The following alphabetical list represents papers published in 2017 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.
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2017 Titles :
Abraham, B.J., Hnisz, D., Weintraub, A.S., Kwiatkowski, N., Li, C.H., Li, Z., Weichert-Leahey, N., Rahman, S., Liu, Y., Etchin, J.,Tong Ihn Lee, and Richard A. Young. (2017). Small genomic insertions form enhancers that misregulate oncogenes. Nature communications 8, 14385.The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers. Full Text
AbuRemaileh, M, Wyant, G.A., Kim, C., Laqtom, N.N., Abbasi, M., Chan, S.H., Freinkman, E., and Sabatini, D.M. (2017). Lysosomal metabolomics reveals V-ATPase and mTOR-dependent regulation of amino acid efflux from lysosomes. Science Epub ahead of print] The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+ATPase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mTOR pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Full Text
Aiello, N.M., Brabletz, T., Kang, Y., Nieto, M.A., Weinberg, R.A., and Stanger, B.Z. (2017). Upholding a role for EMT in pancreatic cancer metastasiss utilize an EMT program or programs during metastatic dissemination. Nature 547, E7-e8 .Full Text
AlvarezDominguez2, J.R., and Lodish, H.F. (2017). Emerging mechanisms of long noncoding RNA function during normal and malignant hematopoiesis. Blood [Epub ahead of print] Long noncoding RNAs (lncRNAs) are increasingly recognized as vital components of gene programs controlling cell differentiation and function. Central to their functions is an ability to act as scaffolds or as decoys that recruit or sequester effector proteins from their DNA, RNA, or protein targets. lncRNA-modulated effectors include regulators of transcription, chromatin organization, RNA processing, and translation, such that lncRNAs can influence gene expression at multiple levels. Here we review the current understanding of how lncRNAs help coordinate gene expression to modulate cell fate in the hematopoietic system. We focus on a growing number of mechanistic studies to synthesize emerging principles of lncRNA function, emphasizing how they facilitate diversification of gene programming during development. We also survey how disrupted lncRNA function can contribute to malignant transformation, highlighting opportunities for therapeutic intervention in specific myeloid and lymphoid cancers. Finally, we discuss challenges and prospects for further elucidation of lncRNA mechanisms.Full Text
Alvarez, S.W., Sviderskiy, V.O., Terzi, E.M., Papagiannakopoulos, T., Moreira, A.L., Adams, S., Sabatini, D.M., Birsoy, K., and Possemato, R. (2017). NFS1 undergoes positive selection in lung tumours and protects cells from ferroptosis. Nature [Epub ahead of print] Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron-sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron-sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage. Full Text
AlvarezDominguez, J.R., Knoll, M., Gromatzky, A.A., and Lodish, H.F. (2017). The Super-Enhancer-Derived alncRNA-EC7/Bloodlinc Potentiates Red Blood Cell Development in trans. Cell reports 19, 2503-2514.Enhancer-derived RNAs are thought to act locally by contributing to their parent enhancer function. Whether large domains of clustered enhancers (super-enhancers) also produce cis-acting RNAs, however, remains unclear. Unlike typical enhancers, super-enhancers form large spans of robustly transcribed chromatin, amassing capped and polyadenylated RNAs that are sufficiently abundant to sustain trans functions. Here, we show that one such RNA, alncRNA-EC7/Bloodlinc, is transcribed from a super-enhancer of the erythroid membrane transporter SLC4A1/BAND3 but diffuses beyond this site. Bloodlinc localizes to trans-chromosomal loci encoding critical regulators and effectors of terminal erythropoiesis and directly binds chromatin-organizing and transcription factors, including the chromatin attachment factor HNRNPU. Inhibiting Bloodlinc or Hnrnpu compromises the terminal erythropoiesis gene program, blocking red cell production, whereas expressing Bloodlinc ectopically stimulates this program and can promote erythroblast proliferation and enucleation in the absence of differentiation stimuli. Thus, Bloodlinc is a trans-acting super-enhancer RNA that potentiates red blood cell development. Full Text
Bachran, C., Schroder, M., Conrad, L., Cragnolini, J.J., Tafesse, F.G., Helming, L., Ploegh, H.L.., and Swee, L.K. (2017). The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent. Scientific reports 7, 17916. The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates. Full Text
Bardhan, N.M., Kumar, P.V., Li, Z., Ploegh, H.L., Grossman, J.C., Belcher, A.M., and Chen, G.Y. (2017). Enhanced Cell Capture on Functionalized Graphene Oxide Nanosheets through Oxygen Clustering. ACS nano Article ASAP With the global rise in incidence of cancer and infectious diseases, there is a need for the development of techniques to diagnose, treat, and monitor these conditions. The ability to efficiently capture and isolate cells and other biomolecules from peripheral whole blood for downstream analyses is a necessary requirement. Graphene oxide (GO) is an attractive template nanomaterial for such biosensing applications. Favorable properties include its two-dimensional architecture and wide range of functionalization chemistries, offering significant potential to tailor affinity toward aromatic functional groups expressed in biomolecules of interest. However, a limitation of current techniques is that as-synthesized GO nanosheets are used directly in sensing applications, and the benefits of their structural modification on the device performance have remained unexplored. Here, we report a microfluidic-free, sensitive, planar device on treated GO substrates to enable quick and efficient capture of Class-II MHC-positive cells from murine whole blood. We achieve this by using a mild thermal annealing treatment on the GO substrates, which drives a phase transformation through oxygen clustering. Using a combination of experimental observations and MD simulations, we demonstrate that this process leads to improved reactivity and density of functionalization of cell capture agents, resulting in an enhanced cell capture efficiency of 92 +/- 7% at room temperature, almost double the efficiency afforded by devices made using as-synthesized GO (54 +/- 3%). Our work highlights a scalable, cost-effective, general approach to improve the functionalization of GO, which creates diverse opportunities for various next-generation device applications. Full Text
Bellott, D.W., Skaletsky, H., Cho, T.J., Brown, L., Locke, D., Chen, N., Galkina, S., Pyntikova, T., Koutseva, N., Graves, T., Page DC, et al. (2017). Avian W and mammalian Y chromosomes convergently retained dosage-sensitive regulators. Nature genetics [Epub ahead of print] After birds diverged from mammals, different ancestral autosomes evolved into sex chromosomes in each lineage. In birds, females are ZW and males are ZZ, but in mammals females are XX and males are XY. We sequenced the chicken W chromosome, compared its gene content with our reconstruction of the ancestral autosomes, and followed the evolutionary trajectory of ancestral W-linked genes across birds. Avian W chromosomes evolved in parallel with mammalian Y chromosomes, preserving ancestral genes through selection to maintain the dosage of broadly expressed regulators of key cellular processes. We propose that, like the human Y chromosome, the chicken W chromosome is essential for embryonic viability of the heterogametic sex. Unlike other sequenced sex chromosomes, the chicken W chromosome did not acquire and amplify genes specifically expressed in reproductive tissues. We speculate that the pressures that drive the acquisition of reproduction-related genes on sex chromosomes may be specific to the male germ line. Full Text
Betancur, P.A., Abraham, B.J., Yiu, Y.Y., Willingham, S.B., Khameneh, F., Zarnegar, M., Kuo, A.H., McKenna, K., Kojima, Y., Leeper, N.J,.Young, RA, et al. (2017). A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer. Nature communications 8, 14802. CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPalpha, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance. Full Text
Bierie, B., Pierce, S.E., Kroeger, C., Stover, D.G., Pattabiraman, D.R., Thiru, P., Liu Donaher, J., Reinhardt, F., Chaffer, C.L., Keckesova, Z, and Robert A. Weinberg (2017). Integrin-beta4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells. Proceedings of the National Academy of Sciences of the United States of America Early Edition Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-beta4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63alpha (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC. Full Text
Boguraev, A.S.,Christensen, H.C., Bonneau, A.R., Pezza, J.A., Nichols, N.M., Giraldez, A.J., Gray, M.M., Wagner, B.M., Aken, J.T., Foley, K.D., et al. (2017). Successful amplification of DNA aboard the International Space Station. NPJ microgravity 3, 26 . As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics. Full Text
Bourne, P.E., Polka, J.K., Vale, R.D., and Kiley, R. (2017). Ten simple rules to consider regarding preprint submission. PLoS computational biology 13, e1005473. As a result of an ASAPbio meeting held in February of 2016, a paper was published that describes the pros and cons of preprints from the perspective of the stakeholders—scientists, publishers, and funders. Here, we formulate the message specifically for scientists in the form of ten simple rules for considering using preprints as a communication mechanism. Full Text
Bradner, J.E., Hnisz, D., and Young, R.A. (2017). Transcriptional Addiction in Cancer. Cell 168, 629-643.Cancer arises from genetic alterations that invariably lead to dysregulated transcriptional programs. These dysregulated programs can cause cancer cells to become highly dependent on certain regulators of gene expression. Here, we discuss how transcriptional control is disrupted by genetic alterations in cancer cells, why transcriptional dependencies can develop as a consequence of dysregulated programs, and how these dependencies provide opportunities for novel therapeutic interventions in cancer. Full Text
Brethour, D., Mehrabian, M., Williams, D., Wang, X., Ghodrati, F., Ehsani, S., Rubie, E.A., Woodgett, J.R., Sevalle, J., Xi, Z., et al. (2017). A ZIP6-ZIP10 heteromer controls NCAM1 phosphorylation and integration into focal adhesion complexes during epithelial-to-mesenchymal transition. Scientific reports 7, 40313.The prion protein (PrP) evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6 and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for understanding the function of PrP. Building on data which suggested PrP and ZIP6 are critical during epithelial-to-mesenchymal transition (EMT), we investigated ZIP6 in an EMT paradigm using ZIP6 knockout cells, mass spectrometry and bioinformatic methods. Reminiscent of PrP, ZIP6 levels are five-fold upregulated during EMT and the protein forms a complex with NCAM1. ZIP6 also interacts with ZIP10 and the two ZIP transporters exhibit interdependency during their expression. ZIP6 contributes to the integration of NCAM1 in focal adhesion complexes but, unlike cells lacking PrP, ZIP6 deficiency does not abolish polysialylation of NCAM1. Instead, ZIP6 mediates phosphorylation of NCAM1 on a cluster of cytosolic acceptor sites. Substrate consensus motif features and in vitro phosphorylation data point toward GSK3 as the kinase responsible, and interface mapping experiments identified histidine-rich cytoplasmic loops within the ZIP6/ZIP10 heteromer as a novel scaffold for GSK3 binding. Our data suggests that PrP and ZIP6 inherited the ability to interact with NCAM1 from their common ZIP ancestors but have since diverged to control distinct posttranslational modifications of NCAM1. Full Text
Bruno, P.M., Liu, Y., Park, G.Y., Murai, J., Koch, C.E., Eisen, T.J., Pritchard, J.R., Pommier, Y., Lippard, S.J., and Hemann, M.T. (2017). A subset of platinum-conthttp://www.pnas.org/content/early/2017/12/05/1711926115.full.pdf?with-ds=yesaining chemotherapeutic agents kills cells by inducing ribosome biogenesis stress. Nature medicine advance online publication. Cisplatin and its platinum analogs, carboplatin and oxaliplatin, are some of the most widely used cancer chemotherapeutics. Although cisplatin and carboplatin are used primarily in germ cell, breast and lung malignancies, oxaliplatin is instead used almost exclusively to treat colorectal and other gastrointestinal cancers. Here we utilize a unique, multi-platform genetic approach to study the mechanism of action of these clinically established platinum anti-cancer agents, as well as more recently developed cisplatin analogs. We show that oxaliplatin, unlike cisplatin and carboplatin, does not kill cells through the DNA-damage response. Rather, oxaliplatin kills cells by inducing ribosome biogenesis stress. This difference in drug mechanism explains the distinct clinical implementation of oxaliplatin relative to cisplatin, and it might enable mechanistically informed selection of distinct platinum drugs for distinct malignancies. These data highlight the functional diversity of core components of front-line cancer therapy and the potential benefits of applying a mechanism-based rationale to the use of our current arsenal of anti-cancer drugs. Full Text
Cantor, J.R., Abu-Remaileh, M., Kanarek, N., Freinkman, E., Gao, X., Louissaint, A., Jr., Lewis, C.A., and Sabatini, D.M. (2017). Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase. Cell 169, 258-272.e217.A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility. Full Text
Caraveo , , G., Soste, M., Cappelleti, V., Fanning, S., van Rossum, D.B., Whitesell, L., Huang, Y., Chung, C.Y., Baru, V., Zaichick, S., Picotti, P., and Lindquist, S. (2017). FKBP12 contributes to alpha-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome. PNAS [Epub ahead of print]Calcineurin is an essential Ca(2+)-dependent phosphatase. Increased calcineurin activity is associated with alpha-synuclein (alpha-syn) toxicity, a protein implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged alpha-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to alpha-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin's activity toward those proteins and protects against the toxic hallmarks of alpha-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD. Full Text
Caron, A., Mouchiroud, M., Gautier, N., Labbe, S.M., Villot, R., Turcotte, L., Secco, B., Lamoureux, G., Shum, M., Gelinas, Y, Sabatini, D.M., et al. (2017). Loss of hepatic DEPTOR alters the metabolic transition to fasting. Molecular metabolism 6, 447-458. OBJECTIVE: The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulates growth and metabolism. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to reduce their activity. Whether DEPTOR loss affects metabolism and organismal growth in vivo has never been tested. METHODS: We have generated a conditional transgenic mouse allowing the tissue-specific deletion of DEPTOR. This model was crossed with CMV-cre mice or Albumin-cre mice to generate either whole-body or liver-specific DEPTOR knockout (KO) mice. RESULTS: Whole-body DEPTOR KO mice are viable, fertile, normal in size, and do not display any gross physical and metabolic abnormalities. To circumvent possible compensatory mechanisms linked to the early and systemic loss of DEPTOR, we have deleted DEPTOR specifically in the liver, a tissue in which DEPTOR protein is expressed and affected in response to mTOR activation. Liver-specific DEPTOR null mice showed a reduction in circulating glucose upon fasting versus control mice. This effect was not associated with change in hepatic gluconeogenesis potential but was linked to a sustained reduction in circulating glucose during insulin tolerance tests. In addition to the reduction in glycemia, liver-specific DEPTOR KO mice had reduced hepatic glycogen content when fasted. We showed that loss of DEPTOR cell-autonomously increased oxidative metabolism in hepatocytes, an effect associated with increased cytochrome c expression but independent of changes in mitochondrial content or in the expression of genes controlling oxidative metabolism. We found that liver-specific DEPTOR KO mice showed sustained mTORC1 activation upon fasting, and that acute treatment with rapamycin was sufficient to normalize glycemia in these mice. CONCLUSION: We propose a model in which hepatic DEPTOR accelerates the inhibition of mTORC1 during the transition to fasting to adjust metabolism to the nutritional status. Full Text
