The following alphabetical list represents papers published in 2012 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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Agarwala, S.D., Blitzblau, H.G., Hochwagen, A., and Fink, G.R. (2012). RNA Methylation by the MIS Complex Regulates a Cell Fate Decision in Yeast. PLoS Genet 8, e1002732.For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions. Full Text
Anguera, M.C., Sadreyev, R., Zhang, Z.Q., Szanto, A., Payer, B., Sheridan, S.D., Kwok, S., Haggarty, S.J., Sur, M., Alvarez, J.,Gimelbrant A, Mitalipova M, Kirby JE, and Lee JTl. (2012). Molecular Signatures of Human Induced Pluripotent Stem Cells Highlight Sex Differences and Cancer Genes. Cell Stem Cell 11, 75-90.Although human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct. Loss of XIST expression is strongly correlated with upregulation of X-linked oncogenes, accelerated growth rate in vitro, and poorer differentiation in vivo. Whereas differences in X-inactivation potential result in epigenetic variability of female hiPSC lines, male hiPSC lines generally resemble each other and do not overexpress the oncogenes. Neither physiological oxygen levels nor HDAC inhibitors offer advantages to culturing female hiPSC lines. We conclude that female hiPSCs may be epigenetically less stable in culture and caution that loss of XIST may result in qualitatively less desirable stem cell lines. Full Text
Anthoni, H., Sucheston, L.E., Lewis, B.A., Tapia-Paez, I., Fan, X.T., Zucchelli, M., Taipale, M., Stein, C.M., Hokkanen, M.E., Castren, E., et al. (2012). The Aromatase Gene CYP19A1: Several Genetic and Functional Lines of Evidence Supporting a Role in Reading, Speech and Language. Behavior Genetics 42, 509-527.Inspired by the localization, on 15q21.2 of the CYP19A1 gene in the linkage region of speech and language disorders, and a rare translocation in a dyslexic individual that was brought to our attention, we conducted a series of studies on the properties of CYP19A1 as a candidate gene for dyslexia and related conditions. The aromatase enzyme is a member of the cytochrome P450 super family, and it serves several key functions: it catalyzes the conversion of androgens into estrogens; during early mammalian development it controls the differentiation of specific brain areas (e.g. local estrogen synthesis in the hippocampus regulates synaptic plasticity and axonal growth); it is involved in sexual differentiation of the brain; and in songbirds and teleost fishes, it regulates vocalization. Our results suggest that variations in CYP19A1 are associated with dyslexia as a categorical trait and with quantitative measures of language and speech, such as reading, vocabulary, phonological processing and oral motor skills. Variations near the vicinity of its brain promoter region altered transcription factor binding, suggesting a regulatory role in CYP19A1 expression. CYP19A1 expression in human brain correlated with the expression of dyslexia susceptibility genes such as DYX1C1 and ROBO1. Aromatase-deficient mice displayed increased cortical neuronal density and occasional cortical heterotopias, also observed in Robo1-/- mice and human dyslexic brains, respectively. An aromatase inhibitor reduced dendritic growth in cultured rat neurons. From this broad set of evidence, we propose CYP19A1 as a candidate gene for human cognitive functions implicated in reading, speech and language. PDF
Anwar, M., Gupta, P.B., Palaniapan, R., and Matsudaira, P. (2012). Protein array patterning by diffusive gel stamping. Plos One 7, e46382.Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or in-vitro protein synthesis, making it widely accessible to researchers. The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications. Full Text
Avalos, A.M., Kirak, O., Oelkers, J.M., Pils, M.C., Kim, Y.M., Ottinger, M., Jaenisch, R., Ploegh, H.L., and Brinkmann, M.M. (2012). Cell-Specific TLR9 Trafficking in Primary APCs of Transgenic TLR9-GFP Mice. Journal of Immunology Dec 12. [Epub ahead of print] .Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum-resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow-derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs. Full Text
Backer, C.B., Gutzman, J.H., Pearson, C.G., and Cheeseman, I.M. (2012). CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development. Mol Biol Cell.Apr 4. [Epub ahead of print] The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here, we identify the microtubule binding protein Centriole and Spindle Associated Protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left-right asymmetry, defects qualitatively similar to those reported previously for depletion of polyglutamylation conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations providing specific functional capabilities to these populations .PDF
BarPeled2, L., and Sabatini, D.M. (2012). SnapShot : mTORC1 Signaling at the Lysosomal Surface. Cell 151, 1390-1390 e1391 In mammals, the mTOR complex 1 (mTORC1) ser/thr kinase regulates cellular and organismal growth in response to a variety of environmental and intracellular stimuli. Amino acid levels mediate the first step in the bipartite activation of mTORC1 by promoting its translocation from a cytosolic compartment to the lysosomal surface. Full Text
BarPeled, L., Schweitzer, L.D., Zoncu, R., and Sabatini, D.M. (2012). Ragulator Is a GEF for the Rag GTPases that Signal Amino Acid Levels to mTORC1. Cell 150, 1196-1208.The mTOR Complex 1 (mTORC1) pathway regulates cell growth in response to numerous cues, including amino acids, which promote mTORC1 translocation to the lysosomal surface, its site of activation. The heterodimeric RagA/B-RagC/D GTPases, the Ragulator complex that tethers the Rags to the lysosome, and the v-ATPase form a signaling system that is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of guanosine triphosphate to RagA and RagB but the factors that regulate Rag nucleotide loading are unknown. Here, we identify HBXIP and C7orf59 as two additional Ragulator components that are required for mTORC1 activation by amino acids. The expanded Ragulator has nucleotide exchange activity toward RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by identifying Ragulator as a guanine nucleotide exchange factor (GEF) for the Rag GTPases. Full Text
Bernstein3, D.A., Vyas, V.K., and Fink, G.R. (2012). Genes come and go: The evolutionarily plastic path of budding yeast RNase III enzymes. RNA Biology 9(9): 1123-1128 .Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III's in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer. As none of the budding yeast species has the canonical Dicer found in many other fungal lineages and most eukaryotes, the extant species must have evolved from an ancestor that lost the canonical Dicer, and evolved a novel Dicer from the essential RNT1 gene. No single, simple model could explain the evolution of RNase III enzymes from this ancestor because existing sequence data are consistent with two equally plausible models. The models share an architecture for RNase III evolution that involves gene duplication, loss, subfunctionalization, and neofunctionalization. This commentary explains our reasoning, and offers the prospect that further genomic data could further resolve the dilemma surrounding the budding yeast RNase III's evolution. PDF
Bernstein2, D.A. (2012). Identification of Small Molecules That Disrupt SSB-Protein Interactions Using a High-Throughput Screen. Methods Mol Biol 922, 183-191.Bacterial single-stranded DNA-binding proteins (SSBs) recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. The essential nature of these SSB-protein interactions makes inhibitors that block SSB-partner complex formation valuable biochemical tools and attractive potential antibacterial agents. However, many of these protein-protein interactions are weak and not amenable to the high-throughput nature of small molecule screens. Here I describe a high-throughput screen to identify small molecules that inhibit the interaction between Exonuclease I (ExoI) and the final 10 amino acids of the SSB C-terminal tail (SSB-Ct). The strength of the binding between ExoI and the SSB-Ct tail is fundamental to the interaction's utility in the high-throughput screen. Full Text
Bernstein, D.A., Vyas, V.K., Weinberg, D.E., Drinnenberg, I.A., Bartel, D.P., and Fink, G.R. (2012). Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation. Proc Natl Acad Sci U S A 109, 523-528.The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts. Full Text.
