D., Faddah, D.A., Jaenisch, R., and van Oudenaarden, A. (2014). Transcriptional profiling of cells sorted by RNA abundance. Nature Methods Published online
30 March 2014. We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of mouse induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins. Full Text
Knight, Z.A., Schmidt, S.F., Birsoy, K., Tan, K., and Friedman, J.M. (2014). A critical role for mTORC1 in erythropoiesis and anemia. Elife, e01913 .Red blood cells (RBC) must coordinate their rate of growth and proliferation with the availability of nutrients, such as iron, but the signaling mechanisms that link nutritional state to RBC growth are incompletely understood. We performed a screen for cell types that have high levels of signaling through mTORC1, a protein kinase that couples nutrient availability to cell growth. This screen revealed that reticulocytes show high levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1. We found that mTORC1 activity in RBCs is regulated by dietary iron, and that genetic activation or inhibition of mTORC1 results in macrocytic or microcytic anemia, respectively. Finally, ATP competitive mTOR inhibitors reduced RBC proliferation and were lethal after treatment with phenylhydrazine, an inducer of hemolysis. These results identify the mTORC1 pathway as a critical regulator of RBC growth and proliferation, and establish that perturbations in this pathway result in anemia.Full Text.
Koenig2, P.A., Nicholls, P.K., Schmidt, F.I., Hagiwara, M., Maruyama, T., Frydman, G.H., Watson, N., Page, D.C., and Ploegh, H.L. (2014). The E2 Ubiquitin Conjugating Enzyme UBE2J1 is Required for Spermiogenesis in Mice. J Biol Chem ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1-/- mice have reduced viability and fail to thrive early after birth. Male Ube2j1-/- mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1-/- elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control. Full Text.
Koenig, P.A., and Ploegh, H.L. (2014). Protein quality control in the endoplasmic reticulum. F1000 Prime Rep 6, 49. THE TOPOLOGICAL BARRIERS DEFINED BY BIOLOGICAL MEMBRANES ARE NOT IMPERMEABLE: from small solutes to intact proteins, specialized transport and translocation mechanisms adjust to the cell's needs. Here, we review the removal of unwanted proteins from the endoplasmic reticulum (ER) and emphasize the need to extend observations from tissue culture models and simple eukaryotes to studies in whole animals. The variation in protein production and composition that characterizes different cell types and tissues requires tailor-made solutions to exert proper control over both protein synthesis and breakdown. The ER is an organelle essential to achieve and maintain such homeostasis .Full Text.
Koubova, J., Hu, Y.C., Bhattacharyya, T., Soh, Y.Q., Gill, M.E., Goodheart, M.L., Hogarth, C.A., Griswold, M.D., and Page, D.C. (2014). Retinoic Acid activates two pathways required for meiosis in mice. PLoS Genet 10, e1004541.In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA), the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function. Full Text.
Kronja2, I., Whitfield, Z.J., Yuan, B., Dzeyk, K., Kirkpatrick, J., Krijgsveld, J., and Orr-Weaver, T.L. (2014). Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition. Proc Natl Acad Sci U S A Oct 27. [Epub ahead of print] The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/CCORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C). Full Text.
Kronja, I., Yuan, B., Eichhorn, S.W., Dzeyk, K., Krijgsveld, J., Bartel, D.P., and Orr-Weaver, T.L. (2014). Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition. Cell Reports Available online 29 May 2014 The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity. Full Text.
Kwiatkowski, N., Zhang, T., Rahl, P.B., Abraham, B.J., Reddy, J., Ficarro, S.B., Dastur, A., Amzallag, A., Ramaswamy, S., Tesar, B, Nancy M. Hannett,.Young RA, et al. (2014). Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature. Published online 22 June 2014. Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state. Full Text.
Lam, F.H., Ghaderi, A., Fink, G.R., and Stephanopoulos, G. (2014). Engineering alcohol tolerance in yeast. Science 346, 71-75. Ethanol toxicity in the yeast Saccharomyces cerevisiae limits titer and productivity in the industrial production of transportation bioethanol. We show that strengthening the opposing potassium and proton electrochemical membrane gradients is a mechanism that enhances general resistance to multiple alcohols. The elevation of extracellular potassium and pH physically bolsters these gradients, increasing tolerance to higher alcohols and ethanol fermentation in commercial and laboratory strains (including a xylose-fermenting strain) under industrial-like conditions. Production per cell remains largely unchanged, with improvements deriving from heightened population viability. Likewise, up-regulation of the potassium and proton pumps in the laboratory strain enhances performance to levels exceeding those of industrial strains. Although genetically complex, alcohol tolerance can thus be dominated by a single cellular process, one controlled by a major physicochemical component but amenable to biological augmentation. Full Text.
Lambert, N., Robertson, A., Jangi, M., McGeary, S., Sharp, P.A., and Burge, C.B. (2014). RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins. Mol Cell Vol. 54, Issue 5, 5 June 2014, Pages 887–900. Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity. Full Text.
Lamming2, D.W., Mihaylova, M.M., Katajisto, P., Baar, E.L., Yilmaz, O.H., Hutchins, A., Gultekin, Y., Gaither, R., and Sabatini, D.M. (2014). Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan. Aging Cell published online: 25 JUL 2014 Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females. PDF
Lamming, D.W., Demirkan, G., Boylan, J.M., Mihaylova, M.M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D.M., and Gruppuso, P.A. (2014). Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2). FASEB Journal 28, 300-315.The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [Rictor-knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo. Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2-mediated events. Hepatic mTORC2 regulated a complex gene expression and post-translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell-autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2-related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.Lamming, D. W., Demirkan, G., Boylan, J. M., Mihaylova, M. M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D. M., Gruppuso, P. A. Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2) .Full Text.
Lancaster, A.K., Nutter-Upham, A., Lindquist, S., and King, O.D. (2014). PLAAC: a web and command-line application to identify proteins with Prion-Like Amino Acid Composition. Bioinformatics(2014) doi: 10.1093/bioinformatics/btu310 SUMMARY: Prions are self-templating protein aggregates that stably perpetuate distinct biological states and are of keen interest to researchers in both evolutionary and biomedical science. The best understood prions are from yeast and have a prion-forming domain with strongly biased amino acid composition, most notably enriched for Q or N. PLAAC is a web application that scans protein sequences for domains with P: rion-L: ike A: mino A: cid C: omposition. Users can upload sequence files, or paste sequences directly into a textbox. PLAAC ranks the input sequences by several summary scores and allows scores along sequences to be visualized. Text output files can be downloaded for further analyses, and visualizations saved in PDF and PNG formats. Availability and Implementation: http://plaac.wi.mit.edu/. The Ruby-based web framework, and the command-line software (implemented in Java, with visualization routines in R) are available at: http://github.com/whitehead/plaac under the MIT license. All software can be run under OS X, Windows, and Unix Full Text.
Lee, C.C., Freinkman, E., Sabatini, D.M., and Ploegh, H.L. (2014). The Protein Synthesis Inhibitor Blasticidin S Enters Mammalian Cells via Leucine-Rich Repeat-Containing Protein 8D. J Biol Chem Papers in Press. Published on April 29, 2014. Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized and no specific function has been assigned to them. There is no consensus on how this family of proteins might function, because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes.Full Text.
Lee2, S., Kang, D., Ra, E.A., Lee, T.A., Ploegh, H.L., and Park, B. (2014). Negative Self-Regulation of TLR9 Signaling by Its N-Terminal Proteolytic Cleavage Product. Journal of Immunology Sep 3. [Epub ahead of print]TLR signaling is essential to innate immunity against microbial invaders and must be tightly controlled. We have previously shown that TLR9 undergoes proteolytic cleavage processing by lysosomal proteases to generate two distinct fragments. The C-terminal cleavage product plays a critical role in activating TLR9 signaling; however, the precise role of the N-terminal fragment, which remains in lysosomes, in the TLR9 response is still unclear. In this article, we report that the N-terminal cleavage product negatively regulates TLR9 signaling. Notably, the N-terminal fragment promotes the aspartic protease-mediated degradation of the C-terminal fragment in endolysosomes. Furthermore, the N-terminal TLR9 fragment physically interacts with the C-terminal product, thereby inhibiting the formation of homodimers of the C-terminal fragment; this suggests that the monomeric C-terminal product is more susceptible to attack by aspartic proteases. Together, these results suggest that the N-terminal TLR9 proteolytic cleavage product is a negative self-regulator that prevents excessive TLR9 signaling activity. Full Text.