Chahal, J.S., Fang, T., Woodham, A.W., Khan, O.F., Ling, J., Anderson, D.G., and Ploegh, H.L. (2017). An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model. Scientific reports 7, 252.The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2Db-restricted epitope to which there was a CD8+ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus. Full Text
Chen, G.R., Sive, H., and Bartel, D.P. (2017). A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially Rescues Severely Impaired Cleavage Found in Fish. Molecular cell 68, 1095-1107.e1095. The RNAi pathway provides both innate immunity and efficient gene-knockdown tools in many eukaryotic species, but curiously not in zebrafish. We discovered that RNAi is less effective in zebrafish at least partly because Argonaute2-catalyzed mRNA slicing is impaired. This defect is due to two mutations that arose in an ancestor of most teleost fish, implying that most fish lack effective RNAi. Despite lacking efficient slicing activity, these fish have retained the ability to produce miR-451, a microRNA generated by a cleavage reaction analogous to slicing. This ability is due to a G-G mismatch within the fish miR-451 precursor, which substantially enhances its cleavage. An analogous G-G mismatch (or sometimes also a G-A mismatch) enhances target slicing, despite disrupting seed pairing important for target binding. These results provide a strategy for restoring RNAi to zebrafish and reveal unanticipated opposing effects of a seed mismatch with implications for mechanism and guide-RNA design. Full Text
Chen, J., Jacox, L.A., Saldanha, F., and Sive, H. (2017). Mouth development. Wiley interdisciplinary reviews Developmental biology [Epub ahead of print]. A mouth is present in all animals, and comprises an opening from the outside into the oral cavity and the beginnings of the digestive tract to allow eating. This review focuses on the earliest steps in mouth formation. In the first half, we conclude that the mouth arose once during evolution. In all animals, the mouth forms from ectoderm and endoderm. A direct association of oral ectoderm and digestive endoderm is present even in triploblastic animals, and in chordates, this region is known as the extreme anterior domain (EAD). Further support for a single origin of the mouth is a conserved set of genes that form a 'mouth gene program' including foxA and otx2. In the second half of this review, we discuss steps involved in vertebrate mouth formation, using the frog Xenopus as a model. The vertebrate mouth derives from oral ectoderm from the anterior neural ridge, pharyngeal endoderm and cranial neural crest (NC). Vertebrates form a mouth by breaking through the body covering in a precise sequence including specification of EAD ectoderm and endoderm as well as NC, formation of a 'pre-mouth array,' basement membrane dissolution, stomodeum formation, and buccopharyngeal membrane perforation. In Xenopus, the EAD is also a craniofacial organizer that guides NC, while reciprocally, the NC signals to the EAD to elicit its morphogenesis into a pre-mouth array. Human mouth anomalies are prevalent and are affected by genetic and environmental factors, with understanding guided in part by use of animal models. For further resources related to this article, please visit the WIREs website. Full Text
Chen, M.B., Whisler, J.A., Frose, J., Yu, C., Shin, Y., and Kamm, R.D. (2017). On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics. Nature protocols 12, 865-880.Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing protocol describing a microfluidic platform that offers additional applications. This protocol describes an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4-5 d, after which the tumor can be perfused and extravasation events are easily tracked over 72 h via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells, combined with high-resolution imaging, can be easily achieved because of the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly as compared with those in thick tissues. The ability to generate large numbers of devices ( approximately 36) per experiment further allows for highly parametric studies, which are required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single-cell extravasation events, allowing both tumor and endothelial morphological dynamics to be observed in high detail with a moderate number of data points. Full Text
Chen, W.W., Freinkman, E., and Sabatini, D.M. (2017). Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites. Nature protocols 12, 2215-2231.Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in similar to 12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week. Full Text
Chezem, W.R., Memon, A., Li, F.S., Weng, J.K., and Clay, N.K. (2017). SG2-type R2R3-MYB transcription factor MYB15 controls defense-induced lignification and basal immunity in Arabidopsis. The Plant cell [Epub ahead of print]. Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops. Full Text
Chmatal, L., Schultz, R.M., Black, B.E., and Lampson, M.A. (2017). Cell Biology of Cheating-Transmission of Centromeres and Other Selfish Elements Through Asymmetric Meiosis. Progress in molecular and subcellular biology 56, 377-396.Mendel's First Law of Genetics states that a pair of alleles segregates randomly during meiosis so that one copy of each is represented equally in gametes. Whereas male meiosis produces four equal sperm, in female meiosis only one cell, the egg, survives, and the others degenerate. Meiotic drive is a process in which a selfish DNA element exploits female meiotic asymmetry and segregates preferentially to the egg in violation of Mendel's First Law, thereby increasing its transmission to the offspring and frequency in a population. In principle, the selfish element can consist either of a centromere that increases its transmission via an altered kinetochore connection to the meiotic spindle or a centromere-like element that somehow bypasses the kinetochore altogether in doing so. There are now examples from eukaryotic model systems for both types of meiotic drive. Although meiotic drive has profound evolutionary consequences across many species, relatively little is known about the underlying mechanisms. We discuss examples in various systems and open questions about the underlying cell biology, and propose a mechanism to explain biased segregation in mammalian female meiosis. Full Text
Christ, B., Hochstrasser, R., Guyer, L., Francisco, R., Aubry, S., Hortensteiner, S., and Weng, J.K. (2017). Non-specific activities of the major herbicide-resistance gene BAR. Nature plants [Epub ahead of print]. Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops (1-4) . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids (1) , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes (5,6) . Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes. Full Text
Chung, C.Y., Khurana, V., Yi, S., Sahni, N., Loh, K.H., Auluck, P.K., Baru, V., Udeshi, N.D., Freyzon, Y., Carr, S.A, .Lindquist S, et al. (2017). In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons. Cell systems 4(2) : 242-250. Synucleinopathies, including Parkinson's disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (alpha-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of alpha-syn in living neurons to shed light on alpha-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with alpha-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial alpha-syn map overlapped with genetic modifiers of alpha-syn toxicity, developed in an accompanying study (Khurana et al., this issue of Cell Systems). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of alpha-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells. Full Text
Chung2, J., Wittig, J.G., Ghamari, A., Maeda, M., Dailey, T.A., Bergonia, H., Kafina, M.D., Coughlin, E.E., Minogue, C.E., Hebert, A.S, Harvey F Lodish., et al. (2017). Erythropoietin signaling regulates heme biosynthesis. eLife 6 : e24767 [Epub ahead of print] Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development. Full Text
ClancyThompson, E., Chen, G.Z., Tyler, P.M., Servos, M.M., Barisa, M., Brennan, P.J., Ploegh, H.L., and Dougan, S.K. (2017). Monoclonal Invariant NKT (iNKT) Cell Mice Reveal a Role for Both Tissue of Origin and the TCR in Development of iNKT Functional Subsets. Journal of immunology [Epub ahead of print]. Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-gamma, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Valpha14Jalpha18 TCRalpha paired with unique Vbeta7 or Vbeta8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORgammat, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity. Full Text
Cohen, A.J., Saiakhova, A., Corradin, O., Luppino, J.M., Lovrenert, K., Bartels, C.F., Morrow, J.J., Mack, S.C., Dhillon, G., Beard, L., et al. (2017). Hotspots of aberrant enhancer activity punctuate the colorectal cancer epigenome. Nature communications 8, 14400.In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival. Full Text
Degn, S.E., van der Poel, C.E., Firl, D.J., Ayoglu, B., Al Qureshah, F.A., Bajic, G., Mesin, L., Reynaud, C.A., Weill, J.C., Utz, P.J.,Victora GD, Carroll MC. (2017). Clonal Evolution of Autoreactive Germinal Centers. Cell 170, 913-926.e919.Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease. Full Text
Dettmer, U., Ramalingam, N., von Saucken, V.E., Kim, T.E., Newman, A.J., Terry-Kantor, E., Nuber, S., Ericsson, M., Fanning, S., Bartels, T., Lindquist S., et al. (2017). Loss of native alpha-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells. Human molecular genetics 26, 3466-3481. alpha-Synuclein (alphaS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of alphaS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that alphaS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered alphaS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant alphaS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the alphaS-associated vesicles were of diverse endocytic and secretory origin. An alphaS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced alphaS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase alphaS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered alphaS variant added hydrophobicity to the hydrophobic half of alphaS helices, thereby stabilizing alphaS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing alphaS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible alphaS amphipathic helix formation and dynamic multimerization regulate a normal function of alphaS at vesicles, and abrogating multimers has pathogenic consequences. Full Text
Dongre, A., Rashidian, M., Reinhardt, F., Bagnato, A., Keckesova, Z., Ploegh, H.L., and Weinberg, R.A. (2017). Epithelial-to-mesenchymal Transition contributes to Immunosuppression in Breast Carcinomas. Cancer research [Epub ahead of print] The Epithelial-to-mesenchymal transition (EMT) is a cell-biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is, unclear, however, whether activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1 and contained within their stroma CD8+ T cells and M1 (anti-tumor) macrophages. In contrast, tumors arising from more-mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1 and contained within their stroma regulatory T cells, M2 (pro-tumor) macrophages and exhausted CD8+ T cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Lastly, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens. Full Text
Doulatov, S., Vo, L.T., Macari, E.R., Wahlster, L., Kinney, M.A., Taylor, A.M., Barragan, J., Gupta, M., McGrath, K., Lee, H.Y., Harvey F. Lodish, et al. (2017). Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors. Science translational medicine 9(376) Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. Full Text
Edupuganti, R.R., Harikumar, A., Aaronson, Y., Biran, A., Sailaja, B.S., Nissim-Rafinia, M., Azad, G.K., Cohen, M.A., Park, J.E., Shivalila, C.S., Markoulaki S, Jaenisch R, et al. (2017). Alternative SET/TAFI Promoters Regulate Embryonic Stem Cell Differentiation. Stem cell reports (Epub) Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETalpha, to the primary isoform in differentiated cells, SETbeta, through alternative promoters. SETalpha is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage. Full Text
Endo , T., Freinkman, E., de Rooij, D.G., and Page, D.C. (2017). Periodic production of retinoic acid by meiotic and somatic cells coordinates four transitions in mouse spermatogenesis. PNAS[Epub ahead of print].Mammalian spermatogenesis is an elaborately organized differentiation process, starting with diploid spermatogonia, which include germ-line stem cells, and ending with haploid spermatozoa. The process involves four pivotal transitions occurring in physical proximity: spermatogonial differentiation, meiotic initiation, initiation of spermatid elongation, and release of spermatozoa. We report how the four transitions are coordinated in mice. Two premeiotic transitions, spermatogonial differentiation and meiotic initiation, were known to be coregulated by an extrinsic signal, retinoic acid (RA). Our chemical manipulations of RA levels in mouse testes now reveal that RA also regulates the two postmeiotic transitions: initiation of spermatid elongation and spermatozoa release. We measured RA concentrations and found that they changed periodically, as also reflected in the expression patterns of an RA-responsive gene, STRA8; RA levels were low before the four transitions, increased when the transitions occurred, and remained elevated thereafter. We found that pachytene spermatocytes, which express an RA-synthesizing enzyme, Aldh1a2, contribute directly and significantly to RA production in testes. Indeed, chemical and genetic depletion of pachytene spermatocytes revealed that RA from pachytene spermatocytes was required for the two postmeiotic transitions, but not for the two premeiotic transitions. We conclude that the premeiotic transitions are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate the two postmeiotic transitions. In combination, these elements underpin the spatiotemporal coordination of spermatogenesis and ensure its prodigious output in adult males. Full Text
Erdmann, R.M., Satyaki, P.R.V., Klosinska, M., and Gehring, M. (2017). A Small RNA Pathway Mediates Allelic Dosage in Endosperm. Cell reports 21, 3364-3372.Balance between maternal and paternal genomes within the triploid endosperm is necessary for normal seed development. The majority of endosperm genes are expressed in a 2:1 maternal:paternal ratio, reflecting genomic DNA content. Here, we find that the 2:1 transcriptional ratio is, unexpectedly, actively regulated. In A. thaliana and A. lyrata, endosperm 24-nt small RNAs are reduced in transposable elements and enriched in genes compared with the embryo. We find an inverse relationship between the parent of origin of sRNAs and mRNAs, with genes more likely to be associated with maternally than paternally biased sRNAs. Disruption of the Pol IV sRNA pathway causes a shift toward maternal allele mRNA expression for many genes. Furthermore, paternal inheritance of an RNA Pol IV mutation is sufficient to rescue seed abortion caused by excess paternal genome dosage. Thus, RNA Pol IV mediates the transcriptional balance between maternally and paternally inherited genomes in endosperm. Full Text
Erdmann, R.M., Hoffmann, A., Walter, H.K., Wagenknecht, H.A., Gross-Hardt, R., and Gehring, M. (2017). Molecular movement in the Arabidopsis thaliana female gametophyte. Plant reproduction [Epub ahead of print] Size limits on molecular movement among female gametes. Cellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.