Birsoy2, K., Wang, T., Possemato, R., Yilmaz, O.H., Koch, C.E., Chen, W.W., Hutchins, A.W., Gultekin, Y., Peterson, T.R., Carette, J.Thijn R Brummelkamp, Clary B Clish & David M Sabatini. (2012). MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors. Nature Genetics Dec 2. [Epub ahead of print] .There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA-resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors. Full Text
Birsoy, K., Sabatini, D.M., and Possemato, R. (2012). Untuning the tumor metabolic machine: Targeting cancer metabolism: a bedside lesson. Nature Medicine 18, 1022-1023. Several decades of scientific observations followed by years of basic and now clinical research support the notion that the metabolic power of tumor cells can provide the long-desired Achilles' heel of cancer. Yet many questions remain as to what defines the true metabolic makeup of a tumor and whether well-known factors and pathways involved in metabolic signaling act as tumor suppressors or oncogenes. In 'Bedside to Bench', Kıvanç Birsoy, David M. Sabatini and Richard Possemato discuss how retrospective studies of diabetic individuals with pancreatic cancer treated with the antidiabetic drug metformin point to a possible anticancer effect for this drug. Further research will need to discern whether this drug acts at the organismal level or by directly targeting the power plant of tumor cells. In 'Bench to Bedside', Regina M. Young and M. Celeste Simon peruse the complex function of a key metabolic factor that mediates the cell's response to low oxygen levels, often found in tumors. This hypoxia-inducible factor (HIF) comes in two flavors, which can be either tumor promoting or tumor suppressive, depending on the type of cancer. Because of this, the therapeutic use of HIF inhibitors must proceed with caution. Further defining the relationship between metabolic regulation of HIF and tumor progression may open up new diagnostic tools and treatments. Full Text
BlakerLee A., Gupta, S., McCammon, J.M., Derienzo, G., and Sive, H. (2012). Zebrafish homologs of 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes. Dis Model Mech.May 1. [Epub ahead of print] Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASD), intellectual disability disorder (IDD), and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes, however, the number of genes that contribute to these phenotypes is not known. Further, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which may associate the CNV with pathologies, have not been identified. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of eleven phenotypic assays, spanning the first five days of development, demonstrates that this set of genes is highly active, such that 21 out of 22 homologs tested show loss of function phenotypes. Most genes are required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes can substitute for the fish homolog, demonstrating orthology, and consistent with conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels are reduced by ~50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which may present a large genetic target, and may explain why multiple brain function and other phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties amongst the 16p11.2 set, which may link this CNV to brain disorders including ASD and IDD. Full Text.
Blitzblau, H.G., Chan, C.S., Hochwagen, A., and Bell, S.P. (2012). Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes PLoS Genet 8(5): e1002643.The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms. Full Text
Bousquet, M., Nguyen, D., Chen, C., Shields, L., and Lodish, H.F. (2012). MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA. Haematologica-the Hematology Journal 97, 1713-1721.Background We previously described a t(2;11)(p21;q23) chromosomal translocation found in patients with myelodysplasia or acute myeloid leukemia that leads to over-expression of the microRNA miR-125b, and we showed that transplantation of mice with murine stem/progenitor cells over-expressing miR-125b is able to induce leukemia. In this study, we investigated the mechanism of myeloid transformation by miR-125b. Design and Methods To investigate the consequences of miR-125b over-expression on myeloid differentiation, apoptosis and proliferation, we used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line. To test whether miR-125b is able to transform myeloid cells, we used the non-tumorigenic and interleukin-3-dependent 32Dclone3 cell line over-expressing miR-125b, in xenograft experiments in nude mice and in conditions of interleukin-3 deprivation. To identify new miR-125b targets, we compared, by RNA-sequencing, the transcriptome of cell lines that do or do not over-express miR-125b. Results We showed that miR-125b over-expression blocks apoptosis and myeloid differentiation and enhances proliferation in both species. More importantly, we demonstrated that miR-125b is able to transform the 32Dclone3 cell line by conferring growth independence from interleukin-3; xenograft experiments showed that these cells form tumors in nude mice. Using RNA-sequencing and quantitative real-time polymerase chain reaction experiments, we identified multiple miR-125b targets. We demonstrated that ABTB1, an anti-proliferative factor, is a new direct target of miR-125b and we confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. MiR-125b controls apoptosis by down-regulating genes involved in the p53 pathway including BAK1 and TP53INP1. Conclusions This study demonstrates that in a myeloid context, miR-125b is an oncomiR able to transform cell lines. miR-125b blocks myeloid differentiation in part by targeting CBFB, blocks apoptosis through down-regulation of multiple genes involved in the p53 pathway, and confers a proliferative advantage to human and mouse myeloid cell lines in part by targeting ABTB1. Full Text
Bryan, A.W., Jr., O'Donnell, C.W., Menke, M., Cowen, L.J., Lindquist, S., and Berger, B. (2012). STITCHER: Dynamic assembly of likely amyloid and prion beta-structures from secondary structure predictions. Proteins-Structure Function and Bioinformatics 80, 410-420.The supersecondary structure of amyloids and prions, proteins of intense clinical and biological interest, are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Previous work has demonstrated that probability-based prediction of discrete beta-strand pairs can offer insight into these structures. Here, we devise a system of energetic rules that can be used to dynamically assemble these discrete beta-strand pairs into complete amyloid beta-structures. The STITCHER algorithm progressively stitches strand-pairs into full beta-sheets based on a novel free-energy model, incorporating experimentally observed amino-acid side-chain stacking contributions, entropic estimates, and steric restrictions for amyloidal parallel beta-sheet construction. A dynamic program computes the top 50 structures and returns both the highest scoring structure and a consensus structure taken by polling this list for common discrete elements. Putative structural heterogeneity can be inferred from sequence regions that compose poorly. Predictions show agreement with experimental models of Alzheimer's amyloid beta peptide and the Podospora anserina Het-s prion. Predictions of the HET-s homolog HET-S also reflect experimental observations of poor amyloid formation. We put forward predicted structures for the yeast prion Sup35, suggesting N-terminal structural stability enabled by tyrosine ladders, and C-terminal heterogeneity. Predictions for the Rnq1 prion and alpha-synuclein are also given, identifying a similar mix of homogenous and heterogeneous secondary structure elements. STITCHER provides novel insight into the energetic basis of amyloid structure, provides accurate structure predictions, and can help guide future experimental studies. Full Text.