Lesch, B.J., and Page, D.C. (2014). Poised chromatin in the mammalian germ line. Development 141, 3619-3626.Poised (bivalent) chromatin is defined by the simultaneous presence of histone modifications associated with both gene activation and repression. This epigenetic feature was first observed at promoters of lineage-specific regulatory genes in embryonic stem cells in culture. More recent work has shown that, in vivo, mammalian germ cells maintain poised chromatin at promoters of many genes that regulate somatic development, and that they retain this state from fetal stages through meiosis and gametogenesis. We hypothesize that the poised chromatin state is essential for germ cell identity and function. We propose three roles for poised chromatin in the mammalian germ line: prevention of DNA methylation, maintenance of germ cell identity and preparation for totipotency. We discuss these roles in the context of recently proposed models for germline potency and epigenetic inheritance. Full Text.
Li, X., Wei, W., Zhao, Q.Y., Widagdo, J., Baker-Andresen, D., Flavell, C.R., D'Alessio, A., Zhang, Y., and Bredy, T.W. (2014). Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation. Proc Natl Acad Sci U S A 111, 7120-7125.5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation. Full Text.
Lim, S.M., Westover, K.D., Ficarro, S.B., Harrison, R.A., Choi, H.G., Pacold, M.E., Carrasco, M., Hunter, J., Kim, N.D., Xie, T., et al. (2014). Therapeutic Targeting of Oncogenic K-Ras by a Covalent Catalytic Site Inhibitor. Angewandte Chemie-International Edition 53, 199-204.We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling. Full Text
Lourido, S., and Moreno, S.N. (2014). The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. Cell calcium Available online 31 December 2014. Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support Apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in Apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites.
Lu, H., Clauser, K.R., Tam, W.L., Frose, J., Ye, X., Eaton, E.N., Reinhardt, F., Donnenberg, V.S., Bhargava, R., Carr, S.A and Robert A. Weinberg. (2014). A breast cancer stem cell niche supported by juxtacrine signalling from monocytes and macrophages. Nat Cell Biol Sep 28. [Epub ahead of print] The cell-biological program termed the epithelial-mesenchymal transition (EMT) confers on cancer cells mesenchymal traits and an ability to enter the cancer stem cell (CSC) state. However, the interactions between CSCs and their surrounding microenvironment are poorly understood. Here we show that tumour-associated monocytes and macrophages (TAMs) create a CSC niche through juxtacrine signalling with CSCs. We performed quantitative proteomic profiling and found that the EMT program upregulates the expression of CD90, also known as Thy1, and EphA4, which mediate the physical interactions of CSCs with TAMs by directly binding with their respective counter-receptors on these cells. In response, the EphA4 receptor on the carcinoma cells activates Src and NF-kappaB. In turn, NF-kappaB in the CSCs induces the secretion of a variety of cytokines that serve to sustain the stem cell state. Indeed, admixed macrophages enhance the CSC activities of carcinoma cells. These findings underscore the significance of TAMs as important components of the CSC niche. Full Text.
Ludwig, L.S., Gazda, H.T., Eng, J.C., Eichhorn, S.W., Thiru, P., Ghazvinian, R., George, T.I., Gotlib, J.R., Beggs, A.H., Sieff, C.A.,Harvey F Lodish &Vijay G Sankaran, et al. (2014). Altered translation of GATA1 in Diamond-Blackfan anemia. Nature Medicine Published online 22 June 2014. Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.Full Text.
Lyons, D.C., Martindale, M.Q., and Srivastava, M. (2014). The Cell's View of Animal Body-Plan Evolution. Integr Comp Biol Aug 8. [Epub ahead of printl An adult animal's form is shaped by the collective behavior of cells during embryonic development. To understand the forces that drove the divergence of animal body-plans, evolutionary developmental biology has focused largely on studying genetic networks operating during development. However, it is less well understood how these networks modulate characteristics at the cellular level, such as the shape, polarity, or migration of cells. We organized the "Cell's view of animal body plan evolution" symposium for the 2014 The Society for Integrative and Comparative Biology meeting with the explicit goal of bringing together researchers studying the cell biology of embryonic development in diverse animal taxa. Using a broad range of established and emerging technologies, including live imaging, single-cell analysis, and mathematical modeling, symposium participants revealed mechanisms underlying cells' behavior, a few of which we highlight here. Shape, adhesion, and movements of cells can be modulated over the course of evolution to alter adult body-plans and a major theme explored during the symposium was the role of actomyosin in coordinating diverse behaviors of cells underlying morphogenesis in a myriad of contexts. Uncovering whether conserved or divergent genetic mechanisms guide the contractility of actomyosin in these systems will be crucial to understanding the evolution of the body-plans of animals from a cellular perspective. Many speakers presented research describing developmental phenomena in which cell division and tissue growth can control the form of the adult, and other presenters shared work on studying cell-fate specification, an important source of novelty in animal body-plans. Participants also presented studies of regeneration in annelids, flatworms, acoels, and cnidarians, and provided a unifying view of the regulation of cellular behavior during different life-history stages. Additionally, several presentations highlighted technological advances that glean mechanistic insights from new and emerging model systems, thereby providing the phylogenetic breadth so essential for studying animal evolution. Thus, we propose that an explicit study of cellular phenomena is now possible for a wide range of taxa, and that it will be highly informative for understanding the evolution of animal body-plans. Full Text.
Maetzel, D., Sarkar, S., Wang, H., Abi-Mosleh, L., Xu, P., Cheng, A.W., Gao, Q., Mitalipova, M., and Jaenisch, R. (2014). Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells. Stem Cell Reports 2, 866-880.Niemann-Pick type C (NPC) disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs) using transcription activator-like effector nucleases (TALENs). We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease. Full Text.
Mansour, M.R., Abraham, B.J., Anders, L., Berezovskaya, A., Gutierrez, A., Durbin, A.D., Etchin, J., Lawton, L., Sallan, S.E., Silverman, L.B,.Richard A Young, et al. (2014). An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science Published Online November 13 In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, which recruit much of the cell's transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, suggesting a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells. Full Text.
Martz, C.A., Ottina, K.A., Singleton, K.R., Jasper, J.S., Wardell, S.E., Peraza-Penton, A., Anderson, G.R., Winter, P.S., Wang, T., Alley, H.M,.David M. Sabatini, et al. (2014). Systematic identification of signaling pathways with potential to confer anticancer drug resistance. Sci Signal 7, ra121.Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a gamma-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts. Full Text.
Matlack, K.E., Tardiff, D.F., Narayan, P., Hamamichi, S., Caldwell, K.A., Caldwell, G.A., and Lindquist, S. (2014). Clioquinol promotes the degradation of metal-dependent amyloid-beta (Abeta) oligomers to restore endocytosis and ameliorate Abeta toxicity. Proc Natl Acad Sci U S A Mar 3. [Epub ahead of print] .Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-beta peptide (Abeta) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Abeta has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Abeta toxicity where the Abeta peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Abeta 1-42 peptide forms more oligomers and is more toxic than Abeta 1-40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of approximately 140,000 compounds for rescue of Abeta toxicity. Of approximately 30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Abeta burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Abeta secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Abeta peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Abeta oligomer formation contributes to Abeta toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Abeta yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform. PDF
McAllister, S.S., and Weinberg, R.A. (2014). The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. Nat Cell Biol 16, 717-727.Recent pre-clinical and clinical research has provided evidence that cancer progression is driven not only by a tumour's underlying genetic alterations and paracrine interactions within the tumour microenvironment, but also by complex systemic processes. We review these emerging paradigms of cancer pathophysiology and discuss how a clearer understanding of systemic regulation of cancer progression could guide development of new therapeutic modalities and efforts to prevent disease relapse following initial diagnosis and treatment. Full Text.
McKinley, K.L., and Cheeseman, I.M. (2014). Polo-like Kinase 1 Licenses CENP-A Deposition at Centromeres. Cell 158, 397-411.To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity. Full Text.