Ersching, J., Efeyan, A., Mesin, L., Jacobsen, J.T., Pasqual, G., Grabiner, B.C., Dominguez-Sola, D., Sabatini, D.M., and Victora, G.D. (2017). Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase. Immunity 46, 1045-1058.e1046.During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. Full Text
Fang, T., Van Elssen, C., Duarte, J.N., Guzman, J.S., Chahal, J.S., Ling, J.J., and Ploegh, H.L. (2017). Targeted antigen delivery by an anti-class II MHC VHH elicits focused alpha MUC1(Tn) immunity. Chemical Science 8, 5591-5597.Unusual patterns of glycosylation on the surface of transformed cells contribute to immune modulation and metastasis of malignant tumors. Active immunization against them requires effective antigen presentation, which is complicated by a lack of access to tumor-specific posttranslational modifications through standard genetic approaches and by the low efficiency of passive antigen sampling. We found that antigen targeted to antigen presenting cells via class II MHC products can elicit a robust immune response against MUC1(Tn) bearing a defined tumor-associated glycoform, Tn. The two-component vaccine construct was prepared by sortase-mediated protein ligation of a synthetic MUC1(Tn) fragment to a class II MHC-binding singledomain antibody fragment (VHH7) as targeting moiety. We show that VHH7 targets antigen presenting cells in vivo, and when conjugated to MUC1(Tn) can elicit a strong alpha MUC1(Tn) immune response in mice. The resulting sera preferentially recognized the MUC1 epitope with the tumor-associated carbohydrate antigen Tn and were capable of killing cancer cells in a complement-mediated cytotoxicity assay. Immunoglobulin isotype analysis and cytokine release assays suggested a favorable Th1 response. A single boost 12 months after primary immunization triggered a recall response of the same quality, suggesting that long-term alpha MUC1(Tn) memory had been achieved. Full Text
Feng, J., Pena, C.J., Purushothaman, I., Engmann, O., Walker, D., Brown, A.N., Issler, O., Doyle, M., Harrigan, E., Mouzon, E., Rudolf Jaenisch, et al. (2017). Tet1 in Nucleus Accumbens Opposes Depression- and Anxiety-Like Behaviors. Neuropsychopharmacology [Epub ahead of print] .Depression is a leading cause of disease burden, yet current therapies fully treat <50% of affected individuals. Increasing evidence implicates epigenetic mechanisms in depression and antidepressant action. Here, we examined a possible role for the DNA dioxygenase, ten eleven translocation protein 1 (TET1), in depression-related behavioral abnormalities. We applied chronic social defeat stress, an ethologically validated mouse model of depression-like behaviors, and examined Tet1 expression changes in nucleus accumbens (NAc), a key brain reward region. We show decreased Tet1 expression in NAc in stress susceptible mice only. Surprisingly, selective knockout of Tet1 in NAc neurons of adult mice produced antidepressant-like effects in several behavioral assays. To identify Tet1 targets that mediate these actions, we performed RNAseq on NAc after conditional deletion of Tet1 and found that immune-related genes are the most highly dysregulated. Moreover, many of these genes are also upregulated in NAc of resilient mice after chronic social defeat stress. These findings reveal a novel role for TET1, an enzyme important for DNA hydroxymethylation, in the brain's reward circuitry in modulating stress responses in mice. We also identify a subset of genes that are regulated by TET1 in this circuitry. These findings provide new insight into the pathophysiology of depression, which can aid in future antidepressant drug discovery efforts.. Full Text
Feng, Y.X., Jin, D.X., Sokol, E.S., Reinhardt, F., Miller, D.H., and Gupta, P.B. (2017). Cancer-specific PERK signaling drives invasion and metastasis through CREB3L1. Nature communications 8, 1079. PERK signaling is required for cancer invasion and there is interest in targeting this pathway for therapy. Unfortunately, chemical inhibitors of PERK's kinase activity cause on-target side effects that have precluded their further development. One strategy for resolving this difficulty would be to target downstream components of the pathway that specifically mediate PERK's pro-invasive and metastatic functions. Here we identify the transcription factor CREB3L1 as an essential mediator of PERK's pro-metastatic functions in breast cancer. CREB3L1 acts downstream of PERK, specifically in the mesenchymal subtype of triple-negative tumors, and its inhibition by genetic or pharmacological methods suppresses cancer cell invasion and metastasis. In patients with this tumor subtype, CREB3L1 expression is predictive of distant metastasis. These findings establish CREB3L1 as a key downstream mediator of PERK-driven metastasis and a druggable target for breast cancer therapy. Full Text
Franzoso, F.D., Seyffert, M., Vogel, R., Yakimovich, A., de Andrade Pereira, B., Meier, A.F., Sutter, S.O., Tobler, K., Vogt, B., Greber, U.F., et al. (2017). Cell cycle-dependent expression of AAV2 Rep in HSV-1 co-infections gives rise to a mosaic of cells replicating either AAV2 or HSV-1. Journal of virology[Epub ahead of print] Adeno-associated virus 2 (AAV2) depends for productive replication on the simultaneous presence of a helper virus such as herpes simplex virus type 1 (HSV-1). At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question how AAV2 and HSV-1 can co-exist in a cell population. Here we show that in co-infected cultures, AAV2 DNA replication takes place almost exclusively in S/G2 cells, while HSV-1 DNA replication is restricted to G1. Live microscopy revealed that not only wtAAV2 replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2 cells, suggesting that the S/G2 preference is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of the S/G2 cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication, and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors, and hence, creates a biological niche for either virus to replicate.IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpes virus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here, we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus type 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent, and gives rise to distinct time controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phases support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 permit HSV-1 replication, but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. Full Text
Frederick, K.K., Michaelis, V.K., Caporini, M.A., Andreas, L.B., Debelouchina, G.T., Griffin, R.G., and Lindquist, S. (2017). Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register. Proceedings of the National Academy of Sciences of the United States of America Early edition. The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a beta-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. Full Text
Gao, X., Lee, H.Y., Li, W., Platt, R.J., Barrasa, M.I., Ma, Q., Elmes, R.R., Rosenfeld, M.G., and Lodish, H.F. (2017). Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print.] An effect of thyroid hormone (TH) on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which TH affects red cell formation is still elusive. Here we demonstrate an essential role of TH during terminal human erythroid cell differentiation; specific depletion of TH from the culture medium completely blocked terminal erythroid differentiation and enucleation. Treatment with TRbeta agonists stimulated premature erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model by regulating erythroid gene expression. To identify factors that cooperate with TRbeta during human erythroid terminal differentiation, we conducted RNA-seq in human reticulocytes and identified nuclear receptor coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in perinatal periods and fail to respond to TH by enhanced erythropoiesis. Genome-wide analysis suggests that TH promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism by which TH functions during red blood cell formation, results that are potentially useful to treat certain anemias. Full Text
Gu X., Orozco, J.M., Saxton, R.A., Condon, K.J., Liu, G.Y., Krawczyk, P.A., Scaria, S.M. , Harper, J.W., Gygi, S.P., and Sabatini, D.M. (2017). SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 358, 813-818. mTOR complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple environmental cues. Nutrients signal via the Rag guanosine triphosphatases (GTPases) to promote the localization of mTORC1 to the lysosomal surface, its site of activation. We identified SAMTOR, a previously uncharacterized protein, which inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. We found that the methyl donor S-adenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 muM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling. Full Text
Guen, V.J., Chavarria, T.E., Kroger, C., Ye, X., Weinberg, R.A.,, and Lees, J.A. (2017). EMT programs promote basal mammary stem cell and tumor-initiating cell stemness by inducing primary ciliogenesis and Hedgehog signaling. PNAS [Epub ahead of print] Tissue regeneration relies on adult stem cells (SCs) that possess the ability to self-renew and produce differentiating progeny. In an analogous manner, the development of certain carcinomas depends on a small subset of tumor cells, called "tumor-initiating cells" (TICs), with SC-like properties. Mammary SCs (MaSCs) reside in the basal compartment of the mammary epithelium, and their neoplastic counterparts, mammary TICs (MaTICs), are thought to serve as the TICs for the claudin-low subtype of breast cancer. MaSCs and MaTICs both use epithelial-mesenchymal transition (EMT) programs to acquire SC properties, but the mechanism(s) connecting EMT programs to stemness remain unclear. Here we show that this depends on primary cilia, which are nonmotile, cell-surface structures that serve as platforms for receiving cues and enable activation of various signaling pathways. We show that MaSC and MaTIC EMT programs induce primary cilia formation and Hedgehog (Hh) signaling, which has previously been implicated in both MaSC and MaTIC function. Moreover, ablation of these primary cilia is sufficient to repress Hh signaling, the stemness of MaSCs, and the tumor-forming potential of MaTICs. Together, our findings establish primary ciliogenesis and consequent Hh signaling as a key mechanism by which MaSC and MaTIC EMT programs promote stemness and thereby support mammary tissue outgrowth and tumors of basal origin. Full Text
Guo, L.Y., Allu, P.K., Zandarashvili, L., McKinley, K.L., Sekulic, N., Dawicki-McKenna, J.M., Fachinetti, D., Logsdon, G.A., Jamiolkowski, R.M., Cleveland, D.W., Cheeseman IM and Black BE. (2017). Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. Nature communications 8, 15775.Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA. Full Text
Hanke, L., Schmidt, F.I., Knockenhauer, K.E., Morin, B., Whelan, S.P., Schwartz, T.U., and Ploegh, H.L. (2017). Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication. EMBO reports [Epub ahead of print]The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHHs. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention. Full Text
Hara, M., Petrova, B., and Orr-Weaver, T.L. (2017). Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation. eLife 6: e22219. The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity we show here to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition. Full Text
Harikumar, A., Edupuganti, R.R., Sorek, M., Azad, G.K., Markoulaki, S ., Sehnalova, P., Legartova, S., Bartova, E., Farkash-Amar, S., Jaenisch, R., et al. (2017). An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells. Stem cell reports 9(4):1304-1314 Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more. Full Text
Harmanci, A.S., Youngblood, M.W., Clark, V.E., Coskun, S., Henegariu, O., Duran, D., Erson-Omay, E.Z., Kaulen, L.D., Lee, T.I., Abraham, B.J.,Young RA, et al. (2017). Integrated genomic analyses of de novo pathways underlying atypical meningiomas. Nature communications 8, 14433.Meningiomas are mostly benign brain tumours, with a potential for becoming atypical or malignant. On the basis of comprehensive genomic, transcriptomic and epigenomic analyses, we compared benign meningiomas to atypical ones. Here, we show that the majority of primary (de novo) atypical meningiomas display loss of NF2, which co-occurs either with genomic instability or recurrent SMARCB1 mutations. These tumours harbour increased H3K27me3 signal and a hypermethylated phenotype, mainly occupying the polycomb repressive complex 2 (PRC2) binding sites in human embryonic stem cells, thereby phenocopying a more primitive cellular state. Consistent with this observation, atypical meningiomas exhibit upregulation of EZH2, the catalytic subunit of the PRC2 complex, as well as the E2F2 and FOXM1 transcriptional networks. Importantly, these primary atypical meningiomas do not harbour TERT promoter mutations, which have been reported in atypical tumours that progressed from benign ones. Our results establish the genomic landscape of primary atypical meningiomas and potential therapeutic targets. Full Text
Hnisz, D., Young, R.A. (2017). New Insights into Genome Structure: Genes of a Feather Stick Together. Molecular cell 67, 730-731.DNA structuring proteins such as CTCF facilitate DNA loop formation and are presumed to be among the major determinants of eukaryotic genome structure. Recent studies, including Rowley et al. (2017), suggest that gene activation and repression play fundamentally important roles in structuring the genome independently of CTCF. Full Text
Hnisz, D., Shrinivas, K., Young, R.A., Chakraborty, A.K., and Sharp, P.A. (2017). A Phase Separation Model for Transcriptional Control. Cell 169, 13-23.Phase-separated multi-molecular assemblies provide a general regulatory mechanism to compartmentalize biochemical reactions within cells. We propose that a phase separation model explains established and recently described features of transcriptional control. These features include the formation of super-enhancers, the sensitivity of super-enhancers to perturbation, the transcriptional bursting patterns of enhancers, and the ability of an enhancer to produce simultaneous activation at multiple genes. This model provides a conceptual framework to further explore principles of gene control in mammals. Full Text
Hua, B.L., and Orr-Weaver, T.L. (2017). DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression. Genetics 207, 29-47.Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. Full Text
Huang, N.J., Pishesha, N., Mukherjee, J., Zhang, S., Deshycka, R., Sudaryo, V., Dong, M., Shoemaker, C.B., and Lodish, H.F. (2017). Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin. Nature communications 8, 423. A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A. Full Text
Ilic, N., Birsoy, K., Aguirre, A.J., Kory, N., Pacold, M.E., Singh, S., Moody, S.E., DeAngelo, J.D., Spardy, N.A., Freinkman, E., Sabatini DM, et al. (2017). PIK3CA mutant tumors depend on oxoglutarate dehydrogenase. Proceedings of the National Academy of Sciences of the United States of America Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. Full Text
Ingram2, J.R., Dougan, M., Rashidian, M., Knoll, M., Keliher, E.J., Garrett, S., Garforth, S., , Blomberg, O.S., Espinosa, C.., Bhan, A.,Almo SC, Weissleder R, Lodish H , , Dougan SK, and Ploegh HL (2017). PD-L1 is an activation-independent marker of brown adipocytes. Nature communications 8, 647. Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or beta-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.Full Text
Ingram, J.R., Blomberg, O.S., Sockolosky, J.T., Ali, L., Schmidt, F.I., Pishesha, N., Espinosa, C., Dougan, S.K., Garcia, K.C., Ploegh, H.L., and Dougan M. (2017). Localized CD47 blockade enhances immunotherapy for murine melanoma. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print]. CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPalpha). Inhibitors of the CD47-SIRPalpha interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPalpha interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy. Full Text
Kalaitzidis, D., Lee, D.J., Efeyan, A., Kfoury, Y., Nayyar, N., Sykes, D.B., Mercier, F.E., Papazian, A., Baryawno, N., Victora, G.D,.David M Sabatini, et al. (2017). Amino acid-insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells. Journal of Clinical Investigation 127, 1405-1413.The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches. Full Text