Buganim3, Y., and Jaenisch, R. (2012). Transdifferentiation by defined factors as a powerful research tool to address basic biological questions. Cell Cycle 11: 24 , 1-2. Ttransdifferentiation of different cell types in vitro may enablethe identification of key regulators of developmental processes more readily than complex and cumbersome in vitro/vivo approaches. Full Text
Buganim2, Y., Faddah, D.A., Cheng, A.W., Itskovich, E., Markoulaki, S., Ganz, K., Klemm, S.L., van Oudenaarden, A., and Jaenisch, R. (2012). Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase. Cell 150, 1209-1222.During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry. Full Text
Buganim, Y., Itskovich, E., Hu, Y.C., Cheng, A.W., Ganz, K., Sarkar, S., Fu, D., Welstead, G.G., Page, D.C., and Jaenisch, R. (2012). Direct Reprogramming of Fibroblasts into Embryonic Sertoli-like Cells by Defined Factors. Cell Stem Cell 11, 373-386.Sertoli cells are considered the "supporting cells" of the testis that play an essential role in sex determination during embryogenesis and in spermatogenesis during adulthood. Their essential roles in male fertility along with their immunosuppressive and neurotrophic properties make them an attractive cell type for therapeutic applications. Here we demonstrate the generation of induced embryonic Sertoli-like cells (ieSCs) by ectopic expression of five transcription factors. We characterize the role of specific transcription factor combinations in the transition from fibroblasts to ieSCs and identify key steps in the process. Initially, transduced fibroblasts underwent a mesenchymal to epithelial transition and then acquired the ability to aggregate, formed tubular-like structures, and expressed embryonic Sertoli-specific markers. These Sertoli-like cells facilitated neuronal differentiation and self-renewal of neural progenitor cells (NPCs), supported the survival of germ cells in culture, and cooperated with endogenous embryonic Sertoli and primordial germ cells in the generation of testicular cords in the fetal gonad. Full Text
Bumgarner, S.L., Neuert, G., Voight, B.F., Symbor-Nagrabska, A., Grisafi, P., van Oudenaarden, A., and Fink, G.R. (2012). Single-Cell Analysis Reveals that Noncoding RNAs Contribute to Clonal Heterogeneity by Modulating Transcription Factor Recruitment. Mol Cell .Jan 18. [Epub ahead of print] Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11's variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter. Full Text.
Cantor, J.R., and Sabatini, D.M. (2012). Cancer cell metabolism: one hallmark, many faces. Cancer Discov 2, 881-898.Cancer cells must rewire cellular metabolism to satisfy the demands of growth and proliferation. Although many of the metabolic alterations are largely similar to those in normal proliferating cells, they are aberrantly driven in cancer by a combination of genetic lesions and nongenetic factors such as the tumor microenvironment. However, a single model of altered tumor metabolism does not describe the sum of metabolic changes that can support cell growth. Instead, the diversity of such changes within the metabolic program of a cancer cell can dictate by what means proliferative rewiring is driven, and can also impart heterogeneity in the metabolic dependencies of the cell. A better understanding of this heterogeneity may enable the development and optimization of therapeutic strategies that target tumor metabolism .PDF
Ceyhan, O., Birsoy, K., and Hoffman, C.S. (2012). Identification of Biologically Active PDE11-Selective Inhibitors Using a Yeast-Based High-Throughput Screen. Chemistry & Biology 19, 155-163.The biological roles of cyclic nucleotide phosphodiesterase 11 (PDE11) enzymes are poorly understood, in part due to the lack of selective inhibitors. To address the need for such compounds, we completed an similar to 200,000 compound high-throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay, and identified 4 potent and selective PDE11 inhibitors. One compound, along with two structural analogs, elevates cAMP and cortisol levels in human adrenocortical cells, consistent with gene association studies that link PDE11 activity to adrenal function. As such, these compounds can immediately serve as chemical tools to study PDE11 function in cell culture, and as leads to develop therapeutics for the treatment of adrenal insufficiencies. Our results further validate this yeast-based HTS platform for the discovery of potent, selective, and biologically active PDE inhibitors. Full Text.
Chang2, J.T., and Sive, H. (2012). Manual drainage of the zebrafish embryonic brain ventricles. J Vis Exp Dec 16;(70). pii: 4243.Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules(1,2). In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles(3-5). In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)(6). Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development(7). eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development(8,9). Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium(10-20). Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation(21-24). However, the complex functions of the eCSF are largely unknown. We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development. Full Text
Chang, J.T., Lowery, L.A., and Sive, H. (2012). Multiple roles for the Na,K-ATPase subunits, Atp1a1 and Fxyd1, during brain ventricle development. Developmental Biology Jun 6. [Epub ahead of print] .Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na(+) increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function - formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF. Full Text
Chen2, D.H., Sun, Y.T., Wei, Y.K., Zhang, P.J., Rezaeian, A.H., Teruya-Feldstein, J., Gupta, S., Liang, H., Lin, H.K., Hung, M.C., et al. (2012). LIFR is a breast cancer metastasis suppressor upstream of the Hippo-YAP pathway and a prognostic marker. Nature Medicine 18, 1511-U1105.There is a pressing need to identify prognostic markers of metastatic disease and targets for treatment. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies and clinical validation, we identify leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor downstream of the microRNA miR-9 and upstream of Hippo signaling. Restoring LIFR expression in highly malignant tumor cells suppresses metastasis by triggering a Hippo kinase cascade that leads to phosphorylation, cytoplasmic retention and functional inactivation of the transcriptional coactivator YES-associated protein (YAP). Conversely, loss of LIFR in nonmetastatic breast cancer cells induces migration, invasion and metastatic colonization through activation of YAP. LIFR is downregulated in human breast carcinomas and inversely correlates with metastasis. Notably, in approximately 1,000 nonmetastatic breast tumors, LIFR expression status correlated with metastasis-free, recurrence-free and overall survival outcomes in the patients. These findings identify LIFR as a metastasis suppressor that functions through the Hippo-YAP pathway and has significant prognostic power. Full Text
Chen, N., Bellott, D.W., Page, D.C., and Clark, A.G. (2012). Identification of avian W-linked contigs by short-read sequencing. BMC Genomics 13: 183 .Background: The female-specific W chromosomes and male-specific Y chromosomes have proven difficult to assemble with whole-genome shotgun methods, creating a demand for new approaches to identify sequence contigs specific to these sex chromosomes. Here, we develop and apply a novel method for identifying sequences that are W-specific. Results: Using the Illumina Genome Analyzer, we generated sequence reads from a male domestic chicken (ZZ) and mapped them to the existing female (ZW) genome sequence. This method allowed us to identify segments of the female genome that are underrepresented in the male genome and are therefore likely to be female specific. We developed a Bayesian classifier to automate the calling of W-linked contigs and successfully identified more than 60 novel W-specific sequences. Conclusions: Our classifier can be applied to improve heterogametic whole-genome shotgun assemblies of the W or Y chromosome of any organism. This study greatly improves our knowledge of the W chromosome and will enhance future studies of avian sex determination and sex chromosome evolution. Full Text