Mihaylova, M.M., Sabatini, D.M., and Yilmaz, O.H. (2014). Dietary and Metabolic Control of Stem Cell Function in Physiology and Cancer. Cell Stem Cell 14, 292-305.Organismal diet has a profound impact on tissue homeostasis and health in mammals. Adult stem cells are a keystone of tissue homeostasis that alters tissue composition by balancing self-renewal and differentiation divisions. Because somatic stem cells may respond to shifts in organismal physiology to orchestrate tissue remodeling and some cancers are understood to arise from transformed stem cells, there is a likely possibility that organismal diet, stem cell function, and cancer initiation are interconnected. Here we will explore the emerging effects of diet on nutrient-sensing pathways active in mammalian tissue stem cells and their relevance to normal and cancerous growth. Full Text
Nam, J.W., Rissland, O.S., Koppstein, D., Abreu-Goodger, C., Jan, C.H., Agarwal, V., Yildirim, M.A., Rodriguez, A., and Bartel, D.P. (2014). Global Analyses of the Effect of Different Cellular Contexts on MicroRNA Targeting. Molecular Cell, Volume 53, Issue 6, 1031-1043. MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development-with its resulting diversity of cellular contexts-impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3' UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3' UTR isoforms revealed that they influence approximately 10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3' UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3' UTR landscape helps predict targeting efficacy and explain differential regulation that is observed. Full Text
Narayan, P., Ehsani, S., and Lindquist, S. (2014). Combating neurodegenerative disease with chemical probes and model systems. Nature Chem Biol 10, 911-920.The disheartening results of recent clinical trials for neurodegenerative disease (ND) therapeutics underscore the need for a more comprehensive understanding of the underlying disease biology before effective therapies can be devised. One hallmark of many NDs is a disruption in protein homeostasis. Therefore, investigating the role of protein homeostasis in these diseases is central to delineating their underlying pathobiology. Here, we review the seminal role that chemical biology has played in furthering the research on and treatment of dysfunctional protein homeostasis in NDs. We also discuss the vital and predictive role of model systems in identifying conserved homeostasis pathways and genes therein that are altered in neurodegeneration. Integrating approaches from chemical biology with the use of model systems yields a powerful toolkit with which to unravel the complexities of ND biology. Full Text.
Nicolae, C.M., Aho, E.R., Vlahos, A.H.S., Choe, K.N., De, S., Karras, G.I., and Moldovan, G.L. (2014). The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen ( PCNA) and Is Required for DNA Damage Tolerance. Journal of Biological Chemistry 289, 13627-13637.Background: PCNA mono-ubiquitination at stalled replication forks recruits translesion synthesis polymerases for fork restart. Results: The mono-ADP-ribosyltransferase PARP10 interacts with PCNA through a PIP-box. PARP10 knockdown results in DNA damage hypersensitivity and defective translesion synthesis. Conclusion: PARP10 participates in PCNA-dependent DNA damage tolerance. Significance: This is the first time that post-translational modification by mono-ADP-ribosylation is implicated in DNA repair. All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation. Full Text.
Nogueira, C., Kashevsky, H., Pinto, B., Clarke, A., and Orr-Weaver, T.L. (2014). Regulation of Centromere Localization of the Drosophila Shugoshin MEI-S332 and Sister-Chromatid Cohesion in Meiosis. G3 (Bethesda)Early Online July 31, 2014 The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. We investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported here by localization and function studies in meiosis. Phospho-resistant and phospho-mimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phospho-mimetic mutation in the AuroraB phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly. PDF
, J.T., Kozhevnikova, E.N., Verrijzer, C.P., Pindyurin, A.V., Andreyeva, E.N., Shloma, V.V., Zhimulev, I.F., and Orr-Weaver, T.L. (2014). DNA Copy-Number Control through Inhibition of Replication Fork Progression. Cell Rep 9, 841-849.Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression. Full Text.
Ntziachristos, P., Tsirigos, A., Welstead, G.G., Trimarchi, T., Bakogianni, S., Xu, L., Loizou, E., Holmfeldt, L., Strikoudis, A., King, B., et al and Rudolph Jaenisch. (2014). Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia. Nature Published online 17 August 2014 T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors. Full Text.
Pattabiraman, D.R., and Weinberg, R.A. (2014). Tackling the cancer stem cells - what challenges do they pose? Nat Rev Drug Discov 13, 497-512. Since their identification in 1994, cancer stem cells (CSCs) have been objects of intensive study. Their properties and mechanisms of formation have become a major focus of current cancer research, in part because of their enhanced ability to initiate and drive tumour growth and their intrinsic resistance to conventional therapeutics. The discovery that activation of the epithelial-to-mesenchymal transition (EMT) programme in carcinoma cells can give rise to cells with stem-like properties has provided one possible mechanism explaining how CSCs arise and presents a possible avenue for their therapeutic manipulation. Here we address recent developments in CSC research, focusing on carcinomas that are able to undergo EMT. We discuss the signalling pathways that create these cells, cell-intrinsic mechanisms that could be exploited for selective elimination or induction of their differentiation, and the role of the tumour microenvironment in sustaining them. Finally, we propose ways to use our current knowledge of the complex biology of CSCs to design novel therapies to eliminate them. Full Text.
Petschnigg, J., Groisman, B., Kotlyar, M., Taipale, M., Zheng, Y., Kurat, C.F., Sayad, A., Sierra, J.R., Usaj, M.M., Snider, J., Irina Krykbaeva, Susan Lindquist, et al. (2014). The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nature Methods. Published online 23 March 2014. Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins. Full Text
Phillips, S., Prat, A., Sedic, M., Proia, T., Wronski, A., Mazumdar, S., Skibinski, A., Shirley, S.H., Perou, C.M., Gill, G.,Piyush B. Gupta, et al. (2014). Cell-State Transitions Regulated by SLUG Are Critical for Tissue Regeneration and Tumor Initiation. Stem Cell Reports 2, 633-647.Perturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis.Full Text.
Pignatta, D., Erdmann, R.M., Scheer, E., Picard, C.L., Bell, G.W., and Gehring, M. (2014). Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting. Elife, e03198.Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds. Full Text.
Pincus, D., Aranda-Diaz, A., Zuleta, I.A., Walter, P., and El-Samad, H. (2014). Delayed Ras/PKA signaling augments the unfolded protein response. Proceedings of the National Academy of Sciences of the United States of America 111, 14800-14805.During environmental, developmental, or genetic stress, the cell's folding capacity can become overwhelmed, and misfolded proteins can accumulate in all cell compartments. Eukaryotes evolved the unfolded protein response (UPR) to counteract proteotoxic stress in the endoplasmic reticulum (ER). Although the UPR is vital to restoring homeostasis to protein folding in the ER, it has become evident that the response to ER stress is not limited to the UPR. Here, we used engineered orthogonal UPR induction, deep mRNA sequencing, and dynamic flow cytometry to dissect the cell's response to ER stress comprehensively. We show that budding yeast augments the UPR with time-delayed Ras/PKA signaling. This second wave of transcriptional dynamics is independent of the UPR and is necessary for fitness in the presence of ER stress, partially due to a reduction in general protein synthesis. This Ras/PKA-mediated effect functionally mimics other mechanisms, such as translational control by PKR-like ER kinase (PERK) and regulated inositol-requiring enzyme 1 (IRE1)-dependent mRNA decay (RIDD), which reduce the load of proteins entering the ER in response to ER stress in metazoan cells. Full Text.
Pishesha, N., Thiru, P., Shi, J., Eng, J.C., Sankaran, V.G., and Lodish, H.F. (2014). Transcriptional divergence and conservation of human and mouse erythropoiesis. Proc Natl Acad Sci U S AMar 3. [Epub ahead of print] .Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease. PDF
Rahl, P.B., and Young, R.A. (2014). MYC and Transcription Elongation. Cold Spring Harbor Perspectives in Medicine 4(1). pii: a020990. Most transcription factors specify the subset of genes that will be actively transcribed in the cell by stimulating transcription initiation at these genes, but MYC has a fundamentally different role. MYC binds E-box sites in the promoters of active genes and stimulates recruitment of the elongation factor P-TEFb and thus transcription elongation. Consequently, rather than specifying the set of genes that will be transcribed in any particular cell, MYC's predominant role is to increase the production of transcripts from active genes. This increase in the transcriptional output of the cell's existing gene expression program, called transcriptional amplification, has a profound effect on proliferation and other behaviors of a broad range of cells. Transcriptional amplification may reduce rate-limiting constraints for tumor cell proliferation and explain MYC's broad oncogenic activity among diverse tissues. Full Text
Reymer, A., Frederick, K.K., Rocha, S., Beke-Somfai, T., Kitts, C.C., Lindquist, S., and Norden, B. (2014). Orientation of aromatic residues in amyloid cores: Structural insights into prion fiber diversity. Proc Natl Acad Sci U S A [Epub ahead of print] Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing. Full Text.