Kalan, S., Amat, R., Schachter, M.M., Kwiatkowski, N., Abraham, B.J, Liang, Y., Zhang, T., Olson, C.M., Larochelle, S., Young, R.A., et al. (2017). Activation of the p53 Transcriptional Program Sensitizes Cancer Cells to Cdk7 Inhibitors. Cell reports 21, 467-481. Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition. Full Text
Karras, G.I., Yi, S., Sahni, N., Fischer, M., Xie, J., Vidal, M., D'Andrea, A.D., Whitesell, L., and Lindquist, S. (2017). HSP90 Shapes the Consequences of Human Genetic Variation. Cell [Epub ahead of print] HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 >/= HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Full Text
Keckesova, Z., Donaher, J.L., De Cock, J., Freinkman, E., Lingrell, S., Bachovchin, D.A., Bierie, B., Tischler, V., Noske, A., Okondo, M.C., Ferenc Reinhardt, Prathapan Thiru, Todd R. Golub, Jean E. Vance& Robert A. Weinberg. (2017). LACTB is a tumour suppressor that modulates lipid metabolism and cell state. Nature Published online Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression. Full Text
Kern, D.M., Monda, J.K., Su, K.C., Wilson-Kubalek, E.M., and Cheeseman, I.M. (2017). Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80. eLife 6. pii: e26866. Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions. Full Text
Kersten , R.D., Lee, S., Fujita, D., Pluskal, T.,, Kram, S., Smith, J.E., Iwai, T., Noel, J.P., Fujita, M., and Weng, JK. (2017). A Red Algal Bourbonane Sesquiterpene Synthase Defined by Microgram-Scale NMR-Coupled Crystalline Sponge X-ray Diffraction Analysis. Journal of the American Chemical Society [Epub ahead of print]. Sesquiterpene scaffolds are the core backbones of many medicinally and industrially important natural products. A plethora of sesquiterpene synthases, widely present in bacteria, fungi, and plants, catalyze the formation of these intricate structures often with multiple stereocenters starting from linear farnesyl diphosphate substrates. Recent advances in next-generation sequencing and metabolomics technologies have greatly facilitated gene discovery for sesquiterpene synthases. However, a major bottleneck limits biochemical characterization of recombinant sesquiterpene synthases: the absolute structural elucidation of the derived sesquiterpene products. Here, we report the identification and biochemical characterization of LphTPS-A, a sesquiterpene synthase from the red macroalga Laurencia pacifica. Using the combination of transcriptomics, sesquiterpene synthase expression in yeast, and microgram-scale nuclear magnetic resonance-coupled crystalline sponge X-ray diffraction analysis, we resolved the absolute stereochemistry of prespatane, the major sesquiterpene product of LphTPS-A, and thereby functionally define LphTPS-A as the first bourbonane-producing sesquiterpene synthase and the first biochemically characterized sesquiterpene synthase from red algae. Our study showcases a workflow integrating multiomics approaches, synthetic biology, and the crystalline sponge method, which is generally applicable for uncovering new terpene chemistry and biochemistry from source-limited living organisms. Full Text
Khurana2, V., Chung, C.Y., and Tardiff, D.F. (2017). From Yeast to Patients: The Audacity and Vision of Susan Lindquist. Cell systems 4, 147-148. In this issue of Cell Systems, we present two papers (Chung et al., 2017 ; Khurana et al., 2017) that bring together 15 years of effort from the laboratory of Susan Lindquist, a visionary biologist we were privileged to call our mentor. Susan passed away from complications of cancer on October 27, 2016, just as the final revisions of these manuscripts were being submitted. Full Text
Khurana, V., Peng, J., Chung, C.Y., Auluck, P.K., Fanning, S., Tardiff, D.F., Bartels, T., Koeva, M., Eichhorn, S.W., Benyamini, H.Lou Y, Nutter-Upham A, Baru V, Freyzon Y, Barrasa MI, Ehsani S, Bartel DP, and Lindquist S, et al. (2017). Genome-Scale Networks Link Neurodegenerative Disease Genes to alpha-Synuclein through Specific Molecular Pathways. Cell systems 4(2) : 157-170. Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (alpha-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact alpha-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked alpha-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy. Full Text
Knoll , M., Winther, S., Natarajan, A., Yang, H., Jiang, M., Thiru, P., Shahsafaei, A., Chavarria, T.E., Lamming, D.W., Sun, L.,,Hansen, J.B.,and Lodish, H. (2017). SYK kinase mediates brown fat differentiation and activation.Nature communications 8, 2115. Brown adipose tissue (BAT) metabolism influences glucose homeostasis and metabolic health in mice and humans. Sympathetic stimulation of beta-adrenergic receptors in response to cold induces proliferation, differentiation, and UCP1 expression in pre-adipocytes and mature brown adipocytes. Here we show that spleen tyrosine kinase (SYK) is upregulated during brown adipocyte differentiation and activated by beta-adrenergic stimulation. Deletion or inhibition of SYK, a kinase known for its essential roles in the immune system, blocks brown and white pre-adipocyte proliferation and differentiation in vitro, and results in diminished expression of Ucp1 and other genes regulating brown adipocyte function in response to beta-adrenergic stimulation. Adipocyte-specific SYK deletion in mice reduces BAT mass and BAT that developed consisted of SYK-expressing brown adipocytes that had escaped homozygous Syk deletion. SYK inhibition in vivo represses beta-agonist-induced thermogenesis and oxygen consumption. These results establish SYK as an essential mediator of brown fat formation and function. Full Text
Krzisch, M., and Toni, N. (2017). Forced neuronal interactions cause poor communication. Neurogenesis 4, e1286424.Post-natal hippocampal neurogenesis plays a role in hippocampal function, and neurons born post-natally participate to spatial memory and mood control. However, a great proportion of granule neurons generated in the post-natal hippocampus are eliminated during the first 3 weeks of their maturation, a mechanism that depends on their synaptic integration. In a recent study, we examined the possibility of enhancing the synaptic integration of neurons born post-natally, by specifically overexpressing synaptic cell adhesion molecules in these cells. Synaptic cell adhesion molecules are transmembrane proteins mediating the physical connection between pre- and post-synaptic neurons at the synapse, and their overexpression enhances synapse formation. Accordingly, we found that overexpressing synaptic adhesion molecules increased the synaptic integration and survival of newborn neurons. Surprisingly, the synaptic adhesion molecule with the strongest effect on new neurons' survival, Neuroligin-2A, decreased memory performances in a water maze task. We present here hypotheses explaining these surprising results, in the light of the current knowledge of the mechanisms of synaptic integration of new neurons in the post-natal hippocampus.
Lambert, A.W., Pattabiraman, D.R., and Weinberg, R.A. (2017). Emerging Biological Principles of Metastasis. Cell 168, 670-691.Metastases account for the great majority of cancer-associated deaths, yet this complex process remains the least understood aspect of cancer biology. As the body of research concerning metastasis continues to grow at a rapid rate, the biological programs that underlie the dissemination and metastatic outgrowth of cancer cells are beginning to come into view. In this review we summarize the cellular and molecular mechanisms involved in metastasis, with a focus on carcinomas where the most is known, and we highlight the general principles of metastasis that have begun to emerge. Full Text
Levin, J.M., Holtzman, S.H., Maraganore, J., Hastings, P.J., Cohen, R., Dahiyat, B., Adams, J., Adams, C., Ahrens, B., Albers, J., Lodish H, et al. (2017). US immigration order strikes against biotech. Nature biotechnology 35, 204-206. We the undersigned, founders and leaders of biotech companies, write to express our deep concern and opposition to the executive order signed by President Donald Trump on January 27, 2017, barring the entry of citizens from seven countries into the United States. Full Text
Li2, C., Singh, B., Graves-Deal, R., Ma, H., Starchenko, A., Fry, W.H., Lu, Y., Wang, Y., Bogatcheva, G., Khan, M.P., et al. (2017). Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print]. We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors. Full Text
Li3, F.S., and Weng, J.K. (2017). Demystifying traditional herbal medicine with modern approach. Nature plants 3, 17109.Plants have long been recognized for their therapeutic properties. For centuries, indigenous cultures around the world have used traditional herbal medicine to treat a myriad of maladies. By contrast, the rise of the modern pharmaceutical industry in the past century has been based on exploiting individual active compounds with precise modes of action. This surge has yielded highly effective drugs that are widely used in the clinic, including many plant natural products and analogues derived from these products, but has fallen short of delivering effective cures for complex human diseases with complicated causes, such as cancer, diabetes, autoimmune disorders and degenerative diseases. While the plant kingdom continues to serve as an important source for chemical entities supporting drug discovery, the rich traditions of herbal medicine developed by trial and error on human subjects over thousands of years contain invaluable biomedical information just waiting to be uncovered using modern scientific approaches. Here we provide an evolutionary and historical perspective on why plants are of particular significance as medicines for humans. We highlight several plant natural products that are either in the clinic or currently under active research and clinical development, with particular emphasis on their mechanisms of action. Recent efforts in developing modern multi-herb prescriptions through rigorous molecular-level investigations and standardized clinical trials are also discussed. Emerging technologies, such as genomics and synthetic biology, are enabling new ways for discovering and utilizing the medicinal properties of plants. We are entering an exciting era where the ancient wisdom distilled into the world's traditional herbal medicines can be reinterpreted and exploited through the lens of modern science. Full Text
Li4, Y., Muffat, J., Omer, A., Bosch, I., Lancaster, M.A., Sur, M., Gehrke, L., Knoblich, J.A., and Jaenisch, R. (2017). Induction of Expansion and Folding in Human Cerebral Organoids. Cell stem cell 20, 385-396.e383. An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex. Full Text
Li, Z., Abraham, B.J., Berezovskaya, A., Farah, N., Liu, Y., Leon, T., Fielding, A., Tan, S.H., Sanda, T., Weintraub, A.S.,Young RA, et al. (2017). APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL. Leukemia [Epub ahead of print] Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells resulting in clonal selection. Although most cancer causing mutations have been detected in the protein-coding regions of the cancer genome, driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well.Leukemia accepted article preview online, 06 March 2017. doi:10.1038/leu.2017.75. Full Text
Liu, F.D., Pishesha, N., Poon, Z., Kaushik, T., and Van Vliet, K.J. (2017). Material Viscoelastic Properties Modulate the Mesenchymal Stem Cell Secretome for Applications in Hematopoietic Recovery. Acs Biomaterials Science & Engineering 3, 3292-3306. Human mesenchymal stem cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum material to which those cells adhere. Such mechanosensitivity has been explored in vitro to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair in vivo through paracrine signaling via secreted biomolecules. Here we leveraged cell-material interactions in vitro to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. Specifically, we varied the viscoelastic properties of cell-culture compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules, including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli <1 kPa) and highest ratio of loss modulus to storage modulus (tan(delta) > 1). A specific subpopulation of these cells, shown previously to express increased osteopontin in vitro and to promote bone marrow recovery in vivo, also exhibited up to a 5-fold increase in osteopontin expression when grown on compliant PDMS relative to heterogeneous MSCs on polystyrene. Importantly, this mechanically modulated increase in protein expression preceded detectable changes in the terminal differentiation capacity of MSCs. In coculture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted in vitro proliferation of HSPCs without altering the multipotency for either myeloid or lymphoid lineages. Cytokine and protein expression by human MSCs can thus be manipulated directly by mechanical cues conferred by the material substrata prior to and instead of tissue lineage differentiation. This approach enables enhanced in vitro production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications. Full Text
Lobbardi, R., Pinder, J., Martinez-Pastor, B., Theodorou, M., Blackburn, J.S., Abraham, B.J., Namiki, Y., Mansour, M., Abdelfattah, N.S., Molodtsov, A., Young RA, et al. (2017). TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.Cancer discovery 7, 1336-1353. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Full Text
LoCascio, S.A., Lapan, S.W., and Reddien, P.W. (2017). Eye Absence Does Not Regulate Planarian Stem Cells during Eye Regeneration. Developmental cell 40, 381-391.e383.Dividing cells called neoblasts contain pluripotent stem cells and drive planarian flatworm regeneration from diverse injuries. A long-standing question is whether neoblasts directly sense and respond to the identity of missing tissues during regeneration. We used the eye to investigate this question. Surprisingly, eye removal was neither sufficient nor necessary for neoblasts to increase eye progenitor production. Neoblasts normally increase eye progenitor production following decapitation, facilitating regeneration. Eye removal alone, however, did not induce this response. Eye regeneration following eye-specific resection resulted from homeostatic rates of eye progenitor production and less cell death in the regenerating eye. Conversely, large head injuries that left eyes intact increased eye progenitor production. Large injuries also non-specifically increased progenitor production for multiple uninjured tissues. We propose a model for eye regeneration in which eye tissue production by planarian stem cells is not directly regulated by the absence of the eye itself. Full Text
Lodish , H.F. (2017). Fifty years of mentoring and advising. Molecular biology of the Cell 28, 2908-2910. Advancement of science depends on thoughtfully mentoring a rare group of scientists that are highly educated, creative, and motivated-and that come from every country in the world. On the basis of my own experiences, I suggest ways to recruit top young scientists of both genders, support their development into leading researchers, and advise them about careers inside and outside of academia. Creating a family-friendly environment within the laboratory and the institution is crucial to these efforts. Full Text
Manier, S., Huynh, D., Shen, Y.J., Zhou, J., Yusufzai, T., Salem, K.Z., Ebright, R.Y., Shi, J., Park, J., Glavey, S.V., Whitesell L, et al. (2017). Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma. Science translational medicine Vol. 9, Issue 389, eaal2668. Multiple myeloma (MM) is a frequently incurable hematological cancer in which overactivity of MYC plays a central role, notably through up-regulation of ribosome biogenesis and translation. To better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small-molecule library for anti-MM activity. The most potent hits identified were rocaglate scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates. Full Text