Cheng, B., Li, T.D., Rahl, P.B., Adamson, T.E., Loudas, N.B., Guo, J.N., Varzavand, K., Cooper, J.J., Hu, X.P., Gnatt, Young RA, Price DH(2012). Functional Association of Gdown1 with RNA Polymerase II Poised on Human Genes. Molecular Cell 45, 38-50.Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pot II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. Full Text.\Chin, B.L., Ryan, O., Lewitter, F., Boone, C., and Fink, G.R. (2012). Genetic Variation in Saccharomyces cerevisiae: Circuit Diversification in a Signal Transduction Network. Genetics 192, 1523-+.The connection between genotype and phenotype was assessed by determining the adhesion phenotype for the same mutation in two closely related yeast strains, S288c and Sigma, using two identical deletion libraries. Previous studies, all in Sigma, had shown that the adhesion phenotype was controlled by the filamentation mitogen-activated kinase (fMAPK) pathway, which activates a set of transcription factors required for the transcription of the structural gene FLO11. Unexpectedly, the fMAPK pathway is not required for FLO11 transcription in S288c despite the fact that the fMAPK genes are present and active in other pathways. Using transformation and a sensitized reporter, it was possible to isolate RPI1, one of the modifiers that permits the bypass of the fMAPK pathway in S288c. RPI1 encodes a transcription factor with allelic differences between the two strains: The RPI1 allele from S288c but not the one from Sigma can confer fMAPK pathway-independent transcription of FLO11. Biochemical analysis reveals differences in phosphorylation between the alleles. At the nucleotide level the two alleles differ in the number of tandem repeats in the ORF. A comparison of genomes between the two strains shows that many genes differ in size due to variation in repeat length. Full Text
Chin2, B.L., Ryan, O., Lewitter, F., Boone, C., and Fink, G.R. (2012). Genetic Variation in Saccharomyces cerevisiae: Circuit Diversification in a Signal Transduction Network. Genetics 192, 1523-+.The connection between genotype and phenotype was assessed by determining the adhesion phenotype for the same mutation in two closely related yeast strains, S288c and Sigma, using two identical deletion libraries. Previous studies, all in Sigma, had shown that the adhesion phenotype was controlled by the filamentation mitogen-activated kinase (fMAPK) pathway, which activates a set of transcription factors required for the transcription of the structural gene FLO11. Unexpectedly, the fMAPK pathway is not required for FLO11 transcription in S288c despite the fact that the fMAPK genes are present and active in other pathways. Using transformation and a sensitized reporter, it was possible to isolate RPI1, one of the modifiers that permits the bypass of the fMAPK pathway in S288c. RPI1 encodes a transcription factor with allelic differences between the two strains: The RPI1 allele from S288c but not the one from Sigma can confer fMAPK pathway-independent transcription of FLO11. Biochemical analysis reveals differences in phosphorylation between the alleles. At the nucleotide level the two alleles differ in the number of tandem repeats in the ORF. A comparison of genomes between the two strains shows that many genes differ in size due to variation in repeat length. Full Text
Chin, B.L., Frizzell, M.A., Timberlake, W.E., and Fink, G.R. (2012). FASTER MT: Isolation of Pure Populations of a and alpha Ascospores from Saccharomycescerevisiae. G3 (Bethesda) 2, 449-452.The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci. These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. To this end, we inserted a gene encoding red fluorescent protein into the MATa locus. Red fluorescent protein expression caused MATa and a/alpha diploid vegetative cells and MATa ascospores to fluoresce; MATalpha cells without the gene did not fluoresce. Heterozygous diploids segregated fluorescent and nonfluorescent ascospores 2:2 in tetrads and bulk populations. The two populations of spores were separable by fluorescence-activated cell sorting with little cross contamination or contamination with diploid vegetative cells. This approach, which we call Fluorescent Ascospore Technique for Efficient Recovery of Mating Type (FASTER MT), should be applicable to laboratory, industrial, and undomesticated, strains. Full Text.
Claessen, J.H.L., Kundrat, L., and Ploegh, H.L. (2012). Protein quality control in the ER: balancing the ubiquitin checkbook. Trends in Cell Biology 22, 22-32.Protein maturation in the endoplasmic reticulum (ER) is subject to stringent quality control. Terminally misfolded polypeptides are usually ejected into the cytoplasm and targeted for destruction by the proteasome. Ubiquitin conjugation is essential for both extraction and proteolysis. We discuss the role of the ubiquitin conjugation machinery in this pathway and focus on the role of ubiquitin ligase complexes as gatekeepers for membrane passage. We then examine the type of ubiquitin modification applied to the misfolded ER protein and the role of de-ubiquitylating enzymes in the extraction of proteins from the ER. Full Text.
Costantino, C.M., Spooner, E., Ploegh, H.L., and Hafler, D.A. (2012). Class II MHC Self-Antigen Presentation in Human B and T Lymphocytes. Plos One 7(1):e29805.Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein. Full Text
Dai, C.K., Santagata, S., Tang, Z.J., Shi, J.Y., Cao, J.X., Kwon, H., Bronson, R.T., Whitesell, L., and Lindquist, S. (2012). Loss of tumor suppressor NF1 activates HSF1 to promote carcinogenesis. Journal of Clinical Investigation 122, 3742-3754.Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1(-/-) cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis. Full Text
Davies, C.W., Chaney, J., Korbel, G., Ringe, D., Petsko, G.A., Ploegh, H., and Das, C. (2012). The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK). Bioorganic & Medicinal Chemistry Letters 22, 3900-3904.UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson's disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the X-ray structure of UCHL1 co-crystallized with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 angstrom resolution. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin's C-terminal tail, thereby occupying the P1' (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of molecular contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency. (C) 2012 Elsevier Ltd. All rights reserved. Full Text
DeRienzo, G., Gutzman, J.H., and Sive, H. (2012). Efficient shRNA-Mediated Inhibition of Gene Expression in Zebrafish. Zebrafish 9(3): 97-107.Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression. Full Text
Donmez, G., Arun, A., Chung, C.Y., McLean, P.J., Lindquist, S., and Guarente, L. (2012). SIRT1 Protects against alpha-Synuclein Aggregation by Activating Molecular Chaperones. J Neurosci 32, 124-132 .alpha-Synuclein is a key molecule in the pathogenesis of synucleinopathy including dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy. Sirtuins are NAD(+)-dependent protein deacetylases that are highly conserved and counter aging in lower organisms. We show that the life span of a mouse model with A53T alpha-synuclein mutation is increased by overexpressing SIRT1 and decreased by knocking out SIRT1 in brain. Furthermore, alpha-synuclein aggregates are reduced in the brains of mice with SIRT1 overexpression and increased by SIRT1 deletion. We show that SIRT1 deacetylates HSF1 (heat shock factor 1) and increases HSP70 RNA and protein levels, but only in the brains of mice with A53T and SIRT1 expression. Thus, SIRT1 responds to alpha-synuclein aggregation-induced stress by activating molecular chaperones to protect against disease. Full Text.
Dougan, S.K., Ogata, S., Hu, C.C., Grotenbreg, G.M., Guillen, E., Jaenisch, R., and Ploegh, H.L. (2012). IgG1+ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells. PNAS Aug 6. [Epub ahead of print].We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1(+) ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1(-/-) mice have exclusively IgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3' of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM(+)IgG1(+) double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3. PDF
Dower, K., Filone, C.M., Hodges, E.N., Bjornson, Z.B., Rubins, K.H., Brown, L.E., Schaus, S., Hensley, L.E., and Connor, J.H. (2012). Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-Oriented Synthesis Library. Journal of Virology 86, 2632-2640.Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential bio-threat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals. Full Text.