Riggi, N., Knoechel, B., Gillespie, S.M., Rheinbay, E., Boulay, G., Suva, M.L., Rossetti, N.E., Boonseng, W.E., Oksuz, O., Cook, E.B., Gymrek M , et al. (2014). EWS-FLI1 Utilizes Divergent Chromatin Remodeling Mechanisms to Directly Activate or Repress Enhancer Elements in Ewing Sarcoma. Cancer Cell 26, 668-681.The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation. Full Text.
Rosowski, E.E., Nguyen, Q.P., Camejo, A., Spooner, E., and Saeij, J.P.J. (2014). Toxoplasma gondii Inhibits Gamma Interferon ( IFN-gamma)- and IFN-beta-Induced Host Cell STAT1 Transcriptional Activity by Increasing the Association of STAT1 with DNA. Infection and Immunity 82, 706-719.The gamma interferon (IFN-gamma) response, mediated by the STAT1 transcription factor, is crucial for host defense against the intracellular pathogen Toxoplasma gondii, but prior infection with Toxoplasma can inhibit this response. Recently, it was reported that the Toxoplasma type II NTE strain prevents the recruitment of chromatin remodeling complexes containing Brahma-related gene 1 (BRG-1) to promoters of IFN-gamma-induced secondary response genes such as Ciita and major histocompatibility complex class II genes in murine macrophages, thereby inhibiting their expression. We report here that a type I strain of Toxoplasma inhibits the expression of primary IFN-gamma response genes such as IRF1 through a distinct mechanism not dependent on the activity of histone deacetylases. Instead, infection with a type I, II, or III strain of Toxoplasma inhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. This leads to increased IFN-gamma-induced binding of STAT1 at the IRF1 promoter in host cells and increased global IFN-gamma-induced association of STAT1 with chromatin. Toxoplasma type I infection also inhibits IFN-beta-induced interferon-stimulated gene factor 3-mediated gene expression, and this inhibition is also linked to increased association of STAT1 with chromatin. The secretion of proteins into the host cell by a type I strain of Toxoplasma without complete parasite invasion is not sufficient to block STAT1-mediated expression, suggesting that the effector protein responsible for this inhibition is not derived from the rhoptries. Full Text
Sarkar S., Maetzel, D., Korolchuk, V.I., and Jaenisch, R. (2014). Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder, Niemann-Pick type C1 disease. Autophagy 10, 1137-1140.Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-beta-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-beta-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.
ScherzShouval, R., Santagata, S., Mendillo, M.L., Sholl, L.M., Ben-Aharon, I., Beck, A.H., Dias-Santagata, D., Koeva, M., Stemmer, S.M., Whitesell, L., and Susan Lindquist (2014). The Reprogramming of Tumor Stroma by HSF1 Is a Potent Enabler of Malignancy. Cell 158, 564-578.Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-beta and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications .Full Text.
Schwartz2, S., Bernstein, D.A., Mumbach, M.R., Jovanovic, M., Herbst, R.H., Leon-Ricardo, B.X., Engreitz, J.M., Guttman, M., Satija, R., Lander, E.S., Gerald Fink, et al. (2014). Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA. Cell. Available online 11 September 2014. Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Psi-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Psi-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function. Full Text.
Schwartz, S., Mumbach, M.R., Jovanovic, M., Wang, T., Maciag, K., Bushkin, G.G., Mertins, P., Ter-Ovanesyan, D., Habib, N., Cacchiarelli, D.,Elizaveta Freinkman, Michael E. Pacold, et al. (2014). Perturbation of m6A Writers Reveals Two Distinct Classes of mRNA Methylation at Internal and 5' Sites. Cell Reports Volume 8, Issue 1, p284–296. N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification. PDF
Scimone2, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-beta-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts. Full Text.
Scimone, M.L., Lapan, S.W., and Reddien, P.W. (2014). A forkhead Transcription Factor Is Wound-Induced at the Planarian Midline and Required for Anterior Pole Regeneration. PLoS Genet 10, e1003999.Planarian regeneration requires positional information to specify the identity of tissues to be replaced as well as pluripotent neoblasts capable of differentiating into new cell types. We found that wounding elicits rapid expression of a gene encoding a Forkhead-family transcription factor, FoxD. Wound-induced FoxD expression is specific to the ventral midline, is regulated by Hedgehog signaling, and is neoblast-independent. FoxD is subsequently expressed within a medial subpopulation of neoblasts at wounds involving head regeneration. Ultimately, FoxD is co-expressed with multiple anterior markers at the anterior pole. Inhibition of FoxD with RNA interference (RNAi) results in the failure to specify neoblasts expressing anterior markers (notum and prep) and in anterior pole formation defects. FoxD(RNAi) animals fail to regenerate a new midline and to properly pattern the anterior blastema, consistent with a role for the anterior pole in organizing pattern of the regenerating head. Our results suggest that wound signaling activates a forkhead transcription factor at the midline and, if the head is absent, FoxD promotes specification of neoblasts at the prior midline for anterior pole regeneration. Full Text
Sexton, A.N., Regalado, S.G., Lai, C.S., Cost, G.J., O'Neil, C.M., Urnov, F.D., Gregory, P.D., Jaenisch, R., Collins, K., and Hockemeyer, D. (2014). Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation. Genes & Development published online August 15, 2014 Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.Full Text.
Shalgi, R., Hurt, J.A., Lindquist, S., and Burge, C.B. (2014). Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock. Cell Reports in Press. During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from approximately 1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes. PDF
Shaul, Y.D., Freinkman, E., Comb, W.C., Cantor, J.R., Tam, W.L., Thiru, P., Kim, D., Kanarek, N., Pacold, M.E., Chen, W.W, .Bierie B, Possemato R, Reinhardt F, Weinberg RA, Yaffe MB ,and Sabatini DM. (2014). Dihydropyrimidine accumulation is required for the epithelial-mesenchymal transition. Cell 158, 1094-1109. It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits. Full Text.
Shi, J., Kundrat, L., Pishesha, N., Bilate, A., Theile, C., Maruyama, T., Dougan, S.K., Ploegh, H.L., and Lodish, H.F. (2014). Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes. Proc Natl Acad Sci U S A Published online before print June 30, 2014. We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes. Full Text.
Sidik, S.M., Hackett, C.G., Tran, F., Westwood, N.J., and Lourido, S. (2014). Efficient Genome Engineering of Toxoplasma gondii Using CRISPR/Cas9. PLoS One 9, e100450.Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes. Full Text.
Skibinski, A., Breindel, J.L., Prat, A., Galvan, P., Smith, E., Rolfs, A., Gupta, P.B., Labaer, J., and Kuperwasser, C. (2014). The Hippo Transducer TAZ Interacts with the SWI/SNF Complex to Regulate Breast Epithelial Lineage Commitment. Cell Reports 06 March 2014 .Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. We identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes. Full Text
Smith, J.E., Alvarez-Dominguez, J.R., Kline, N., Huynh, N.J., Geisler, S., Hu, W., Coller, J., and Baker, K.E. (2014). Translation of Small Open Reading Frames within Unannotated RNA Transcripts in Saccharomyces cerevisiae. Cell Rep 7, 1858-1866.High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10-96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.Full Text.
Soh, Y.Q., Alfoldi, J., Pyntikova, T., Brown, L.G., Graves, T., Minx, P.J., Fulton, R.S., Kremitzki, C., Koutseva, N., Mueller, J.L., Steve Rozen, Jennifer F. Hughes, Helen Skaletsky, and David C. Page, et al. (2014). Sequencing the mouse y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes. Cell 159, 800-813.We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism. Full Text.