Markowitz, T.E., Suarez, D., Blitzblau, H.G., Patel, N.J., Markhard, A.L., MacQueen, A.J., and Hochwagen, A. (2017). Reduced dosage of the chromosome axis factor Red1 selectively disrupts the meiotic recombination checkpoint in Saccharomyces cerevisiae. PLoS genetics 13, e1006928.Meiotic chromosomes assemble characteristic "axial element" structures that are essential for fertility and provide the chromosomal context for meiotic recombination, synapsis and checkpoint signaling. Whether these meiotic processes are equally dependent on axial element integrity has remained unclear. Here, we investigated this question in S. cerevisiae using the putative condensin allele ycs4S. We show that the severe axial element assembly defects of this allele are explained by a linked mutation in the promoter of the major axial element gene RED1 that reduces Red1 protein levels to 20-25% of wild type. Intriguingly, the Red1 levels of ycs4S mutants support meiotic processes linked to axis integrity, including DNA double-strand break formation and deposition of the synapsis protein Zip1, at levels that permit 70% gamete survival. By contrast, the ability to elicit a meiotic checkpoint arrest is completely eliminated. This selective loss of checkpoint function is supported by a RED1 dosage series and is associated with the loss of most of the cytologically detectable Red1 from the axial element. Our results indicate separable roles for Red1 in building the structural axis of meiotic chromosomes and mounting a sustained recombination checkpoint response. Full Text
Mathis, R.A., Sokol, E.S., and Gupta, P.B. (2017). Cancer cells exhibit clonal diversity in phenotypic plasticity. Open biology 7(2) : 160283 Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone-with a heritability of 0.89. To estimate the effects of combination therapies on phenotypically heterogeneous tumours, we generated quantitative simulations incorporating empirical data from our barcoding experiments. These analyses indicated that combination therapies which alternate between epithelial- and mesenchymal-specific treatments eventually select for clones with increased phenotypic plasticity. However, this selection could be minimized by increasing the frequency of alternation between treatments, identifying designs that may minimize selection for increased phenotypic plasticity. These findings establish new insights into phenotypic plasticity in cancer, and suggest design principles for optimizing the effectiveness of combination therapies for phenotypically heterogeneous tumours. Full Text
McCammon, J.M., Blaker-Lee, A., Chen, X., and Sive, H. (2017). The 16p11.2 homologs fam57ba and doc2a generate certain brain and body phenotypes. Human molecular genetics 26, 3699-3712 .Deletion of the 16p11.2 CNV affects 25 core genes and is associated with multiple symptoms affecting brain and body, including seizures, hyperactivity, macrocephaly, and obesity. Available data suggest that most symptoms are controlled by haploinsufficiency of two or more 16p11.2 genes. To identify interacting 16p11.2 genes, we used a pairwise partial loss of function antisense screen for embryonic brain morphology, using the accessible zebrafish model. fam57ba, encoding a ceramide synthase, was identified as interacting with the doc2a gene, encoding a calcium-sensitive exocytosis regulator, a genetic interaction not previously described. Using genetic mutants, we demonstrated that doc2a+/- fam57ba+/- double heterozygotes show hyperactivity and increased seizure susceptibility relative to wild-type or single doc2a-/- or fam57ba-/- mutants. Additionally, doc2a+/- fam57ba+/- double heterozygotes demonstrate the increased body length and head size. Single doc2a+/- and fam57ba+/- heterozygotes do not show a body size increase; however, fam57ba-/- homozygous mutants show a strongly increased head size and body length, suggesting a greater contribution from fam57ba to the haploinsufficient interaction between doc2a and fam57ba. The doc2a+/- fam57ba+/- interaction has not been reported before, nor has any 16p11.2 gene previously been linked to increased body size. These findings demonstrate that one pair of 16p11.2 homologs can regulate both brain and body phenotypes that are reflective of those in people with 16p11.2 deletion. Together, these findings suggest that dysregulation of ceramide pathways and calcium sensitive exocytosis underlies seizures and large body size associated with 16p11.2 homologs in zebrafish. The data inform consideration of mechanisms underlying human 16p11.2 deletion symptoms. Full Text
McLellan ,C.A., Vincent, B.M., Solis, N.V., Lancaster, A.K., Sullivan, L.B., Hartland, C.L., Youngsaye, W., Filler, S.G., Whitesell, L., and Lindquist, S.. (2017). Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy. Nature chemical biology [Epub ahead of print]. The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.Full Text
McKinley, K.L., and Cheeseman, I.M. (2017). Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects. Developmental cell [Epub ahead of print] Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression. Full Text
Mekhdjian, A.H., Kai, F., Rubashkin, M.G., Prahl, L.S., Przybyla, L.M., McGregor, A.L., Bell, E.S., Barnes, J.M., DuFort, C.C., Ou, G., Ye, X, et al. (2017). Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix. Molecular biology of the cell [Epub ahead of print] Metastasis requires tumor cells to navigate through a stiff stroma, and to squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data showed that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exerted higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model where motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also displayed an altered integrin adhesion molecular organization at the nanoscale, and recruited a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, was similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics revealed an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus, adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome. Full Text
Mellios, N., Feldman, D.A., Sheridan, S.D., Ip, J.P.K., Kwok, S., Amoah, S.K., Rosen, B., Rodriguez, B.A., Crawford, B., Swaminathan, R.,Li, Y., Jaenisch R., et al. (2017). MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling. Molecular psychiatry [Epub ahead of print] Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders. Full Text
Micheletti, R., Plaisance, I., Abraham, B.J., Sarre, A., Ting, C.C., Alexanian, M., Maric, D., Maison, D., Nemir, M., Young, R.A., et al. (2017). The long noncoding RNA Wisper controls cardiac fibrosis and remodeling. Science translational medicine 9 (395). pii: eaai9118. Long noncoding RNAs (lncRNAs) are emerging as powerful regulators of cardiac development and disease. However, our understanding of the importance of these molecules in cardiac fibrosis is limited. Using an integrated genomic screen, we identified Wisper (Wisp2 super-enhancer-associated RNA) as a cardiac fibroblast-enriched lncRNA that regulates cardiac fibrosis after injury. Wisper expression was correlated with cardiac fibrosis both in a murine model of myocardial infarction (MI) and in heart tissue from human patients suffering from aortic stenosis. Loss-of-function approaches in vitro using modified antisense oligonucleotides (ASOs) demonstrated that Wisper is a specific regulator of cardiac fibroblast proliferation, migration, and survival. Accordingly, ASO-mediated silencing of Wisper in vivo attenuated MI-induced fibrosis and cardiac dysfunction. Functionally, Wisper regulates cardiac fibroblast gene expression programs critical for cell identity, extracellular matrix deposition, proliferation, and survival. In addition, its association with TIA1-related protein allows it to control the expression of a profibrotic form of lysyl hydroxylase 2, implicated in collagen cross-linking and stabilization of the matrix. Together, our findings identify Wisper as a cardiac fibroblast-enriched super-enhancer-associated lncRNA that represents an attractive therapeutic target to reduce the pathological development of cardiac fibrosis in response to MI and prevent adverse remodeling in the damaged heart. Full Text
Miller, D.H., Sokol, E.S., and Gupta, P.B. (2017). 3D Primary Culture Model to Study Human Mammary Development. Methods in molecular biology (Clifton, NJ) 1612, 139-147.We present a protocol for expanding human mammary tissues from primary patient-derived cells in three-dimensional (3D) cultures. The primary epithelial cells are seeded into 3D hydrogels with defined components, which include both proteins and carbohydrates present in mammary tissue. Over a span of 10-14 days, the seeded cells form mammary tissues with complex ductal-lobular topologies and include luminal and basal cells in the correct orientation, together with cells that stain positively for stem cell markers. In addition to recapitulating key architectural features of human mammary tissue, the expanded tissues also respond to lactogenic hormones including estrogen, progesterone, and prolactin. We anticipate that these cultures will prove useful for studies of mammary development and breast cancer. Full Text
Monda, J.K., Whitney, I.P., Tarasovetc, E.V., Wilson-Kubalek, E., Milligan, R.A., Grishchuk, E.L., and Cheeseman, I.M.(2017). Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces. Current biology : CB [Epub ahead of print]. The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation. Full Text
Muir, A., Danai, L.V., Gui, D.Y., Waingarten, C.Y., Lewis, C.A., and Vander Heiden, M.G. (2017). Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition. eLife 6: e27713. Many mammalian cancer cell lines depend on glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation. However, some cell lines that depend on glutamine anaplerosis in culture rely less on glutamine catabolism to proliferate in vivo. We sought to understand the environmental differences that cause differential dependence on glutamine for anaplerosis. We find that cells cultured in adult bovine serum, which better reflects nutrients available to cells in vivo, exhibit decreased glutamine catabolism and reduced reliance on glutamine anaplerosis compared to cells cultured in standard tissue culture conditions. We find that levels of a single nutrient, cystine, accounts for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11. Thus, xCT/SLC7A11 expression, in conjunction with environmental cystine, is necessary and sufficient to increase glutamine catabolism, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer. Full Text
Newby , G.A., and Lindquist, S. (2017). Pioneer cells established by the [SWI+] prion can promote dispersal and out-crossing in yeast. PLoS biology 15, e2003476. To thrive in an ever-changing environment, microbes must widely distribute their progeny to colonize new territory. Simultaneously, they must evolve and adapt to the stresses of unpredictable surroundings. In both of these regards, diversity is key-if an entire population moved together or responded to the environment in the same way, it could easily go extinct. Here, we show that the epigenetic prion switch [SWI+] establishes a specialized subpopulation with a "pioneer" phenotypic program in Saccharomyces cerevisiae. Cells in the pioneer state readily disperse in water, enabling them to migrate and colonize new territory. Pioneers are also more likely to find and mate with genetically diverse partners, as inhibited mating-type switching causes mother cells to shun their own daughters. In the nonprion [swi-] state, cells instead have a "settler" phenotype, forming protective flocs and tending to remain in their current position. Settler cells are better able to withstand harsh conditions like drought and alkaline pH. We propose that these laboratory observations reveal a strategy employed in the wild to rapidly diversify and grant distinct, useful roles to cellular subpopulations that benefit the population as a whole. Full Text
Newby, G.A., Kiriakov, S., Hallacli, E., Kayatekin, C., Tsvetkov, P., Mancuso, C.P., Bonner, J.M.., Hesse, W.R., Chakrabortee, S.., Manogaran, A.L., Lindquist S., et al. (2017). A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance. Cell[Epub ahead of print] Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells. Full Text
Newman, S.A., Klepac-Ceraj, V., Mariotti, G., Pruss, S.B., Watson, N., and Bosak, T. (2017). Experimental fossilization of mat-forming cyanobacteria in coarse-grained siliciclastic sediments. Geobiology (Early View) Microbial fossils and textures are commonly preserved in Ediacaran and early Cambrian coarse-grained siliciclastic sediments that were deposited in tidal and intertidal marine settings. In contrast, the fossilization of micro-organisms in similar marine environments of post-Cambrian age is less frequently reported. Thus, temporal discrepancies in microbial preservation may have resulted from the opening and closing of a unique taphonomic window during the terminal Proterozoic and early Phanerozoic, respectively. Here, we expand upon previous work to identify environmental factors which may have facilitated the preservation of cyanobacteria growing on siliciclastic sand, by experimentally determining the ability of microbial mats to trap small, suspended mineral grains, and precipitate minerals from ions in solution. We show that (i) fine grains coat the sheaths of filamentous cyanobacteria (e.g., Nodosilinea sp.) residing within the mat, after less than 1 week of cell growth under aerobic conditions, (ii) clay minerals do not coat sterile cellulose fibers and rarely coat unsheathed cyanobacterial cells (e.g., Nostoc sp.), (iii) stronger disturbances (where culture jars were agitated at 170 rpm; 3 mm orbital diameter) produce the smoothest and most extensive mineral veneers around cells, compared with those agitated at slower rotational speeds (150 and 0 rpm), and (iv) mineral veneers coating cyanobacterial cells are ~1 mum in width. These new findings suggest that sheathed filamentous cyanobacteria may be preferentially preserved under conditions of high fluid energy. We integrate these results into a mechanistic model that explains the preservation of microbial fossils and textures in Ediacaran sandstones and siltstones, and in fine-grained siliciclastic deposits that contain exceptionally preserved microbial mats. Full Text
Oderberg, I.M., Li, D.J., Scimone, M.L., Gavino, M.A., and Reddien, P.W. (2017). Landmarks in Existing Tissue at Wounds Are Utilized to Generate Pattern in Regenerating Tissue. Current biology : CB [Epub ahead of print] Regeneration in many organisms involves the formation of a blastema, which differentiates and organizes into the appropriate missing tissues. How blastema pattern is generated and integrated with pre-existing tissues is a central question in the field of regeneration. Planarians are free-living flatworms capable of rapidly regenerating from small body fragments [1]. A cell cluster at the anterior tip of planarian head blastemas (the anterior pole) is required for anterior-posterior (AP) and medial-lateral (ML) blastema patterning [2-4]. Transplantation of the head tip into tails induced host tissues to grow patterned head-like outgrowths containing a midline. Given the important patterning role of the anterior pole, understanding how it becomes localized during regeneration would help explain how wounds establish pattern in new tissue. Anterior pole progenitors were specified at the pre-existing midline of regenerating fragments, even when this location deviated from the ML median plane of the wound face. Anterior pole progenitors were specified broadly on the dorsal-ventral (DV) axis and subsequently formed a cluster at the DV boundary of the animal. We propose that three landmarks of pre-existing tissue at wounds set the location of anterior pole formation: a polarized AP axis, the pre-existing midline, and the dorsal-ventral median plane. Subsequently, blastema pattern is organized around the anterior pole. This process, utilizing positional information in existing tissue at unpredictably shaped wounds, can influence the patterning of new tissue in a manner that facilitates integration with pre-existing tissue in regeneration. Full Text
Oh, C.K., Sultan, A., Platzer, J., Dolatabadi, N., Soldner, F, McClatchy, D.B., Diedrich, J.K., Yates, J.R., 3rd, Ambasudhan, R., Nakamura, T., Jaenisch R, and Lipton SA. (2017). S-Nitrosylation of PINK1 Attenuates PINK1/Parkin-Dependent Mitophagy in hiPSC-Based Parkinson's Disease Models. Cell reports 21, 2171-2182. Mutations in PARK6 (PINK1) and PARK2 (Parkin) are linked to rare familial cases of Parkinson's disease (PD). Mutations in these genes result in pathological dysregulation of mitophagy, contributing to neurodegeneration. Here, we report that environmental factors causing a specific posttranslational modification on PINK1 can mimic these genetic mutations. We describe a molecular mechanism for impairment of mitophagy via formation of S-nitrosylated PINK1 (SNO-PINK1). Mitochondrial insults simulating age- or environmental-related stress lead to increased SNO-PINK1, inhibiting its kinase activity. SNO-PINK1 decreases Parkin translocation to mitochondrial membranes, disrupting mitophagy in cell lines and human-iPSC-derived neurons. We find levels of SNO-PINK1 in brains of alpha-synuclein transgenic PD mice similar to those in cell-based models, indicating the pathophysiological relevance of our findings. Importantly, SNO-PINK1-mediated deficits in mitophagy contribute to neuronal cell death. These results reveal a direct molecular link between nitrosative stress, SNO-PINK1 formation, and mitophagic dysfunction that contributes to the pathogenesis of PD. Full Text