Edwards, M.D., and Gifford, D.K. (2012). High-resolution genetic mapping with pooled sequencing. BMC Bioinformatics 13(Suppl 6):S8 . Background: Modern genetics has been transformed by high-throughput sequencing. New experimental designs in model organisms involve analyzing many individuals, pooled and sequenced in groups for increased efficiency. However, the uncertainty from pooling and the challenge of noisy sequencing data demand advanced computational methods. Results: We present MULTIPOOL, a computational method for genetic mapping in model organism crosses that are analyzed by pooled genotyping. Unlike other methods for the analysis of pooled sequence data, we simultaneously consider information from all linked chromosomal markers when estimating the location of a causal variant. Our use of informative sequencing reads is formulated as a discrete dynamic Bayesian network, which we extend with a continuous approximation that allows for rapid inference without a dependence on the pool size. MULTIPOOL generalizes to include biological replicates and case-only or case-control designs for binary and quantitative traits. Conclusions: Our increased information sharing and principled inclusion of relevant error sources improve resolution and accuracy when compared to existing methods, localizing associations to single genes in several cases. MULTIPOOL is freely available at http://cgs.csail.mit.edu/multipool/. Full Text
Efeyan2, A., Zoncu, R., Chang, S., Gumper, I., Snitkin, H., Wolfson, R.L., Kirak, O., Sabatini, D.D., and Sabatini, D.M. (2012). Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature. Published online 23 December 2012 The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors. Cell-based studies showed that mTORC1 senses amino acids through the RagA-D family of GTPases (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagA(GTP)) from its endogenous promoter. RagA(GTP/GTP) mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagA(GTP/GTP) neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-RagA(GTP/GTP) neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagA(GTP/GTP) mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagA(GTP/GTP) neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagA(GTP/GTP) neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagA(GTP/GTP) fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability. Full Text
Efeyan, A., Zoncu, R., and Sabatini, D.M. (2012). Amino acids and mTORC1: from lysosomes to disease. Trends Mol Medicine Available online 28 June 2012 .The mechanistic target of rapamycin (mTOR) kinase controls growth and metabolism, and its deregulation underlies the pathogenesis of many diseases, including cancer, neurodegeneration, and diabetes. mTOR complex 1 (mTORC1) integrates signals arising from nutrients, energy, and growth factors, but how exactly these signals are propagated await to be fully understood. Recent findings have placed the lysosome, a key mediator of cellular catabolism, at the core of mTORC1 regulation by amino acids. A multiprotein complex that includes the Rag GTPases, Ragulator, and the v-ATPase forms an amino acid-sensing machinery on the lysosomal surface that affects the decision between cell growth and catabolism at multiple levels. The involvement of a catabolic organelle in growth signaling may have important implications for our understanding of mTORC1-related pathologies. Full Text
ElHaibi, C.P., Bell, G.W., Zhang, J.W., Collmann, A.Y., Wood, D., Scherber, C.M., Csizmadia, E., Mariani, O., Zhu, C.H., Campagne, A., et al. (2012). Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy. Proceedings of the National Academy of Sciences of the United States of America 109, 17460-17465. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into multiple mesoderm lineages in the course of normal tissue homeostasis or during injury. We have previously shown that MSCs migrate to sites of tumorigenesis, where they become activated by cancer cells to promote metastasis. However, the molecular and phenotypic attributes of the MSC-induced metastatic state of the cancer cells remained undetermined. Here, we show that bone marrow-derived human MSCs promote de novo production of lysyl oxidase (LOX) from human breast carcinoma cells, which is sufficient to enhance the metastasis of otherwise weakly metastatic cancer cells to the lungs and bones. We also show that LOX is an essential component of the CD44-Twist signaling axis, in which extracellular hyaluronan causes nuclear translocation of CD44 in the cancer cells, thus triggering LOX transcription by associating with its promoter. Processed and enzymatically active LOX, in turn, stimulates Twist transcription, which mediates the MSC-triggered epithelial-to-mesenchymal transition (EMT) of carcinoma cells. Surprisingly, although induction of EMT in breast cancer cells has been tightly associated with the generation of cancer stem cells, we find that LOX, despite being critical for EMT, does not contribute to the ability of MSCs to promote the formation of cancer stem cells in the carcinoma cell populations. Collectively, our studies highlight a critical role for LOX in cancer metastasis and indicate that the signaling pathways controlling stroma-induced EMT are distinct from pathways regulating the development of cancer stem cells. Full Text
Engler, D.A., Gupta, S., Growdon, W.B., Drapkin, R.I., Nitta, M., Sergent, P.A., Allred, S.F., Gross, J., Deavers, M.T., Kuo, W.L., et al. (2012). Genome Wide DNA Copy Number Analysis of Serous Type Ovarian Carcinomas Identifies Genetic Markers Predictive of Clinical Outcome. Plos One ;7(2):e30996. Ovarian cancer is the fifth leading cause of cancer death in women. Ovarian cancers display a high degree of complex genetic alterations involving many oncogenes and tumor suppressor genes. Analysis of the association between genetic alterations and clinical endpoints such as survival will lead to improved patient management via genetic stratification of patients into clinically relevant subgroups. In this study, we aim to define subgroups of high-grade serous ovarian carcinomas that differ with respect to prognosis and overall survival. Genome-wide DNA copy number alterations (CNAs) were measured in 72 clinically annotated, high-grade serous tumors using high-resolution oligonucleotide arrays. Two clinically annotated, independent cohorts were used for validation. Unsupervised hierarchical clustering of copy number data derived from the 72 patient cohort resulted in two clusters with significant difference in progression free survival (PFS) and a marginal difference in overall survival (OS). GISTIC analysis of the two clusters identified altered regions unique to each cluster. Supervised clustering of two independent large cohorts of high-grade serous tumors using the classification scheme derived from the two initial clusters validated our results and identified 8 genomic regions that are distinctly different among the subgroups. These 8 regions map to 8p21.3, 8p23.2, 12p12.1, 17p11.2, 17p12, 19q12, 20q11.21 and 20q13.12; and harbor potential oncogenes and tumor suppressor genes that are likely to be involved in the pathogenesis of ovarian carcinoma. We have identified a set of genetic alterations that could be used for stratification of high-grade serous tumors into clinically relevant treatment subgroups. Full Text
Gascoigne, K.E., and Cheeseman, I.M. (2012). T time for point centromeres. Nature Cell Biol. Published online 06 May 2012 The diverse nature of eukaryotic centromere structure has led to a prevailing view that the kinetochore-chromatin interface is fundamentally different in distinct species. Two studies now challenge this dogma with the identification of budding yeast homologues of the vertebrate centromere DNA-binding proteins CENP-T and CENP-W. Full Text.
Germain, A.R., Carmody, L.C., Morgan, B., Fernandez, C., Forbeck, E., Lewis, T.A., Nag, P.P., Ting, A., VerPlank, L., Feng, Y.X .Jose R. Perez, Sivaraman Dandapani ,Michelle Palmer, Eric S. Lander, Piyush B. Gupta, Stuart L.Schreiber and Benito Munozl. (2012). Identification of a selective small molecule inhibitor of breast cancer stem cells. Bioorganic & Medicinal Chemistry Letters 22, 3571-3574.A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP). (C) 2012 Elsevier Ltd. All rights reserved. Full Text
Giraud, M., Yoshida, H., Abramson, J., Rahl, P.B., Young, R.A., Mathis, D., and Benoist, C. (2012). Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. Proceedings of the National Academy of Sciences of the United States of America 109, 535-540. Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases. Full Text.