Soliman, M.A., Rahman, A.M.A., Lamming, D.A., Birsoy, K., Pawling, J., Frigolet, M.E., Lu, H.G., Fantus, I.G., Pasculescu, A., Zheng, Y., David M. Sabatini, et al. (2014). The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism. Science Signaling Vol. 7, Issue 313, p. ra17. Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism. Full Text
Srivastava, M., Mazza-Curll, K.L., van Wolfswinkel, J.C., and Reddien, P.W. (2014). Whole-Body Acoel Regeneration Is Controlled by Wnt and Bmp-Admp Signaling. Current Biology Apr 22. [Epub ahead of print] .Whole-body regeneration is widespread in the Metazoa, yet little is known about how underlying molecular mechanisms compare across phyla. Acoels are an enigmatic phylum of invertebrate worms that can be highly informative about many questions in bilaterian evolution, including regeneration. We developed the three-banded panther worm, Hofstenia miamia, as a new acoelomorph model system for molecular studies of regeneration. Hofstenia were readily cultured, with accessible embryos, juveniles, and adults for experimentation. We developed molecular resources and tools for Hofstenia, including a transcriptome and robust systemic RNAi. We report the identification of molecular mechanisms that promote whole-body regeneration in Hofstenia. Wnt signaling controls regeneration of the anterior-posterior axis, and Bmp-Admp signaling controls regeneration of the dorsal-ventral axis. Perturbation of these pathways resulted in regeneration-abnormal phenotypes involving axial feature duplication, such as the regeneration of two heads following Wnt perturbation or the regeneration of ventral cells in place of dorsal ones following bmp or admp RNAi. Hofstenia regenerative mechanisms are strikingly similar to those guiding regeneration in planarians. However, phylogenetic analyses using the Hofstenia transcriptome support an early branching position for acoels among bilaterians, with the last common ancestor of acoels and planarians being the ancestor of the Bilateria. Therefore, these findings identify similar whole-body regeneration mechanisms in animals separated by more than 550 million years of evolution. Full Text
Strijbis2, K., Yilmaz, O.H., Dougan, S.K., Esteban, A., Grone, A., Kumamoto, C.A., and Ploegh, H.L. (2014). Intestinal Colonization by Candida albicans Alters Inflammatory Responses in Bruton's Tyrosine Kinase-Deficient Mice. PLoS One 9, e112472.The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a "pathobiont", a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton's tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-beta were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-beta and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis. Full Text.
Strijbis, K., and Ploegh, H.L. (2014). Secretion of circular proteins using sortase. Methods Mol Biol 1174, 73-83.Circular proteins occur naturally and have been found in microorganisms, plants, and eukaryotes where they are commonly involved in host defense. Properties of circular proteins include enhanced resistance to exoproteases, increased thermostability, longer life spans, and increased activity. Using an enzymatic approach based on the bacterial sortase A (SrtA) transpeptidase, N- and C-termini of conventional linear proteins can be linked resulting in a circular protein. Circularization of bioengineered linear substrate proteins can indeed confer the desirable properties associated with circular proteins. Here, we describe how cells can be manipulated to secrete circularized proteins for substrates of choice via sortase-mediated circularization in the lumen of the endoplasmic reticulum. Full Text.
Subtelny, A.O., Eichhorn, S.W., Chen, G.R., Sive, H., and Bartel, D.P. (2014). Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature.Published online 29 January 2014. Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization. Full Text
Swee, L.K., Lourido, S., Bell, G.W., Ingram, J.R., and Ploegh, H.L. (2014). One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism. ACS chemical biologyNov 10. [Epub ahead of print]. Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells. Full Text.
Tafesse3,F.G., Strijbis, K., and Ploegh, H.L. (2014). Quantitative Analysis of Cellular Diacylglycerol Content. Bio-protocol 5;4(15). pii: e1202. Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid. This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to phosphatidic acid (1,2-diacyl- sn-glycerol-3-phosphate) in conjunction with the incorporation of a radiolabeled phosphate group by DAG kinase (Figure 1). This assay was described in (Strijbis et al., 2013). Full Text
Tafesse2, F.G., Guimaraes, C.P., Maruyama, T., Carette, J.E., Lory, S., Brummelkamp, T.R., and Ploegh, H.L. (2014). GPR107, a GPCR Essential for Intoxication by P. aeruginosa Exotoxin A, Localizes to the Golgi and is Cleaved by Furin. J Biol Chemistry Published on July 16, 2014 as Manuscript. A number of toxins, including Exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan GPCR. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport. Full Text.
Tafesse, F.G., Vacaru, A.M., Bosma, E.F., Hermansson, M., Jain, A., Hilderink, A., Somerharju, P., and Holthuis, J.C.M. (2014). Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis. Journal of Cell Science 127, 445-454.Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile alpha-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis. Full Text
Taipale, M., Tucker, G., Peng, J., Krykbaeva, I., Lin, Z.Y., Larsen, B., Choi, H., Berger, B., Gingras, A.C., and Lindquist, S. (2014). A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell 158, 434-448.Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct beta-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. Full Text.
Tam, W.L., and Ng, H.H. (2014). Sox2: Masterminding the Root of Cancer. Cancer Cell 26, 3-5.The transcription factor Sox2 is a master regulator that maintains stemness in embryonic stem cells and neural stem cells. Using elegant lineage tracing strategies and genetic reporter mouse models, two studies (one of which is by Vanner and colleagues in this issue of Cancer Cell) now demonstrate that rare Sox2-expressing cells are the founding cancer stem cell population driving tumor initiation and therapy resistance .Full Text.
Tardiff, D.F., Khurana, V., Chung, C.Y., and Lindquist, S. (2014). From yeast to patient neurons and back again: A powerful new discovery platform. Movement Disordors published online: 14 AUG 2014 No disease-modifying therapies are available for synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). The lack of therapies has been impeded by a paucity of validated drug targets and problematic cell-based model systems. New approaches are therefore needed to identify genes and compounds that directly target the underlying cellular pathologies elicited by the pathological protein, alpha-synuclein (alpha-syn). This small, lipid-binding protein impinges on evolutionarily conserved processes such as vesicle trafficking and mitochondrial function. For decades, the genetically tractable, single-cell eukaryote, budding yeast, has been used to study nearly all aspects of cell biology. More recently, yeast has revealed key insights into the underlying cellular pathologies caused by alpha-syn. The robust cellular toxicity caused by alpha-syn expression facilitates unbiased high-throughput small-molecule screening. Critically, one must validate the discoveries made in yeast in disease-relevant neuronal models. Here, we describe two recent reports that together establish a yeast-to-human discovery platform for synucleinopathies. In this exemplar, genes and small molecules identified in yeast were validated in patient-derived neurons that present the same cellular phenotypes initially discovered in yeast. On validation, we returned to yeast, where unparalleled genetic approaches facilitated the elucidation of a small molecule's mode of action. This approach enabled the identification and neuronal validation of a previously unknown "druggable" node that interfaces with the underlying, precipitating pathologies caused by alpha-syn. Such platforms can provide sorely needed leads and fresh ideas for disease-modifying therapy for these devastating diseases. (c) 2014 International Parkinson and Movement Disorder Society. Full Text.
Theunissen, T.W., and Jaenisch, R. (2014). Molecular Control of Induced Pluripotency. Cell Stem Cell 14, 720-734.Deciphering the mechanisms of epigenetic reprogramming provides fundamental insights into cell fate decisions, which in turn reveal strategies to make the reprogramming process increasingly efficient. Here we review recent advances in epigenetic reprogramming to pluripotency with a focus on the principal molecular regulators. We examine the trajectories connecting somatic and pluripotent cells, genetic and chemical methodologies for inducing pluripotency, the role of endogenous master transcription factors in establishing the pluripotent state, and functional interactions between reprogramming factors and epigenetic regulators. Defining the crosstalk among the diverse molecular actors implicated in cellular reprogramming presents a major challenge for future inquiry. Full Text.
Theunissen2,T.W., Powell, B.E., Wang, H., Mitalipova, M., Faddah, D.A., Reddy, J., Fan, Z.P., Maetzel, D., Ganz, K., Shi, L.,Tenzin Lungjangwa, Sumeth Imsoonthornruksa,Yonatan Stelzer,Sudharshan Rangarajan,Ana D’Alessio, Jianming Zhang, Qing Gao,Meelad M. Dawlaty, Richard A. Young, Nathanael S. Gray ,and Rudolf Jaenisch. (2014). Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency. Cell Stem Cell Jul 24 [Epub ahead of print] Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells. Full Text.
Thiru, P., Kern, D.M., McKinley, K.L., Monda, J.K., Rago, F., Su, K.C., Tsinman, T., Yarar, D., Bell, G.W., and Cheeseman, I.M. (2014). Kinetochore genes are coordinately upregulated in human tumors as part of a FoxM1-related cell division program. Mol Biol Cell May 14.[Epub ahead of print] The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA-microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here, we analyzed the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the Forkhead Transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program, and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis. PDF
Tsang, S.H., Chan, L., Tsai, Y.T., Wu, W.H., Hsu, C.W., Yang, J., Tosi, J., Wert, K.J., Davis, R.J., and Mahajan, V.B. (2014). Silencing of tuberin enhances photoreceptor survival and function in a preclinical model of retinitis pigmentosa (an american ophthalmological society thesis). Transactions of the American Ophthalmological Society 112, 103-115.PURPOSE: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. METHODS: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival. RESULTS: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. CONCLUSIONS: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP. Full Text.