Olson, C.M., Jiang, B., Erb, M.A., Liang, Y., Doctor, Z.M., Zhang, Z., Zhang, T., Kwiatkowski, N., Boukhali, M., Green, J.L., Young, R.A., et al. (2017). Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nature chemical biology [Epub ahead of print]. Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition. Full Text
Oulhen, N., Swartz, S.Z., Laird, J., Mascaro, A., and Wessel, G. (2017). Transient translational quiescence in primordial germ cells. Development Advance Online Articles. Stem cells in animals often exhibit a slow cell cycle and/or low transcriptional activity referred to as quiescence. Here we report that the translational activity in the primordial germ cells (PGCs) of the sea urchin embryo (Strongylocentrotus purpuratus) is quiescent. We measured new protein synthesis with O-propargyl-puromycin, and L-homopropargylglycine, Click-iT technologies and determined that these cells synthesize protein at only 6% the level of their adjacent somatic cells. Knock-down of translation of the RNA-binding protein Nanos2 by morpholino anti-sense oligonucleotides, or knock-out of the Nanos2 gene by CRISPR/Cas9 resulted in a significant, but partial increase (47%) in general translation specifically in the PGCs. We found that the mRNA of the translation factor eEF1A is excluded from the PGCs in a Nanos2-dependent manner, a consequence of a Nanos/Pumilio response element (PRE) in its 3'UTR. In addition to eEF1A, the cytoplasmic pH of the PGCs appears to repress translation and simply increasing the pH also significantly restores translation selectively in the PGCs. We conclude that the PGCs of this sea urchin institute parallel pathways to quiesce translation thoroughly but transiently. Full Text
Pattabiraman, D.R., and Weinberg, R.A. (2017). Targeting the Epithelial-to-Mesenchymal Transition: The Case for Differentiation-Based Therapy. Cold Spring Harbor symposia on quantitative biology[Epub ahead of print] Although important strides have been made in targeted therapy for certain leukemias and subtypes of breast cancer, the standard of care for most carcinomas still involves chemotherapy, radiotherapy, surgery, or a combination of these. Two processes serve as obstacles to the successful treatment of carcinomas. First, a majority of deaths from these types of cancers occurs as a result of distant metastases and not the primary tumors themselves. Second, subsets of cells that are able to survive conventional therapy drive the aggressive relapse of the tumors, often in forms that are resistant to treatment. A frequently observed feature of malignant carcinomas is the loss of epithelial traits and the gain of certain mesenchymal ones that are programmed by the cell-biological program termed the epithelial-to-mesenchymal transition (EMT). The EMT program can confer (i) an ability to disseminate, (ii) an ability to become stem-like tumor-initiating cells, (iii) an ability to found new tumor colonies at distant anatomical sites, and (iv) an elevated resistance to therapy. These multiple powers of the EMT program explain why it has become an attractive target for therapeutic intervention. Recent work has revealed the variable nature of the EMT, with multiple versions of the program being observed depending on the tissue context and the stage of tumor progression. In this review, we attempt to crystallize emerging concepts in the research on EMT and stemness and discuss the benefits of using a differentiation-based therapeutic strategy for the eradication of stem-like populations that have adopted various versions of the EMT program. Full Text
Picard, C.L., and Gehring, M. (2017). Proximal methylation features associated with nonrandom changes in gene body methylation. Genome biology 18, 73. BACKGROUND: Gene body methylation at CG dinucleotides is a widely conserved feature of methylated genomes but remains poorly understood. The Arabidopsis thaliana strain Cvi has depleted gene body methylation relative to the reference strain Col. Here, we leverage this natural epigenetic difference to investigate gene body methylation stability. RESULTS: Recombinant inbred lines derived from Col and Cvi were used to examine the transmission of distinct gene body methylation states. The vast majority of genic CG methylation patterns are faithfully transmitted over nine generations according to parental genotype, with only 1-4% of CGs either losing or gaining methylation relative to the parent. Genic CGs that fail to maintain the parental methylation state are shared among independent lines, suggesting that these are not random occurrences. We use a logistic regression framework to identify features that best predict sites that fail to maintain parental methylation state. Intermediate levels of CG methylation around a dynamic CG site and high methylation variability across many A. thaliana strains at that site are the strongest predictors. These data suggest that the dynamic CGs we identify are not specific to the Col-Cvi recombinant inbred lines but have an epigenetic state that is inherently less stable within the A. thaliana species. Extending this, variably methylated genic CGs in maize and Brachypodium distachyon are also associated with intermediate local CG methylation. CONCLUSIONS: These results provide new insights into the features determining the inheritance of gene body methylation and demonstrate that two different methylation equilibria can be maintained within single individuals. Full Text
Pincus, D., Resnekov, O., and Reynolds, K. (2017). An evolution-based strategy for engineering allosteric regulation. Physical biology [Epub ahead of print] Allosteric regulation provides a way to control protein activity at the time scale of milliseconds to seconds inside the cell. An ability to engineer synthetic allosteric systems would be of practical utility for the development of novel biosensors, creation of synthetic cell signaling pathways, and design of small molecule pharmaceuticals with regulatory impact. To this end, we outline a general approach - termed Rational Engineering of Allostery at Conserved Hotspots (REACH) - to introduce novel regulation into a protein of interest by exploiting latent allostery that has been hard-wired by evolution into its structure. REACH entails the use of statistical coupling analysis (SCA) to identify "allosteric hotspots" on protein surfaces, the development and implementation of experimental assays to test hotspots for functionality, and a toolkit of allosteric modulators to impinge on endogenous cellular circuitry. REACH can be broadly applied to rewire cellular processes to respond to novel inputs. Full Text
Pinto,B.S., and Orr-Weaver, T.L. (2017). Drosophila protein phosphatases 2A B' Wdb and Wrd regulate meiotic centromere localization and function of the MEI-S332 Shugoshin. PNAS [Epub ahead of print]. Proper segregation of chromosomes in meiosis is essential to prevent miscarriages and birth defects. This requires that sister chromatids maintain cohesion at the centromere as cohesion is released on the chromatid arms when the homologs segregate at anaphase I. The Shugoshin proteins preserve centromere cohesion by protecting the cohesin complex from cleavage, and this has been shown in yeasts to be mediated by recruitment of the protein phosphatase 2A B' (PP2A B'). In metazoans, delineation of the role of PP2A B' in meiosis has been hindered by its myriad of other essential roles. The Drosophila Shugoshin MEI-S332 can bind directly to both of the B' regulatory subunits of PP2A, Wdb and Wrd, in yeast two-hybrid experiments. Exploiting experimental advantages of Drosophila spermatogenesis, we found that the Wdb subunit localizes first along chromosomes in meiosis I, becoming restricted to the centromere region as MEI-S332 binds. Wdb and MEI-S332 show colocalization at the centromere region until release of sister-chromatid cohesion at the metaphase II/anaphase II transition. MEI-S332 is necessary for Wdb localization, but, additionally, both Wdb and Wrd are required for MEI-S332 localization. Thus, rather than MEI-S332 being hierarchical to PP2A B', these proteins reciprocally ensure centromere localization of the complex. We analyzed functional relationships between MEI-S332 and the two forms of PP2A by quantifying meiotic chromosome segregation defects in double or triple mutants. These studies revealed that both Wdb and Wrd contribute to MEI-S332's ability to ensure accurate segregation of sister chromatids, but, as in centromere localization, they do not act solely downstream of MEI-S332. Full Text
Pishesha, N., Bilate, A.M., Wibowo, M.C., Huang, N.J., Li, Z., Dhesycka, R., Bousbaine, D., Li, H., Patterson, H.C., Dougan, S.K.,Maruyama T, Lodish HF, and Ploegh HL (2017). Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes. Full Text
Pluskal, T., and Weng, J.K. (2017). Natural product modulators of human sensations and mood: molecular mechanisms and therapeutic potential. Chemical Society Reviews Online Humans perceive physical information about the surrounding environment through their senses. This physical information is registered by a collection of highly evolved and finely tuned molecular sensory receptors. A multitude of bioactive, structurally diverse ligands have evolved in nature that bind these molecular receptors. The complex, dynamic interactions between the ligands and the receptors lead to changes in our sensory perception or mood. Here, we review our current knowledge of natural products and their derived analogues that interact specifically with human G protein-coupled receptors, ion channels, and nuclear hormone receptors to modulate the sensations of taste, smell, temperature, pain, and itch, as well as mood and its associated behaviour. We discuss the molecular and structural mechanisms underlying such interactions and highlight cases where subtle differences in natural product chemistry produce drastic changes in functional outcome. We also discuss cases where a single compound triggers complex sensory or behavioural changes in humans through multiple mechanistic targets. Finally, we comment on the therapeutic potential of the reviewed area of research and draw attention to recent technological developments in genomics, metabolomics, and metabolic engineering that allow us to tap the medicinal properties of natural product chemistry without taxing nature.Full Text
Rahman, S., Magnussen, M., Leon, T.E., Farah, N., Li, Z., Abraham, B.J., Alapi, K.Z., Mitchell, R.J., Naughton, T., Fielding, A.K., Young RA, et al. (2017). Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia. Blood [Epub ahead of print] Somatic mutations within non-coding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines, in addition to 3.7% (6/160) of pediatric and 5.5% (9/163) of adult T-ALL patient samples. The majority of indels harbour putative de novo MYB, ETS1 or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provide important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy induced T-ALL, suggesting that such events occur at preferential sites in the non-coding genome. Full Text
Rashidian, M., Ingram, J.R., Dougan, M., Dongre, A., Whang, K.A., LeGall, C., Cragnolini, J.J., Bierie, B., Gostissa, M., Gorman, J.,Weinberg, RA ,et al. (2017). Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells. The Journal of experimental medicine[Epub ahead of print]. Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies. Full Text
Raz , A.A., Srivastava, M., Salvamoser, R., and Reddien, P.W. (2017). Acoel regeneration mechanisms indicate an ancient role for muscle in regenerative patterning. Nature communications 8, 1260. Positional information is required for animal regeneration, yet how it is harbored in adult tissues is poorly understood. In planarians, positional control genes (PCGs) control regeneration outcomes and are regionally expressed predominately in the musculature. Acoels are early diverging bilaterally symmetric animals, having separated from other bilaterians > 550 million years ago. Here, we find that PCGs in the acoel Hofstenia miamia are expressed together and specifically in a primary differentiated cell type: muscle. The vast majority of Hofstenia muscle cells in regions tested express PCGs, suggesting positional information is a major feature of muscle. PCG expression domains are dynamic in muscle after injury, consistent with known PCG roles in guiding regeneration. These data demonstrate an instructive positional role for Hofstenia muscle and this similarity with planarians suggests mesodermal muscle originated at the base of the Bilateria not only for contraction, but also as the source of positional information guiding regeneration. Full Text
Reczek, C.R., Birsoy, K., Kong, H., Martinez-Reyes, I., Wang, T, Gao, P., Sabatini, D.M., and Chandel, N.S. (2017). A CRISPR screen identifies a pathway required for paraquat-induced cell death. Nature chemical biology [Epub ahead of print] Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology. Full Text
Rissland , A.A., O.S., Subtelny, A.O..., Wang, M., Lugowski, A., Nicholson, B., Laver, J.D., Sidhu, S.S., Smibert, C.A., Lipshitz, H.D., and Bartel, D.P. (2017). The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.Genome biology 18, 211. All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. RESULTS: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length. Full Text
Rusan, M., Li, K., Li, Y., Christensen, C.L., Abraham, B.J., Kwiatkowski, N., Buczkowski, K.A., Bockorny, B., Chen, T., Li, S., Young RA, et al. (2017). Suppression of adaptive responses to targeted cancer therapy by transcriptional repression. Cancer discovery [Epub ahead of print]. Acquired drug resistance is a major factor limiting the effectiveness of targeted cancer therapies. Targeting tumors with kinase inhibitors induces complex adaptive programs that promote the persistence of a fraction of the original cell population, facilitating the eventual outgrowth of inhibitor-resistant tumor clones. We show that the addition of a newly identified CDK7/12 inhibitor, THZ1, to targeted therapy enhances cell killing and impedes the emergence of drug-resistant cell populations in diverse cellular and in vivo cancer models. We propose that targeted therapy induces a state of transcriptional dependency in a subpopulation of cells poised to become drug tolerant, which THZ1 can exploit by blocking dynamic transcriptional responses, remodeling of enhancers and key signalling outputs required for tumor cell survival in the setting of targeted therapy. These findings suggest that the addition of THZ1 to targeted therapies is a promising broad-based strategy to hinder the emergence of drug-resistant cancer cell populations. Full Text
Russell, J.J., Theriot, J.A., Sood, P., Marshall, W.F., Landweber, L.F., Fritz-Laylin, L., Polka, J.K., Oliferenko, S., Gerbich, T., Gladfelter, A., et al. (2017). Non-model model organisms. BMC biology 15, 55.Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future. Full Text
Sabatini, D.M. (2017). Twenty-five years of mTOR: Uncovering the link from nutrients to growth.PNAS [Epub ahead of print] In my PNAS Inaugural Article, I describe the development of the mTOR field, starting with efforts to understand the mechanism of action of the drug rapamycin, which approximately 25 y ago led to the discovery of the mTOR protein kinase. I focus on insights that we have contributed and on work that has been particularly influential to me, as well as provide some personal reflections and stories. We now appreciate that, as part of two distinct complexes, mTORC1 and mTORC2, mTOR is the major regulator of growth (mass accumulation) in animals and is the key link between the availability of nutrients in the environment and the control of most anabolic and catabolic processes. Nutrients signal to mTORC1 through the lysosome-associated Rag GTPases and their many regulators and associated cytosolic and lysosomal nutrient sensors. mTOR signaling is deregulated in common diseases, like cancer and epilepsy, and mTORC1 is a well-validated modulator of aging in multiple model organisms. There is significant excitement around using mTORC1 inhibitors to treat cancer and neurological disease and, potentially, to improve healthspan and lifespan. Full Text