Golan, D., Erlich, Y., and Rosset, S. (2012). Weighted pooling--practical and cost-effective techniques for pooled high-throughput sequencing. Bioinformatics 28, i197-i206.MOTIVATION: Despite the rapid decline in sequencing costs, sequencing large cohorts of individuals is still prohibitively expensive. Recently, several sophisticated pooling designs were suggested that can identify carriers of rare alleles in large cohorts with a significantly smaller number of pools, thus dramatically reducing the cost of such large-scale sequencing projects. These approaches use combinatorial pooling designs where each individual is either present or absent from a pool. One can then infer the number of carriers in a pool, and by combining information across pools, reconstruct the identity of the carriers. RESULTS: We show that one can gain further efficiency and cost reduction by using 'weighted' designs, in which different individuals donate different amounts of DNA to the pools. Intuitively, in this situation, the number of mutant reads in a pool does not only indicate the number of carriers, but also their identity. We describe and study a powerful example of such weighted designs, using non-overlapping pools. We demonstrate that this approach is not only easier to implement and analyze but is also competitive in terms of accuracy with combinatorial designs when identifying rare variants, and is superior when sequencing common variants. We then discuss how weighting can be incorporated into existing combinatorial designs to increase their accuracy and demonstrate the resulting improvement using simulations. Finally, we argue that weighted designs have enough power to facilitate detection of common alleles, so they can be used as a cornerstone of whole-exome sequencing projects. CONTACT: saharon@post.tau.ac.il. Full Text
Guenther, M.G., and Young, R.A. (2012). Grounded: Transcriptional Pausing in Naive mESCs. Cell Stem Cell 10, 484-485.Growing mouse pluripotent stem cells in defined media with signaling inhibitors establishes a naive ground state different from that of cells grown in serum. Recently in Cell,Marks et al. (2012) reported differences in the transcriptional and epigenomic landscapes of naive and serum-exposed mouse stem cells that improve our understanding of the pluripotent ground state. Full Text.
Guo, W., Keckesova, Z., Donaher, J.L., Shibue, T., Tischler, V., Reinhardt, F., Itzkovitz, S., Noske, A., Zurrer-Hardi, U., Bell, G.,Tam WL, Mani SA, van Oudenaarden A, and Weinberg RA. (2012). Slug and sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015-1028.Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs. Full Text.
Gymrek, M., Golan, D., Rosset, S., and Erlich, Y. (2012). lobSTR: A short tandem repeat profiler for personal genomes. Genome Research Apr 20. [Epub ahead of print] .Short tandem repeats (STRs) have a wide range of applications, including medical genetics, forensics, and genetic genealogy. High-throughput sequencing (HTS) has the potential to profile hundreds of thousands of STR loci. However, mainstream bioinformatics pipelines are inadequate for the task. These pipelines treat STR mapping as gapped alignment, which results in cumbersome processing times and a biased sampling of STR alleles. Here, we present lobSTR, a novel method for profiling STRs in personal genomes. lobSTR harnesses concepts from signal processing and statistical learning to avoid gapped alignment and to address the specific noise patterns in STR calling. The speed and reliability of lobSTR exceed the performance of current mainstream algorithms for STR profiling. We validated lobSTR's accuracy by measuring its consistency in calling STRs from whole-genome sequencing of two biological replicates from the same individual, by tracing Mendelian inheritance patterns in STR alleles in whole-genome sequencing of a HapMap trio, and by comparing lobSTR results to traditional molecular techniques. Encouraged by the speed and accuracy of lobSTR, we used the algorithm to conduct a comprehensive survey of STR variations in a deeply sequenced personal genome. We traced the mutation dynamics of close to 100,000 STR loci and observed more than 50,000 STR variations in a single genome. lobSTR's implementation is an end-to-end solution. The package accepts raw sequencing reads and provides the user with the genotyping results. It is written in C/C++, includes multi-threading capabilities, and is compatible with the BAM format. Full Text.
Halfmann, R., Wright, J., Alberti, S., Lindquist, S., and Rexach, M. (2012). Prion formation by a yeast GLFG nucleoporin. Prion Sep 1;6(4). [Epub ahead of print].The self-assembly of proteins into higher order structures is both central to normal biology and a dominant force in disease. Certain glutamine/asparagine (Q/N)-rich proteins in the budding yeast Saccharomyces cerevisiae assemble into self-replicating amyloid-like protein polymers, or prions, that act as genetic elements in an entirely protein-based system of inheritance. The nuclear pore complex (NPC) contains multiple Q/N-rich proteins whose self-assembly has also been proposed to underlie structural and functional properties of the NPC. Here we show that an essential sequence feature of these proteins-repeating GLFG motifs-strongly promotes their self-assembly into amyloids with characteristics of prions. Furthermore, we demonstrate that Nup100 can form bona fide prions, thus establishing a previously undiscovered ability of yeast GLFG nucleoporins to adopt this conformational state in vivo. Full Text.
Halfmann, R., Jarosz, D.F., Jones, S.K., Chang, A., Lancaster, A.K., and Lindquist, S. (2012). Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature 482, 363-368.The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI(+)] or [MOT3(+)], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities. Full Text.
Haque, A., and Tonks, N.K. (2012). The use of phage display to generate conformation-sensor recombinant antibodies. Nature Protocols 7, 2127-2143.We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that specifically recognize and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (PTP1B). We use a solution-based panning and screening strategy conducted in the presence of reduced active PTP1B, which enriches antibodies to epitopes unique to the oxidized form while excluding antibodies that recognize epitopes common to oxidized and reduced forms of PTP1B. This strategy avoids conventional solid-phase immobilization owing to its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects single-chain variable fragments (scFvs) directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. These conformation-specific scFvs illustrate that stabilization of oxidized PTP1B is an effective strategy to inhibit PTP1B function; it is possible that this approach may be applicable to the protein tyrosine phosphatase (PTP) family as a whole. With this protocol, isolation and characterization of specific scFvs from immune responsive animals should take approximately 6 weeks. Full Text
Heaney, J.D., Anderson, E.L., Michelson, M.V., Zechel, J.L., Conrad, P.A., Page, D.C., and Nadeau, J.H. (2012). Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice. Development 139, 1577-1586. Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19(MOLF/Ei)), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation. Full Text.