Tsapogas, P., Swee, L.K., Nusser, A., Nuber, N., Kreuzaler, M., Capoferri, G., Rolink, H., Ceredig, R., and Rolink, A. (2014). In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development. Haematologica 99, 638-646.Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential. Full Text.
Tsvetkov, P., Myers, N., Eliav, R., Adamovich, Y., Hagai, T., Adler, J., Navon, A., and Shaul, Y. (2014). NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP. Journal of Biological Chemistry Mar 4. [Epub ahead of print].The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH, binds the 26S PC and is sufficient to maintain 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1. We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the DeltaGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state. Full Text.
Unternaehrer, J.J., Zhao, R., Kim, K., Cesana, M., Powers, J.T., Ratanasirintrawoot, S., Onder, T., Shibue, T., Weinberg, R.A., and Daley, G.Q. (2014). The Epithelial-Mesenchymal Transition Factor SNAIL Paradoxically Enhances Reprogramming. Stem Cell Reports Oct 11. [Epub ahead of print] Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency. Full Text.
Valastyan2, J.S., Termine, D.J., and Lindquist, S. (2014). Splice isoform and pharmacological studies reveal that sterol depletion relocalizes alpha-synuclein and enhances its toxicity. Proc Natl Acad Sci U S A Feb 10. [Epub ahead of print].Synucleinopathies are neurodegenerative diseases associated with toxicity of the lipid-binding protein alpha-synuclein (alpha-syn). When expressed in yeast, alpha-syn associates with membranes at the endoplasmic reticulum and traffics with vesicles out to the plasma membrane. At higher levels it elicits a number of phenotypes, including blocking vesicle trafficking. The expression of alpha-syn splice isoforms varies with disease, but how these isoforms affect protein function is unknown. We investigated two of the most abundant isoforms, resulting in deletion of exon four (alpha-synDelta4) or exon six (alpha-synDelta6). alpha-SynDelta4, missing part of the lipid-binding domain, had reduced toxicity and membrane binding. alpha-SynDelta6, missing part of the protein-protein interaction domain, had reduced toxicity but no reduction in membrane binding. To compare the mechanism by which the splice isoforms exert toxicity, equally toxic strains were probed with genetic modifiers of alpha-syn-induced toxicity. Most modifiers equally altered the toxicity induced by the splice isoforms and full-length alpha-syn (alpha-synFL). However, the splice isoform strains responded differently to a sterol-binding protein, leading us to examine the effect of sterols on alpha-syn-induced toxicity. Upon inhibition of sterol synthesis, alpha-synFL and alpha-synDelta6, but not alpha-synDelta4, showed decreased plasma membrane association, increased vesicular association, and increased cellular toxicity. Thus, higher membrane sterol concentrations favor plasma membrane binding of alpha-synFL and alpha-synDelta6 and may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and these potential effects on membrane binding proteins, further investigation of how sterol concentration and alpha-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted. Full Text
Valastyan, J.S., and Lindquist, S. (2014). Mechanisms of protein-folding diseases at a glance. Disease Models and Mechanisms 7, 9-14. For a protein to function appropriately, it must first achieve its proper conformation and location within the crowded environment inside the cell. Multiple chaperone systems are required to fold proteins correctly. In addition, degradation pathways participate by destroying improperly folded proteins. The intricacy of this multisystem process provides many opportunities for error. Furthermore, mutations cause misfolded, nonfunctional forms of proteins to accumulate. As a result, many pathological conditions are fundamentally rooted in the protein-folding problem that all cells must solve to maintain their function and integrity. Here, to illustrate the breadth of this phenomenon, we describe five examples of protein-misfolding events that can lead to disease: improper degradation, mislocalization, dominant-negative mutations, structural alterations that establish novel toxic functions, and amyloid accumulation. In each case, we will highlight current therapeutic options for battling such diseases. Full Text
vanWolfswinkel2, J.C., Wagner, D.E., and Reddien, P.W. (2014). Single-Cell Analysis Reveals Functionally Distinct Classes within the Planarian Stem Cell Compartment. Cell Stem CellJul 9 [Epub ahead of print] Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named zeta (zeta) and sigma (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage, including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings indicate that planarian neoblasts comprise two major and functionally distinct cellular compartments. Full Text.
vanWolfswinkel, J.C. (2014). Piwi and Potency: PIWI Proteins in Animal Stem Cells and Regeneration. Integrative and Comparative Biology Advance Access. PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels. Full Text.
Victora3, G.D. (2014). SnapShot: The Germinal Center Reaction. Cell 159, 700-700.e701.To produce high-affinity antibodies, B cells must undergo iterative cycles of targeted mutagenesis and affinity-based selection. These cycles take place in a structure known as the germinal center, which forms in secondary lymphoid organs upon exposure to antigenic stimuli. Here, we summarize our current understanding of the germinal center reaction. Full Text.
Victora2, G.D., and Mesin, L. (2014). Clonal and cellular dynamics in germinal centers. Curr Opin Immunol 28C, 90-96.Germinal centers (GCs) are the site of antibody affinity maturation, a process that involves complex clonal and cellular dynamics. Selection of B cells bearing higher-affinity immunoglobulins proceeds via a stereotyped pattern whereby B cells migrate cyclically between the GC's two anatomical compartments. This process occurs in a timeframe that is well suited to analysis by intravital microscopy, and much has been learned in recent years by use of these techniques. On a longer time scale, the diversity of B cell clones and variants within individual GCs is also thought to change as affinity maturation progresses; however, our understanding of clonal dynamics in individual GCs is limited. We discuss recent progress in the elucidation of clonal and cellular dynamics patterns. Full Text
Victora, G.D. (2014). ILCs in the zone. Nature Immunology 15, 313-314. Innate lymphoid cells, marginal reticular cells and B cell–helper neutrophils interact to promote antibody secretion by B cells in the marginal zone of the spleen in humans and mice. Full Text
Wagle2, N., Grabiner, B.C., Van Allen, E.M., Amin-Mansour, A., Taylor-Weiner, A., Rosenberg, M., Gray, N., Barletta, J.A., Guo, Y., Swanson, S ,.David M. Sabatini, et al. (2014). Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med 371, 1426-1433.Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors. Full Text.
Wagle, N., Grabiner, B.C., Van Allen, E.M., Hodis, E., Jacobus, S., Supko, J.G., Stewart, M., Choueiri, T.K., Gandhi, L., Cleary, J.M.,David Sabatini, et al. (2014). Activating mTOR Mutations in a Patient with an Extraordinary Response on a Phase I Trial of Everolimus and Pazopanib. Cancer Discovery Published OnlineFirst March 13, 2014. Understanding the genetic mechanisms of sensitivity to targeted anticancer therapies may improve patient selection, response to therapy, and rational treatment designs. One approach to increase this understanding involves detailed studies of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder in a phase I study of pazopanib and everolimus in advanced solid tumors. Whole-exome sequencing of a patient with a 14-month complete response on this trial revealed two concurrent mutations in mTOR, the target of everolimus. In vitro experiments demonstrate that both mutations are activating, suggesting a biologic mechanism for exquisite sensitivity to everolimus in this patient. The use of precision (or "personalized") medicine approaches to screen patients with cancer for alterations in the mTOR pathway may help to identify subsets of patients who may benefit from targeted therapies directed against mTOR. Full Text
Wagner, K., Kwakkenbos, M.J., Claassen, Y.B., Maijoor, K., Bohne, M., van der Sluijs, K.F., Witte, M.D., van Zoelen, D.J., Cornelissen, L.A., Beaumont, T., Hidde L Ploegh et al. (2014). Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc Natl Acad Sci U S A Vol. 111 no. 47 16820–16825 Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners. Full Text.