Sahakyan, A., Kim, R., Chronis, C., Sabri, S., Bonora, G., Theunissen, T.W., Kuoy, E., Langerman, J., Clark, A.T., Jaenisch, R., et al. (2017). Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation. Cell stem cell 20, 87-101.Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications. Full Text
Sanderson, N.S., Zimmermann, M., Eilinger, L., Gubser, C., Schaeren-Wiemers, N., Lindberg, R.L., Dougan, S.K., Ploegh, H.L., Kappos, L., and Derfuss, T. (2017). Cocapture of cognate and bystander antigens can activate autoreactive B cells. Proceedings of the National Academy of Sciences of the United States of America[Epub ahead of print] Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed "bystander" antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity. Full Text
Satyaki, P.R., and Gehring, M. (2017). DNA methylation and imprinting in plants: machinery and mechanisms. Critical reviews in biochemistry and molecular biology, 1-13. Latest articles Imprinting is an epigenetic phenomenon in which genes are expressed selectively from either the maternal or paternal alleles. In plants, imprinted gene expression is found in a tissue called the endosperm. Imprinting is often set by a unique epigenomic configuration in which the maternal chromosomes are less DNA methylated than their paternal counterparts. In this review, we synthesize studies that paint a detailed molecular portrait of the distinctive endosperm methylome. We will also discuss the molecular machinery that shapes and modifies this methylome, and the role of DNA methylation in imprinting. Full Text
Saxton, R.A., and Sabatini, D.M. (2017). mTOR Signaling in Growth, Metabolism, and Disease. Cell 168, 960-976.The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic. Full Text
Scimone, M.L., Cote, L.E., and Reddien, P.W. (2017). Orthogonal muscle fibres have different instructive roles in planarian regeneration. Nature [Epub ahead of print]. The ability to regenerate missing body parts exists throughout the animal kingdom. Positional information is crucial for regeneration, but how it is harboured and used by differentiated tissues is poorly understood. In planarians, positional information has been identified from study of phenotypes caused by RNA interference in which the wrong tissues are regenerated. For example, inhibition of the Wnt signalling pathway leads to regeneration of heads in place of tails. Characterization of these phenotypes has led to the identification of position control genes (PCGs)-genes that are expressed in a constitutive and regional manner and are associated with patterning. Most PCGs are expressed within planarian muscle; however, how muscle is specified and how different muscle subsets affect regeneration is unknown. Here we show that different muscle fibres have distinct regulatory roles during regeneration in the planarian Schmidtea mediterranea. myoD is required for formation of a specific muscle cell subset: the longitudinal fibres, oriented along the anterior-posterior axis. Loss of longitudinal fibres led to complete regeneration failure because of defects in regeneration initiation. A different transcription factor-encoding gene, nkx1-1, is required for the formation of circular fibres, oriented along the medial-lateral axis. Loss of circular fibres led to a bifurcated anterior-posterior axis with fused heads forming in single anterior blastemas. Whereas muscle is often viewed as a strictly contractile tissue, these findings reveal that different muscle types have distinct and specific regulatory roles in wound signalling and patterning to enable regeneration. Full Text
Semrau,S., Goldman, JE. , Soumillon, M., Mikkelsen, T.S., Jaenisch, R., and van Oudenaarden, A. (2017). Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. Nature communications 8(1), 1096. Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.Full Text
Seyffert, M., Glauser, D.L., Schraner, E.M., de Oliveira, A.P., Mansilla-Soto, J., Vogt, B., Buning, H., Linden, R.M., Ackermann, M., and Fraefel, C. (2017). Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication. PloS one 12, e0170908.As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity. Full Text
Shah, R., Murthy, V., Pacold, M., Danielson, K., Tanriverdi, K., Larson, M.G., Hanspers, K., Pico, A., Mick, E., Reis, J., et al. (2017). Extracellular RNAs Are Associated With Insulin Resistance and Metabolic Phenotypes. Diabetes care [Epub ahead of print] OBJECTIVE: Insulin resistance (IR) is a hallmark of obesity and metabolic disease. Circulating extracellular RNAs (ex-RNAs), stable RNA molecules in plasma, may play a role in IR, though most studies on ex-RNAs in IR are small. We sought to characterize the relationship between ex-RNAs and metabolic phenotypes in a large community-based human cohort. RESEARCH DESIGN AND METHODS: We measured circulating plasma ex-RNAs in 2,317 participants without diabetes in the Framingham Heart Study (FHS) Offspring Cohort at cycle 8 and defined associations between ex-RNAs and IR (measured by circulating insulin level). We measured association between candidate ex-RNAs and markers of adiposity. Sensitivity analyses included individuals with diabetes. In a separate cohort of 90 overweight/obese youth, we measured selected ex-RNAs and metabolites. Biology of candidate microRNAs was investigated in silico. RESULTS: The mean age of FHS participants was 65.8 years (56% female), with average BMI 27.7 kg/m2; participants in the youth cohort had a mean age of 15.5 years (60% female), with mean BMI 33.8 kg/m2. In age-, sex-, and BMI-adjusted models across 391 ex-RNAs in FHS, 18 ex-RNAs were associated with IR (of which 16 were microRNAs). miR-122 was associated with IR and regional adiposity in adults and IR in children (independent of metabolites). Pathway analysis revealed metabolic regulatory roles for miR-122, including regulation of IR pathways (AMPK, target of rapamycin signaling, and mitogen-activated protein kinase). CONCLUSIONS: These results provide translational evidence in support of an important role of ex-RNAs as novel circulating factors implicated in IR.
Shen, K., Choe, A., and Sabatini, D.M. (2017). Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 to Respond Rapidly to Amino Acid Availability. Molecular cell [Epub ahead of print]. mTOR complex I (mTORC1) is a central growth regulator that senses amino acids through a pathway that converges on the Rag GTPases, an obligate heterodimer of two related GTPases. Despite their central role in amino acid sensing, it is unknown why the Rag GTPases are heterodimeric and whether their subunits communicate with each other. Here, we find that the binding of guanosine triphosphate (GTP) to one subunit inhibits the binding and induces the hydrolysis of GTP by the other. This intersubunit communication pushes the Rag GTPases into either of two stable configurations, which represent active "on" or "off" states that interconvert via transient intermediates. Subunit coupling confers on the mTORC1 pathway its capacity to respond rapidly to the amino acid level. Thus, the dynamic response of mTORC1 requires intersubunit communication by the Rag GTPases, providing a rationale for why they exist as a dimer and revealing a distinct mode of control for a GTP-binding protein. Full Text
Shibue, T., and Weinberg, R.A. (2017). EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nature reviews Clinical oncology[Epub ahead of print] The success of anticancer therapy is usually limited by the development of drug resistance. Such acquired resistance is driven, in part, by intratumoural heterogeneity - that is, the phenotypic diversity of cancer cells co-inhabiting a single tumour mass. The introduction of the cancer stem cell (CSC) concept, which posits the presence of minor subpopulations of CSCs that are uniquely capable of seeding new tumours, has provided a framework for understanding one dimension of intratumoural heterogeneity. This concept, taken together with the identification of the epithelial-to-mesenchymal transition (EMT) programme as a critical regulator of the CSC phenotype, offers an opportunity to investigate the nature of intratumoural heterogeneity and a possible mechanistic basis for anticancer drug resistance. In fact, accumulating evidence indicates that conventional therapies often fail to eradicate carcinoma cells that have entered the CSC state via activation of the EMT programme, thereby permitting CSC-mediated clinical relapse. In this Review, we summarize our current understanding of the link between the EMT programme and the CSC state, and also discuss how this knowledge can contribute to improvements in clinical practice. Full Text
Silber, S.J., Kato, K., Aoyama, N., Yabuuchi, A., Skaletsky, H., Fan, Y., Shinohara, K., Yatabe, N., and Kobayashi, T. (2017). Intrinsic fertility of human oocytes. Fertility and sterility[Epub ahead of print] OBJECTIVE: To study the intrinsic fertility of the human oocyte. DESIGN: A large retrospective study of natural cycle single embryo transfer (ET) IVF cycles. SETTING: Private IVF clinic, university, and private hospital. PATIENT(S): Patients were enrolled consecutively over an 8-year period in a single ET natural cycle protocol. INTERVENTION(S): A total of 13,949 oocyte retrievals with natural IVF single ET. Software package R (version 3.2.5) was used for statistical calculations. MAIN OUTCOME MEASURE(S): Live baby rate per oocyte according to age. RESULT(S): A total of 14,185 natural cycle oocytes resulted in 1,913 live babies from single ET. The number of oocytes required to make one live baby in this large series varied with the age of the female partner. For those under 35, the live baby born per oocyte was 26%. For over age 42 it decreased to 1%. These results fit very robustly with a logistic function curve, which is at first steady (horizontal), followed by a linear decline after age 35 with a 10% loss every year until age 43, and then a flattening out (horizontal) by age 44. CONCLUSION(S): The intrinsic fertility per oocyte in natural cycle is far greater than reported in hyperstimulated cycles, varying robustly from 26% to 4% with age from <35 to 42 years. The curve is relatively flat until age 34, and then declines rapidly 10% per year thereafter. Full Text
Silvers, R., Colvin, M.T., Frederick, K.K., Jacavone, A.C., Lindquist, S., Linse, S., and Griffin, R.G. (2017). Aggregation and Fibril Structure of AbetaM01-42 and Abeta1-42. Biochemistry Article ASAP A mechanistic understanding of Abeta aggregation and high-resolution structures of Abeta fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Abeta peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Abeta1-42, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Abeta1-42 fusion protein in reasonable yield, with which we compared aggregation kinetics with AbetaM01-42 containing the additional methionine residue. The data revealed that Abeta1-42 and AbetaM01-42 aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both AbetaM01-42 and Abeta1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of AbetaM01-42 in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail. Full Text
Soh, Y.Q., Mikedis, M.M., Kojima, M., Godfrey, A.K., de Rooij, D.G., and Page, D.C. (2017). Meioc maintains an extended meiotic prophase I in mice. PLoS genetics 13, e1006704.The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase-as early as the preleptotene stage-proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts. Full Text
Sokol, E.S., Feng, Y.X., Jin, D.X., Tizabi, M.D., Miller, D.H., Cohen, M.A., Sanduja, S., Reinhardt, F., Pandey, J., Superville, D.A., Rudolf Jaenisch and Piyush B. Gupta (2017). SMARCE1 is required for the invasive progression of in situ cancers. Proceedings of the National Academy of Sciences of the United States of America [Epub ahead of print] Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors. Full Text
SotoFeliciano, Y.M., Bartlebaugh, J.M.E., Liu, Y., Sanchez-Rivera, F.J., Bhutkar, A., Weintraub, A.S., Buenrostro, J.D., Cheng, C.S., Regev, A., Jacks, T.E,. Richard A. Young, et al. (2017). PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes. Genes & development [Epub ahead of print] Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. Full Text
Suzuki, H.I., Young, R.A., and Sharp, P.A. (2017). Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis. Cell 168, 1000-1014.e1015.Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription. Full Text
Tardiff, D.F., Brown, L.E., Yan, X., Trilles, R., Jui, N.T., Barrasa, M.I., Caldwell, K.A., Caldwell, G.A., Schaus, S.E., and Lindquist, S. (2017). Dihydropyrimidine-Thiones and Clioquinol Synergize To Target beta-Amyloid Cellular Pathologies through a Metal-Dependent Mechanism. ACS chemical neuroscience[Epub ahead of print] The lack of therapies for neurodegenerative diseases arises from our incomplete understanding of their underlying cellular toxicities and the limited number of predictive model systems. It is critical that we develop approaches to identify novel targets and lead compounds. Here, a phenotypic screen of yeast proteinopathy models identified dihydropyrimidine-thiones (DHPM-thiones) that selectively rescued the toxicity caused by beta-amyloid (Abeta), the peptide implicated in Alzheimer's disease. Rescue of Abeta toxicity by DHPM-thiones occurred through a metal-dependent mechanism of action. The bioactivity was distinct, however, from that of the 8-hydroxyquinoline clioquinol (CQ). These structurally dissimilar compounds strongly synergized at concentrations otherwise not competent to reduce toxicity. Cotreatment ameliorated Abeta toxicity by reducing Abeta levels and restoring functional vesicle trafficking. Notably, these low doses significantly reduced deleterious off-target effects caused by CQ on mitochondria at higher concentrations. Both single and combinatorial treatments also reduced death of neurons expressing Abeta in a nematode, indicating that DHPM-thiones target a conserved protective mechanism. Furthermore, this conserved activity suggests that expression of the Abeta peptide causes similar cellular pathologies from yeast to neurons. Our identification of a new cytoprotective scaffold that requires metal-binding underscores the critical role of metal phenomenology in mediating Abeta toxicity. Additionally, our findings demonstrate the valuable potential of synergistic compounds to enhance on-target activities, while mitigating deleterious off-target effects. The identification and prosecution of synergistic compounds could prove useful for developing AD therapeutics where combination therapies may be required to antagonize diverse pathologies. Full Text
Theunissen , T.W., and Jaenisch, R. (2017). Mechanisms of gene regulation in human embryos and pluripotent stem cells. Development 144, 4496-4509. Pluripotent stem cells have broad utility in biomedical research and their molecular regulation has thus garnered substantial interest. While the principles that establish and regulate pluripotency have been well defined in the mouse, it has been difficult to extrapolate these insights to the human system due to species-specific differences and the distinct developmental identities of mouse versus human embryonic stem cells. In this Review, we examine genome-wide approaches to elucidate the regulatory principles of pluripotency in human embryos and stem cells, and highlight where differences exist in the regulation of pluripotency in mice and humans. We review recent insights into the nature of human pluripotent cells in vivo, obtained by the deep sequencing of pre-implantation embryos. We also present an integrated overview of the principal layers of global gene regulation in human pluripotent stem cells. Finally, we discuss the transcriptional and epigenomic remodeling events associated with cell fate transitions into and out of human pluripotency. Full Text