Hess, G.T., Cragnolini, J.J., Popp, M.W., Allen, M.A., Dougan, S.K., Spooner, E., Ploegh, H.L., Belcher, A.M., and Guimaraes, C.P. (2012). M13 Bacteriophage Display Framework That Allows Sortase-Mediated Modification of Surface-Accessible Phage Proteins. Bioconjugate Chemistry 23, 1478-1487.We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100 fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool. Full Text
Hu2, W., Alvarezxs-Dominguez, J.R., and Lodish, H.F. (2012). Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO Reports Oct 16. [Epub ahead of print].Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non-coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non-coding species can modulate gene-expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non-coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non-coding RNAs contribute to the regulation of mammalian development. Full Text
Hu, W., and Coller, J. (2012). What comes first: translational repression or mRNA degradation? The deepening mystery of microRNA function .Cell Research (advance online publication 22 May 2012). While many mechanisms have been proposed for microRNAs (miRNAs) function, most ultimately cause message degradation. A view has emerged that miRNAs silence gene expression by promoting the association of mRNA decay factors. Recent research results, however, suggest that in both zebrafish and fruit fly, translational inhibition is the initiating event of miRNA-mediated gene silencing. Full Text
Hughes3, J.F., Skaletsky, H., and Page, D.C. (2012). Sequencing of rhesus macaque Y chromosome clarifies origins and evolution of the DAZ (Deleted in AZoospermia) genes. Bioessays Oct 10. [Epub ahead of print] .Studies of Y chromosome evolution often emphasize gene loss, but this loss has been counterbalanced by addition of new genes. The DAZ genes, which are critical to human spermatogenesis, were acquired by the Y chromosome in the ancestor of Old World monkeys and apes. We and our colleagues recently sequenced the rhesus macaque Y chromosome, and comparison of this sequence to human and chimpanzee enables us to reconstruct much of the evolutionary history of DAZ. We report that DAZ arrived on the Y chromosome about 38 million years ago via the transposition of at least 1.1 megabases of autosomal DNA. This transposition also brought five additional genes to the Y chromosome, but all five genes were subsequently lost through mutation or deletion. As the only surviving gene, DAZ experienced extensive restructuring, including intragenic amplification and gene duplication, and has been the target of positive selection in the chimpanzee lineage. PDF
Hughes2, J.F., and Rozen, S. (2012). Genomics and Genetics of Human and Primate Y Chromosomes.Annual Review of Genomics and Human GeneticsVol. 13 (most recent).In mammals, the Y chromosome plays the pivotal role in male sex determination and is essential for normal sperm production. Yet only three Y chromosomes have been completely sequenced to date-those of human, chimpanzee, and rhesus macaque. While Y chromosomes are notoriously difficult to sequence owing to their highly repetitive genomic landscapes, these dedicated sequencing efforts have generated tremendous yields in medical, biological, and evolutionary insight. Knowledge of the complex structural organization of the human Y chromosome and a complete catalog of its gene content have provided a deeper understanding of the mechanisms that generate disease-causing mutations and large-scale rearrangements. Variation among human Y-chromosome sequences has been an invaluable tool for understanding relationships among human populations. Comprehensive comparisons of the human Y-chromosome sequence with those of other primates have illuminated aspects of Y-chromosome evolutionary dynamics over much longer timescales (>25 million years compared with 100,000 years). The future sequencing of additional Y chromosomes will provide a basis for a more comprehensive understanding of the evolution of Y chromosomes and their roles in reproductive biology. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 13 is August 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates .PDF.
Hughes, J.F., Skaletsky, H., Brown, L.G., Pyntikova, T., Graves, T., Fulton, R.S., Dugan, S., Ding, Y., Buhay, C.J., Kremitzki, C.,Qiaoyan Wang ,Hua Shen, Michael Holder ,Donna Villasana ,Lynne V. Nazareth ,Andrew Cree ,Laura Courtney ,Joelle Veizer, Holland Kotkiewicz,Ting-Jan Cho, Natalia Koutseva, Steve Rozen, Donna M. Muzny, Wesley C. Warren, Richard A. Gibbs, Richard K. Wilson & David C. Page (2012). Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes. Nature 483, 82-86.The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200-300 million years. The human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes' genes owing to genetic decay. This evolutionary decay was driven by a series of five 'stratification' events. Each event suppressed X-Y crossing over within a chromosome segment or 'stratum', incorporated that segment into the MSY and subjected its genes to the erosive forces that attend the absence of crossing over. The last of these events occurred 30 million years ago, 5 million years before the human and Old World monkey lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome, remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the Old World monkey lineage. To investigate this question, we sequenced the MSY of the rhesus macaque, an Old World monkey, and compared it to the human MSY. We discovered that during the last 25 million years MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. In the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 million years ago. Likewise, the rhesus MSY has not lost any older genes (from strata 1-4) during the past 25 million years, despite its major structural differences to the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection. Full Text.
Jaenisch, R. (2012). Nuclear cloning and direct reprogramming: the long and the short path to stockholm. Cell Stem Cell 11, 744-747.The two winners of the 2012 Nobel Prize in Physiology or Medicine share more than just this honor; they are both also fearless adventurers, in science and beyond. Full Text
Jansen, A., van der Zande, E., Meert, W., Fink, G.R., and Verstrepen, K.J. (2012). Distal chromatin structure influences local nucleosome positions and gene expression. Nucleic Acids Res Jan 12. [Epub ahead of print].The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'antinucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression. Full Text.
Ji, P., and Lodish, H.F. (2012). Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation. Biochemical and Biophysical Research Communications 417, 1188-1192.During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex. . Full Text.
Kern, D.M., and Cheeseman, I.M. (2012). Kinetochore structure: pulling answers from yeast. Curr Biol 22, R842-844. Despite the identification of multiple kinetochore proteins, their structure and organization has remained unclear. New work uses electron microscopy to visualize isolated budding yeast kinetochore particles and reveal the kinetochore structure on microtubules. Full Text
Kiyomitsu, T., and Cheeseman, I.M. (2012). Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation. Nature Cell Biology. [Epub ahead of print] Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation. Full Text.
Klionsky, D.J., Abdalla, F.C., Abeliovich, H., Abraham, R.T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso,J Sabatini DM , Sarkar S., et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4), 445-544. In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Krishnan, R., Goodman, J.L., Mukhopadhyay, S., Pacheco, C.D., Lemke, E.A., Deniz, A.A., and Lindquist, S. (2012). Conserved features of intermediates in amyloid assembly determine their benign or toxic states. Proc Natl Acad Sci U S A.Jun 27. [Epub ahead of print] Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Abeta, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Abeta, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike. Full Text
Kuhn, D.M., and Vyas, V.K. (2012). The Candida glabrata adhesin Epa1p causes adhesion, phagocytosis, and cytokine secretion by innate immune cells. FEMS Yeast Res. Jan 6. [Epub ahead of print] While Candida albicans is the most significant fungal pathogen for humans, C. glabrata accounts for an increasing number of infections. Little is known about how C. glabrata interacts with the innate immune system, the first line of defense against such organisms. The C. glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized Epa1p mediates unique, non-opsonic binding to macrophages, leading to induction of immune responses. We found Epa1p mediated adhesion by both C. glabrata (Cg) and transformed S. cerevisiae (Sc(EPA) (1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S. cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA) (1) induced inflammatory cytokine production (IL-8 and TNF-alpha) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA) (1) results in phagocytosis and subsequent cytokine production. Full Text.