Walters, B.J., Hallengren, J.J., Theile, C.S., Ploegh, H.L., Wilson, S.M., and Dobrunz, L.E. (2014). A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short-term plasticity and vesicle number. Journal of Physiology-London 592, 571-586.Key points Mice carrying the ataxia (ax(J)) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in ax(J) mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in ax(J) mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity. Full Text
Wang2, B.L., Ghaderi, A., Zhou, H., Agresti, J., Weitz, D.A., Fink, G.R., and Stephanopoulos, G. (2014). Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nature Biotechnology Published online 06 April 2014 .Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 104 cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. We also enriched L-lactate-producing Escherichia coli clones 5,800x from a population containing one L-lactate producer per 104 D-lactate producers. Our approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals and pharmaceuticals. Full Text
Wang3, H., Geng, J., Wen, X., Bi, E., Kossenkov, A.V., Wolf, A.I., Tas, J., Choi, Y.S., Takata, H., Day, T. J, Gabriel D Victora., et al. (2014). The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells. Nat Immunol Published online 25 May 2014 CD4+ follicular helper T cells (TFH cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4+ T cells deficient in the transcription factor Foxp1 'preferentially' differentiated into TFH cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of TFH cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4+ T cells partially resistant to blockade of the ICOS ligand (ICOSL) during TFH cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of TFH cell differentiation. Full Text.
Wang, T., Wei, J.J., Sabatini, D.M., and Lander, E.S. (2014). Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science 343, 80-84.The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells. Full Text
Watnick, R.S., Rodriguez, R.K., Wang, S., Blois, A.L., Rangarajan, A., Ince, T., and Weinberg, R.A. (2014). Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells. Oncogene advance online publication, 11 August Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation. .Full Text.
Weinberg, R.A. (2014). Coming full circle-from endless complexity to simplicity and back again. Cell 157, 267-271. Cell has celebrated the powers of reductionist molecular biology and its major successes for four decades. Those who have participated in cancer research during this period have witnessed wild fluctuations from times where endless inexplicable phenomenology reigned supreme to periods of reductionist triumphalism and, in recent years, to a move back to confronting the endless complexity of this disease. Full Text
Welch, L., Lewitter, F., Schwartz, R., Brooksbank, C., Radivojac, P., Gaeta, B., and Schneider, M.V. (2014). Bioinformatics curriculum guidelines: toward a definition of core competencies. PLoS Comput Biol 10, e1003496 Full Text
Wen, J., Duan, H., Bejarano, F., Okamura, K., Fabian, L., Brill, J.A., Bortolamiol-Becet, D., Martin, R., Ruby, J.G., and Lai, E.C. (2014). Adaptive Regulation of Testis Gene Expression and Control of Male Fertility by the Drosophila Harpin RNA Pathway. Mol Cell Articles in Press .Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-beta in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs. Full Text.
Whitesell, L., Santagata, S., Mendillo, M.L., Lin, N.U., Proia, D.A., and Lindquist, S. (2014). HSP90 empowers evolution of resistance to hormonal therapy in human breast cancer models. Proc Natl Acad Sci U S A Dec 8. . [Epub ahead of print] The efficacy of hormonal therapies for advanced estrogen receptor-positive breast cancers is limited by the nearly inevitable development of acquired resistance. Efforts to block the emergence of resistance have met with limited success, largely because the mechanisms underlying it are so varied and complex. Here, we investigate a new strategy aimed at the very processes by which cancers evolve resistance. From yeast to vertebrates, heat shock protein 90 (HSP90) plays a unique role among molecular chaperones by promoting the evolution of heritable new traits. It does so by regulating the folding of a diverse portfolio of metastable client proteins, many of which mediate adaptive responses that allow organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs. Guided by our previous work in pathogenic fungi, in which very modest HSP90 inhibition impairs resistance to mechanistically diverse antifungals, we examined the effect of similarly modest HSP90 inhibition on the emergence of resistance to antiestrogens in breast cancer models. Even though this degree of inhibition fell below the threshold for proteotoxic activation of the heat-shock response and had no overt anticancer activity on its own, it dramatically impaired the emergence of resistance to hormone antagonists both in cell culture and in mice. Our findings strongly support the clinical testing of combined hormone antagonist-low-level HSP90 inhibitor regimens in the treatment of metastatic estrogen receptor-positive breast cancer. At a broader level, they also provide promising proof of principle for a generalizable strategy to combat the pervasive problem of rapidly emerging resistance to molecularly targeted therapeutics. Full Text.
Wijeratne, E.M., Xu, Y.M., Scherz-Shouval, R., Marron, M.T., Rocha, D.D., Liu, M.X., Costa-Lotufo, L.V., Santagata, S., Lindquist, S., Whitesell, L., et al. (2014). Structure-Activity Relationships for Withanolides as Inducers of the Cellular Heat-Shock Response. J Medicinal Chemistry. Article ASAP March 13, 2014 To understand the relationship of structure to the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37), and compared their cytotoxicity to cytoprotective heat shock-inducing activity (HSA). Analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced, while introduction of beta-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of beta-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36) it had minimal effect. Importantly, acetylation of 27-OH yielding 15 from 1, 16 from 14, and 35 from 34 enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to selectively enhance HSA, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms. Full Text
Willems, T.F., Gymrek, M., Highnam, G., Mittelman, D., and Erlich, Y. (2014). The landscape of human STR variation. Genome Research published online August 18, 2014 Short tandem repeats are among the most polymorphic loci in the human genome. These loci play a role in the etiology of a range of genetic diseases and have been frequently utilized in forensics, population genetics, and genetic genealogy. Despite this plethora of applications, little is known about the variation of most STRs in the human population. Here, we report the largest-scale analysis of human STR variation to date. We collected information for nearly 700,000 STR loci across over 1,000 individuals in phase 1 of the 1000 Genomes Project. Extensive quality controls show that reliable allelic spectra can be obtained for close to 90% of the STR loci in the genome. We utilize this call set to analyze determinants of STR variation, assess the human reference genome's representation of STR alleles, find STR loci with common loss-of-function alleles, and obtain initial estimates of the linkage disequilibrium between STRs and common SNPs. Overall, these analyses further elucidate the scale of genetic variation beyond classical point mutations. Full Text.
Wood, K.B., Wood, K.C., Nishida, S., and Cluzel, P. (2014). Uncovering Scaling Laws to Infer Multidrug Response of Resistant Microbes and Cancer Cells. Cell Reports 06 March 2014.Drug resistance in bacterial infections and cancers constitutes a major threat to human health. Treatments often include several interacting drugs, but even potent therapies can become ineffective in resistant mutants. Here, we simplify the picture of drug resistance by identifying scaling laws that unify the multidrug responses of drug-sensitive and -resistant cells. On the basis of these scaling relationships, we are able to infer the two-drug response of resistant mutants in previously unsampled regions of dosage space in clinically relevant microbes such as E. coli, E. faecalis, S. aureus, and S. cerevisiae as well as human non-small-cell lung cancer, melanoma, and breast cancer stem cells. Importantly, we find that scaling relations also apply across evolutionarily close strains. Finally, scaling allows one to rapidly identify new drug combinations and predict potent dosage regimes for targeting resistant mutants without any prior mechanistic knowledge about the specific resistance mechanism. Full Text
Wu, X., Scott, D.A., Kriz, A.J., Chiu, A.C., Hsu, P.D., Dadon, D.B., Cheng, A.W., Trevino, A.E., Konermann, S., Chen, S, .Rudolp Jaenisch , et al. (2014). Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nature biotechnology Published online 20 April 2014 Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage. Full Text
Wuethrich2, I., Peeters, J.G., Blom, A.E., Theile, C.S., Li, Z., Spooner, E., Ploegh, H.L., and Guimaraes, C.P. (2014). Site-specific chemoenzymatic labeling of aerolysin enables the identification of new aerolysin receptors. PLoS One 9, e109883.Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins. Full Text.
Wuethrich, I., Guillen, E., and Ploegh, H.L. (2014). A mouse monoclonal antibody against alexa fluor 647. Monoclon Antib Immunodiagn Immunother 33, 109-120.Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.
Xiao, S., Yosef, N., Yang, J., Wang, Y., Zhou, L., Zhu, C., Wu, C., Baloglu, E., Schmidt, D., Ramesh, R., Peter B. Rahl, Richard A. Young, et al. (2014). Small-Molecule RORgammat Antagonists Inhibit T Helper 17 Cell Transcriptional Network by Divergent Mechanisms. Immunity 40, 477-489.We identified three retinoid-related orphan receptor gamma t (RORgammat)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORgammat binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORgammat acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORalpha binding motif. RORgammat is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORgammat-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORgammat from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. Full Text
Yang, H., Wang, H., and Jaenisch, R. (2014). Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nature Protocols 9, 1956-1968 .Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available. Full Text.