Torrens-Spence , M.P., Pluskal, T., Li, F.S., Carballo, V., and Weng, J.K. (2017). Complete pathway elucidation and heterologous reconstitution of Rhodiola salidroside biosynthesis. Molecular plant [Epub ahead of print]. Salidroside is a bioactive tyrosine-derived phenolic natural product found in medicinal plants under the Rhodiola genus. In addition to their anti-fatigue and anti-anoxia roles in traditional medicine, Rhodiola total extract and salidroside have also displayed medicinal properties as anti-cardiovascular diseases and anti-cancer agents. The resulting surge in global demand of Rhodiola plants and salidroside has driven some species close to extinction. Here, we report the full elucidation of Rhodiola salidroside biosynthetic pathway utilizing the first comprehensive transcriptomics and metabolomics datasets for Rhodiola rosea. Unlike the previously proposed pathway involving separate decarboxylation and deamination enzymatic steps from tyrosine to the key intermediate 4-hydroxyphenylacetaldehyde (4-HPAA), Rhodiola contains a pyridoxal phosphate (PLP)-dependent 4-hydroxyphenylacetaldehyde synthase (4HPAAS) that directly converts tyrosine to 4-HPAA. We further identified genes encoding the subsequent 4-HPAA reductase (4HPAR) and tyrosol:UDP-glucose 8-O-glucosyltransferase (T8GT), respectively, to complete salidroside biosynthesis in Rhodiola. We show that heterologous production of salidroside can be achieved in yeast Saccharomyces cerevisiae as well as in plant Nicotiana benthamiana through transgenic expression of Rhodiola salidroside biosynthetic genes. This study provides new tools for engineering sustainable production of salidroside in heterologous hosts. Full Text
Vaeth, M., Maus, M., Klein-Hessling, S., Freinkman, E., Yang, J., Eckstein, M., Cameron, S., Turvey, S.E., Serfling, E., Berberich-Siebelt, F., et al. (2017). Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming. Immunity 47,(4), p664–679.e6 Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses. Full Text
VanElssen, C.H., Rashidian, M., Vrbanac, V., Wucherpfennig, K.W., El Habre, Z., Sticht, J., Freund, C., Jacobsen, J., Cragnolini, J., Ingram, J., Ploegh H., et al. (2017). Noninvasive imaging of human immune responses in a human xenograft model of graft-versus-host disease. Journal of nuclear medicine 58, 1003-1008. The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer or auto-immunity. Immuno-PET is a non-invasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. We aimed to develop a method to noninvasively image human immune responses in a relevant humanized mouse model. We developed a camelid-derived single domain antibody (VHH) specific for human class II MHC products and used it to non-invasively image human immune cell reconstitution in NSG (NOD-scid gamma) mice reconstituted with human fetal thymus, liver and liver-derived hematopoitic stem cells (BLT mice). We show imaging of infiltrating immunocytes in BLT mice that spontaneously develop a graft versus host (GvHD)-like condition, characterized by alopecia, blepharitis and target organ infiltration by activated human T cells. In diseased animals, we show an increased PET signal in the liver, attributable to infiltration of activated Class II MHC-positive T cells. Non-invasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of GvHD and holds promise for other diseases characterized by inflammation. Full Text
Vu, L.P., Prieto, C., Amin, E.M., Chhangawala, S., Krivtsov, A., Calvo-Vidal, M.N., Chou, T., Chow, A., Minuesa, G., Park, S.M, Kwon, JA, et al. (2017). Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells. Nature genetics advance online publication. The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia. Full Text
Wang, T., Yu, H., Hughes, N.W., Liu, B., Kendirli, A., Klein, K., Chen, W.W., Lander, E.S., and Sabatini, D.M. (2017). Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras. Cell [Epub ahead of print] The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells. Full Text
Weintraub , A.S., Li, C.H., Zamudio, A.V., Sigova, A.A., Hannett, N.M., Day, D.S., Abraham, B.J., Cohen, M.A., Nabet, B., Buckley, D.L., Yang Eric Guo, Denes Hnisz , Rudolf Jaenisch, Richard A. Young, et al. (2017). YY1 Is a Structural Regulator of Enhancer-Promoter Loops. Cell[Epub ahead of print]. There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control. Full Text
Williams , B.P., and Gehring, M.(2017). Stable transgenerational epigenetic inheritance requires a DNA methylation-sensing circuit. Nature communications 8, 2124. Epigenetic states are stably propagated in eukaryotes. In plants, DNA methylation patterns are faithfully inherited over many generations but it is unknown how the dynamic activities of cytosine DNA methyltransferases and 5-methylcytosine DNA glycosylases interact to maintain epigenetic homeostasis. Here we show that a methylation-sensing gene regulatory circuit centered on a 5-methylcytosine DNA glycosylase gene is required for long-term epigenetic fidelity in Arabidopsis. Disrupting this circuit causes widespread methylation losses and abnormal phenotypes that progressively worsen over generations. In heterochromatin, these losses are counteracted such that methylation returns to a normal level over four generations. However, thousands of loci in euchromatin progressively lose DNA methylation between generations and remain unmethylated. We conclude that an actively maintained equilibrium between methylation and demethylation activities is required to ensure long-term stable inheritance of epigenetic information. Full Text
Willighagen, E.L., Mayfield, J.W., Alvarsson, J., Berg, A., Carlsson, L., Jeliazkova, N., Kuhn, S., Pluskal, T.., Rojas-Cherto, M., Spjuth, O., et al. (2017). The Chemistry Development Kit (CDK) v2.0: atom typing, depiction, molecular formulas, and substructure searching. Journal of Cheminformatics vol 9, 33 The Chemistry Development Kit (CDK) is a widely used open source cheminformatics toolkit, providing data structures to represent chemical concepts along with methods to manipulate such structures and perform computations on them. The library implements a wide variety of cheminformatics algorithms ranging from chemical structure canonicalization to molecular descriptor calculations and pharmacophore perception. It is used in drug discovery, metabolomics, and toxicology. Over the last 10 years, the code base has grown significantly, however, resulting in many complex interdependencies among components and poor performance of many algorithms. Full Text
Wolfson2, R.L., and Sabatini, D.M. (2017).(mTORC1) is a master regulator of cell growth that responds to a diverse set of environmental inputs, including amino acids. Over the past 10 years, a number of proteins have been identified that help transmit amino acid availability to mTORC1. However, amino acid sensors for this pathway have only recently been discovered. Here, we review these recent advances and highlight the variety of unexplored questions that emerge from the identification of these sensors. Full Text
Wolfson, R.L., Chantranupong, L., Wyant, G.A., Gu, X., Orozco, J.M., Shen, K., Condon, K.J., Petri, S., Kedir, J., Scaria, S.M.,Abu-Remaileh M, Frankel WN, and Sabatini DM (2017). KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543(7645):438-442. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease. Full Text
Wu, Q., Ploegh, H.L., and Truttmann, M.C. (2017). Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans. ACS chemical biology Article ASAP In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo. Full Text
Wu3, X., and Bartel, D.P. (2017). Widespread Influence of 3'-End Structures on Mammalian mRNA Processing and Stability. Cell 169, 905-917.e911.The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, we investigate the possibility that mRNA structures facilitate the 3'-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. We find that RNA structures are predicted to be more prevalent within these extended 3'-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism. Full Text
Wu2, X., and Bartel, D.P. (2017). kpLogo: positional k-mer analysis reveals hidden specificity in biological sequences. Nucleic acids research [Epub ahead of print] Motifs of only 1-4 letters can play important roles when present at key locations within macromolecules. Because existing motif-discovery tools typically miss these position-specific short motifs, we developed kpLogo, a probability-based logo tool for integrated detection and visualization of position-specific ultra-short motifs from a set of aligned sequences. kpLogo also overcomes the limitations of conventional motif-visualization tools in handling positional interdependencies and utilizing ranked or weighted sequences increasingly available from high-throughput assays. Full Text
Wurtzel, O., Oderberg, I.M., and Reddien, P.W. (2017). Planarian Epidermal Stem Cells Respond to Positional Cues to Promote Cell-Type Diversity. Developmental cell 40, 491-504.e495.Successful regeneration requires that progenitors of different lineages form the appropriate missing cell types. However, simply generating lineages is not enough. Cells produced by a particular lineage often have distinct functions depending on their position within the organism. How this occurs in regeneration is largely unexplored. In planarian regeneration, new cells arise from a proliferative cell population (neoblasts). We used the planarian epidermal lineage to study how the location of adult progenitor cells results in their acquisition of distinct functional identities. Single-cell RNA sequencing of epidermal progenitors revealed the emergence of distinct spatial identities as early in the lineage as the epidermal neoblasts, with further pre-patterning occurring in their post-mitotic migratory progeny. Establishment of dorsal-ventral epidermal identities and functions, in response to BMP signaling, required neoblasts. Our work identified positional signals that activate regionalized transcriptional programs in the stem cell population and subsequently promote cell-type diversity in the epidermis. Full Text
Wyant, G.A., Abu-Remaileh, M., Wolfson, R.L., Chen, W.W., Freinkman, E., Danai, L.V., Vander Heiden, M.G., and Sabatini, D.M. (2017). mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient. Cell 171, 642-654.e612. The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Full Text
Yuskaitis, C.J., Jones, B.M., Wolfson, R.L., Super, C.E., Dhamne, S.C., Rotenberg, A., Sabatini, D.M., Sahin, M., and Poduri, A. (2017). A mouse model of DEPDC5-related epilepsy: Neuronal loss of Depdc5 causes dysplastic and ectopic neurons, increased mTOR signaling, and seizure susceptibility. Neurobiology of disease Vol. 111, Pages 91–101. DEPDC5 is a newly identified epilepsy-related gene implicated in focal epilepsy, brain malformations, and Sudden Unexplained Death in Epilepsy (SUDEP). In vitro, DEPDC5 negatively regulates amino acid sensing by the mTOR complex 1 (mTORC1) pathway, but the role of DEPDC5 in neurodevelopment and epilepsy has not been described. No animal model of DEPDC5-related epilepsy has recapitulated the neurological phenotypes seen in patients, and germline knockout rodent models are embryonic lethal. Here, we establish a neuron-specific Depdc5 conditional knockout mouse by cre-recombination under the Synapsin1 promotor. Depdc5(flox/flox)-Syn1(Cre) (Depdc5cc+) mice survive to adulthood with a progressive neurologic phenotype that includes motor abnormalities (i.e., hind limb clasping) and reduced survival compared to littermate control mice. Depdc5cc+ mice have larger brains with increased cortical neuron size and dysplastic neurons throughout the cortex, comparable to the abnormal neurons seen in human focal cortical dysplasia specimens. Depdc5 results in constitutive mTORC1 hyperactivation exclusively in neurons as measured by the increased phosphorylation of the downstream ribosomal protein S6. Despite a lack of increased mTORC1 signaling within astrocytes, Depdc5cc+ brains show reactive astrogliosis. We observed two Depdc5cc+ mice to have spontaneous seizures, including a terminal seizure. We demonstrate that as a group Depdc5cc+ mice have lowered seizure thresholds, as evidenced by decreased latency to seizures after chemoconvulsant injection and increased mortality from pentylenetetrazole-induced seizures. In summary, our neuron-specific Depdc5 knockout mouse model recapitulates clinical, pathological, and biochemical features of human DEPDC5-related epilepsy and brain malformations. We thereby present an important model in which to study targeted therapeutic strategies for DEPDC5-related conditions.Full Text
Ye, X, and Weinberg, R.A. (2017). The SUMO guards for SNAIL.(2017) Oncotarget. 8(58): 97701–97702. Snail, also known as Snai1, is involved in the malignant progression of mouse mammary carcinoma cells in a transgenic model of human mammary adenocarcinoma pathogenesis. Thus, its ongoing functions appear to be critical to the invasiveness of high-grade carcinoma cells and their ability to seed new tumors, i.e., to function as cancer stem cells. Full Text
Ye, X., Brabletz, T., Kang, Y., Longmore, G.D., Nieto, M.A., Stanger, B.Z., Yang, J., and Weinberg, R.A. (2017). Upholding a role for EMT in breast cancer metastasis. Nature 547, E1-E3. Full Text
Zaytouni, T., Tsai, P.Y., Hitchcock, D.S., DuBois, C.D., Freinkman, E., Lin, L., Morales-Oyarvide, V., Lenehan, P.J., Wolpin, B.M., Mino-Kenudson, M., et al. (2017). Critical role for arginase 2 in obesity-associated pancreatic cancer. Nature communications 8, 242.Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Despite recent identification of metabolic alterations in this lethal malignancy, the metabolic dependencies of obesity-associated PDA remain unknown. Here we show that obesity-driven PDA exhibits accelerated growth and a striking transcriptional enrichment for pathways regulating nitrogen metabolism. We find that the mitochondrial form of arginase (ARG2), which hydrolyzes arginine into ornithine and urea, is induced upon obesity, and silencing or loss of ARG2 markedly suppresses PDA. In vivo infusion of 15N-glutamine in obese mouse models of PDA demonstrates enhanced nitrogen flux into the urea cycle and infusion of 15N-arginine shows that Arg2 loss causes significant ammonia accumulation that results from the shunting of arginine catabolism into alternative nitrogen repositories. Furthermore, analysis of PDA patient tumors indicates that ARG2 levels correlate with body mass index (BMI). The specific dependency of PDA on ARG2 rather than the principal hepatic enzyme ARG1 opens a therapeutic window for obesity-associated pancreatic cancer.Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Here the authors show that obesity induces the expression of the mitochondrial form of arginase ARG2 in PDA and that ARG2 silencing or loss results in ammonia accumulation and suppression of obesity-driven PDA tumor growth. Full Text
Zhao, Q., Cui, M.Y., Levsh, O., Yang, D., Liu, J., Li, J., Hill, L., Yang, L., Hu, Y., Weng, J.K., et al. (2017). Two CYP82D enzymes function as flavone hydroxylases in the biosynthesis of root-specific 4'-deoxyflavones in Scutellaria baicelensis. Molecular plant (in press) Baicalein, wogonin and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as anti-oxidant, antiviral, and liver-protective properties. We describe isolation and characterization of two CYP450 enzymes (SbCYP82D1.1 and SbCYP82D2) that function as the flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H), respectively, in S. baicalensis. SbCYP82D1.1 has a broad specificity for flavones like chrysin and apigenin and is responsible for biosynthesis of baicalein and scutellarein in roots and aerial parts of S. baicalensis, respectively. When expression of SbCYP82D1.1 is knocked-down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. CYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, while minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that CYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed neofunctionalization, where the ancestral F6H activity is partially retained in the derived CYP82D2. Full Text
Zheng , X., and Pincus, D. (2017). Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins. Bio-protocol 7(12) : e2348. This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured from the first eluate by anti-V5-conjugated magnetic beads and eluted with ionic detergent. In this manner, the majority of abundant, nonspecific proteins remain either bound to the first beads or in the first eluate, allowing pure, high affinity complexes to be obtained. This approach can be used to show specific interactions with notoriously 'sticky' chaperone proteins.Full Text
Zimmerman, M.W., Liu, Y., He, S., Durbin, A.D., Abraham, B.J., Easton, J., Shao, Y., Xu, B., Zhu, S., Zhang, X., Young, RA, et al. (2017). c-MYC drives a subset of high-risk pediatric neuroblastomas and is activated through mechanisms including enhancer hijacking and focal enhancer amplification. Cancer discovery [Epub ahead of print]. The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that the family member c-MYC is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas.Full Text
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