Yesilaltay, A., Dokshin, G.A., Busso, D., Wang, L., Galiani, D., Chavarria, T., Vasile, E., Quilaqueo, L., Orellana, J.A., Walzer, D ,.David C. Page, et al. (2014). Excess cholesterol induces mouse egg activation and may cause female infertility. Proc Natl Acad Sci U S A Epub 2014 Nov 3. The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-beta-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology. Full Text.
Yien, Y.Y., Robledo, R.F., Schultz, I.J., Takahashi-Makise, N., Gwynn, B., Bauer, D.E., Dass, A., Yi, G., Li, L.T., Hildick-Smith, G.J, Shilpa M. Hattangadi , Harvey F. Lodish., et al. (2014). TMEM14C is required for erythroid mitochondrial heme metabolism. Journal of Clinical Investigation 124, 4294-4304.The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias. Full Text.
Yuan, A.H., Garrity, S.J., Nako, E., and Hochschild, A. (2014). Prion propagation can occur in a prokaryote and requires the ClpB chaperone. Elife 3, e02949.Prions are self-propagating protein aggregates that are characteristically transmissible. In mammals, the PrP protein can form a prion that causes the fatal transmissible spongiform encephalopathies. Prions have also been uncovered in fungi, where they act as heritable, protein-based genetic elements. We previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichia coli. Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the disaggregase activity of the ClpB chaperone. Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process. Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.DOI: http://dx.doi.org/10.7554/eLife.02949.001. Full Text.
Zhang, Y., and Wang, X.F. (2014). Post-transcriptional regulation of MTA family by microRNAs in the context of cancer. Cancer Metastasis Rev [Epub ahead of print] MicroRNAs (miRNAs) are a class of 20-24 nt small non-coding RNAs that regulate a wide range of biological processes through changing the stability and translation of their target messenger RNA (mRNA) genes. Shortly after their identification, many miRNA genes have been found dysregulated in a variety of human cancers, indicating a pathological function of this gene class in mediating cancer progression. Over the past decade, accumulated literature has shown that miRNAs participate in numerous cancer-relevant processes including cell proliferation, apoptosis, differentiation, metabolism, and importantly, metastasis, which accounts for the mortality of approximately 90 % of cancer patients. Several recent publications have linked miRNAs with metastasis-associated protein (MTA) family members. Given the fact that the MTA family members are widely overexpressed in human cancers and their nature of serving as both corepressor and coactivator in gene regulation, it is intriguing to study whether certain miRNAs regulate cancer progression through modulating the expression of MTA family members. In this review, we will focus on recent advances in understanding the regulatory relationship between certain miRNAs and MTA family members. Full Text.
Zhao, B.B., Keerthivasan, G., Mei, Y., Yang, J., McElherne, J., Wong, P., Doench, J.G., Feng, G., Root, D.E., and Ji, P. (2014). Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis. Haematologica 99, 1157-1167.Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional screening strategy, we identified a critical role of pleckstrin-2 in actin dynamics and protection of early stage terminal erythroblasts from oxidative damage. Knockdown of pleckstrin-2 in the early stage of terminal erythropoiesis disrupted the actin cytoskeleton and led to differentiation inhibition and apoptosis. This pro-survival and differentiation function of pleckstrin-2 was mediated through its interaction with cofilin, by preventing cofilin's mitochondrial entry when the intracellular level of reactive oxygen species was higher in the early stage of terminal erythropoiesis. Treatment of the cells with a scavenger of reactive oxygen species rescued cofilin's mitochondrial entry and differentiation inhibition induced by pleckstrin-2 knockdown. In contrast, pleckstrin-2 knockdown in late stage terminal erythroblasts had no effect on survival or differentiation but blocked enucleation due to disorganized actin cytoskeleton. Thus, our study identified a dual function of pleckstrin-2 in the early and late stages of terminal erythropoiesis through its regulations of actin dynamics and cofilin's mitochondrial localization, which reflects intracellular level of reactive oxygen species in different developmental stages. Full Text.
Zhou2, H., Wang, J., Jiang, J., Stavrovskaya, I.G., Li, M., Li, W., Wu, Q., Zhang, X., Luo, C., Zhou, S.,Ana C. Sirianni, Sovan Sarkar, Bruce S. Kristal, Robert M. Friedlander, and Xin Wang (2014). N-Acetyl-Serotonin Offers Neuroprotection through Inhibiting Mitochondrial Death Pathways and Autophagic Activation in Experimental Models of Ischemic Injury. J Neurosci 34, 2967-2978.N-acetylserotonin (NAS) is an immediate precursor of melatonin, which we have reported is neuroprotective against ischemic injury. Here we test whether NAS is a potential neuroprotective agent in experimental models of ischemic injury. We demonstrate that NAS inhibits cell death induced by oxygen-glucose deprivation or H2O2 in primary cerebrocortical neurons and primary hippocampal neurons in vitro, and organotypic hippocampal slice cultures ex vivo and reduces hypoxia/ischemia injury in the middle cerebral artery occlusion mouse model of cerebral ischemia in vivo. We find that NAS is neuroprotective by inhibiting the mitochondrial cell death pathway and the autophagic cell death pathway. The neuroprotective effects of NAS may result from the influence of mitochondrial permeability transition pore opening, mitochondrial fragmentation, and inhibition of the subsequent release of apoptogenic factors cytochrome c, Smac, and apoptosis-inducing factor from mitochondria to cytoplasm, and activation of caspase-3, -9, as well as the suppression of the activation of autophagy under stress conditions by increasing LC3-II and Beclin-1 levels and decreasing p62 level. However, NAS, unlike melatonin, does not provide neuroprotection through the activation of melatonin receptor 1A. We demonstrate that NAS reaches the brain subsequent to intraperitoneal injection using liquid chromatography/mass spectrometry analysis. Given that it occurs naturally and has low toxicity, NAS, like melatonin, has potential as a novel therapy for ischemic injury. Full Text
Zhou, P., Shaffer, D.R., Alvarez Arias, D.A., Nakazaki, Y., Pos, W., Torres, A.J., Cremasco, V., Dougan, S.K., Cowley, G.S., Elpek, K,.Hidde L. Ploegh, et al. (2014). In vivo discovery of immunotherapy targets in the tumour microenvironment. Nature. Published online 29 January 2014 Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments. Full Text
Zielinski2, D., Gordon, A., Zaks, B.L., and Erlich, Y. (2014). iPipet: sample handling using a tablet. Nature Methods 11,784–785. Here we present iPipet, a highly adaptable solution for semi-automated liquid handling using a tablet computer. Our solution is a free web service that allows users to upload and execute complex pipetting patterns in 96-well or 384-well plates with either a single-channel or multichannel pipet. The application is hosted on our public lab website (http://ipipet.teamerlich.org/) and requires only a CSV file detailing the experimental design. The website includes several examples and a video to demonstrate how to use iPipet. The website also allows users to publicly share pipetting designs, which can enhance experiment reproducibility and create easy-to-follow protocols. The entire iPipet project is freely available for further development by the community on GitHub under a GNU General Public License (GPLv3). Full Text.
Zielinski, D., Markus, B., Sheikh, M., Gymrek, M., Chu, C., Zaks, M., Srinivasan, B., Hoffman, J.D., Aizenbud, D., and Erlich, Y. (2014). OTX2 Duplication Is Implicated in Hemifacial Microsomia. PLoS One 9, e96788.Hemifacial microsomia (HFM) is the second most common facial anomaly after cleft lip and palate. The phenotype is highly variable and most cases are sporadic. We investigated the disorder in a large pedigree with five affected individuals spanning eight meioses. Whole-exome sequencing results indicated the absence of a pathogenic coding point mutation. A genome-wide survey of segmental variations identified a 1.3 Mb duplication of chromosome 14q22.3 in all affected individuals that was absent in more than 1000 chromosomes of ethnically matched controls. The duplication was absent in seven additional sporadic HFM cases, which is consistent with the known heterogeneity of the disorder. To find the critical gene in the duplicated region, we analyzed signatures of human craniofacial disease networks, mouse expression data, and predictions of dosage sensitivity. All of these approaches implicated OTX2 as the most likely causal gene. Moreover, OTX2 is a known oncogenic driver in medulloblastoma, a condition that was diagnosed in the proband during the course of the study. Our findings suggest a role for OTX2 dosage sensitivity in human craniofacial development and raise the possibility of a shared etiology between a subtype of hemifacial microsomia and medulloblastoma. PDF
2.4.15