Kurley, S.J., Bierie, B., Carnahan, R.H., Lobdell, N.A., Davis, M.A., Hofmann, I., Moses, H.L., Muller, W.J., and Reynolds, A.B. (2012). p120-catenin is essential for terminal end bud function and mammary morphogenesis. Development 139, 1754-1764.Although p120-catenin (p120) is crucial for E-cadherin function, ablation experiments in epithelial tissues from different organ systems reveal markedly different effects. Here, we examine for the first time the consequences of p120 knockout during mouse mammary gland development. An MMTV-Cre driver was used to target knockout to the epithelium at the onset of puberty. p120 ablation was detected in approximately one-quarter of the nascent epithelium at the forth week post-partum. However, p120 null cells were essentially nonadherent, excluded from the process of terminal end bud (TEB) morphogenesis and lost altogether by week six. This elimination process caused a delay in TEB outgrowth, after which the gland developed normally from cells that had retained p120. Mechanistic studies in vitro indicate that TEB dysfunction is likely to stem from striking E-cadherin loss, failure of cell-cell adhesion and near total exclusion from the collective migration process. Our findings reveal an essential role for p120 in mammary morphogenesis. Full Text
Lamming, D.W., Ye, L., Katajisto, P., Goncalves, M.D., Saitoh, M., Stevens, D.M., Davis, J.G., Salmon, A.B., Richardson, A., Ahima, R.S,David A. Guertin, David M. Sabatini ,and Joseph A. Baur. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science 335, 1638-1643.Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo. Full Text.
Lapan, S.W., and Reddien, P.W. (2012). Transcriptome Analysis of the Planarian Eye Identifies ovo as a Specific Regulator of Eye Regeneration. Cell Reports Aug 2. [Epub ahead of print] .Among the millions of invertebrate species with visual systems, the genetic basis of eye development and function is well understood only in Drosophila melanogaster. We describe an eye transcriptome for the planarian Schmidtea mediterranea. Planarian photoreceptors expressed orthologs of genes required for phototransduction and microvillus structure in Drosophila and vertebrates, and optic pigment cells expressed solute transporters and melanin synthesis enzymes similar to those active in the vertebrate retinal pigment epithelium. Orthologs of several planarian eye genes, such as bestrophin-1 and Usher syndrome genes, cause eye defects in mammals when perturbed and were not previously described to have roles in invertebrate eyes. Five previously undescribed planarian eye transcription factors were required for normal eye formation during head regeneration. In particular, a conserved, transcription-factor-encoding ovo gene was expressed from the earliest stages of eye regeneration and was required for regeneration of all cell types of the eye. Full Text
Laplante, M., Horvat, S., Festuccia, W.T., Birsoy, K., Prevorsek, Z., Efeyan, A., and Sabatini, D.M. (2012). DEPTOR Cell-Autonomously Promotes Adipogenesis, and Its Expression Is Associated with Obesity. Cell Metab 16, 202-212.DEP domain-containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR), but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice, with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected, considering the positive role of mTOR in adipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest-priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furthermore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the proadipogenic Akt/PKB-PPAR-gamma axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis. Full Text
Laplante, M., and Sabatini, D.M. (2012). mTOR Signaling in Growth Control and Disease. Cell 149, 274-293.The mechanistic target of rapamycin (mTOR) signaling pathway senses and integrates a variety of environmental cues to regulate organismal growth and homeostasis. The pathway regulates many major cellular processes and is implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes, and neurodegeneration. Here, we review recent advances in our understanding of the mTOR pathway and its role in health, disease, and aging. We further discuss pharmacological approaches to treat human pathologies linked to mTOR deregulation. Full Text.
Lee, C.C., Avalos, A.M., and Ploegh, H.L. (2012). Accessory molecules for Toll-like receptors and their function .Nature Reviews Immunology [Epub ahead of print] .Toll-like receptors (TLRs) are essential components of the innate immune system. Accessory proteins are required for the biosynthesis and activation of TLRs. Here, we summarize recent findings on TLR accessory proteins that are required for cell-surface and endosomal TLR function, and we classify these proteins based on their function as ligand-recognition and delivery cofactors, chaperones and trafficking proteins. Because of their essential roles in TLR function, targeting of such accessory proteins may benefit strategies aimed at manipulating TLR activation for therapeutic applications. Full Text
Lehmann, S.M., Kruger, C., Park, B., Derkow, K., Rosenberger, K., Baumgart, J., Trimbuch, T., Eom, G., Hinz, M., Kaul, D.,Habbel P, Kälin R, Franzoni E, Rybak A, Nguyen D, Veh R, Ninnemann O, Peters O, Nitsch R, Heppner FL, Golenbock D, Schott E, Ploegh HL, Wulczyn FG, Lehnardt S. (2012). An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nature Neuroscience 15, 827-U844.Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimer's disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7(-/-) fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage. Full Text
Lesch, B.J., and Page, D.C. (2012). Genetics of germ cell development. Nature Reviews Genetics .Oct 9. [Epub ahead of print]The germ line represents a continuous cellular link between generations and between species, but the germ cells themselves develop in a specialized, organism-specific context. The model organisms Caenorhabditis elegans, Drosophila melanogaster and the mouse display striking similarities, as well as major differences, in the means by which they control germ cell development. Recent developments in genetic technologies allow a more detailed comparison of the germ cells of these three organisms than has previously been possible, shedding light not only on universal aspects of germline regulation, but also on the control of the pluripotent state in vivo and on the earliest steps of embryogenesis. Here, we highlight themes from the comparison of these three alternative strategies for navigating the fundamental cycle of sexual reproduction. Full Text
Li, H.J., Reinhardt, F., Herschman, H.R., and Weinberg, R.A. (2012). Cancer-Stimulated Mesenchymal Stem Cells Create a Carcinoma Stem Cell Niche via Prostaglandin E-2 Signaling. Cancer Discovery 2, 840-855.Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell-derived interleukin-1 (IL-1) induces prostaglandin E-2 (PGE(2)) secretion by MSCs. The resulting PGE(2) operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE(2) and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of beta-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE(2) and cytokines. SIGNIFICANCE: Although PGE(2) has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE(2) axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell-like phenotype in the carcinoma cells. Cancer Discov; 2(9); 840-55. Full Text
Lin, C.Y., Loven, J., Rahl, P.B., Paranal, R.M., Burge, C.B., Bradner, J.E., Lee, T.I., and Young, R.A. (2012). Transcriptional Amplification in Tumor Cells with Elevated c-Myc. Cell 151, 56-67 .Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation. Full Text
Liu, Q., Yuan, B., Lo, K.A., Patterson, H.C., Sun, Y., and Lodish, H.F. (2012). Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism. Proc Natl Acad Sci U S A Aug 16. [Epub ahead of print].The effects of adiponectin on hepatic glucose and lipid metabolism at transcriptional level are largely unknown. We profiled hepatic gene expression in adiponectin knockout (KO) and wild-type (WT) mice by RNA sequencing. Compared with WT mice, adiponectin KO mice fed a chow diet exhibited decreased mRNA expression of rate-limiting enzymes in several important glucose and lipid metabolic pathways, including glycolysis, tricarboxylic acid cycle, fatty-acid activation and synthesis, triglyceride synthesis, and cholesterol synthesis. In addition, binding of the transcription factor Hnf4a to DNAs encoding several key metabolic enzymes was reduced in KO mice, suggesting that adiponectin might regulate hepatic gene expression via Hnf4a. Phenotypically, adiponectin KO mice possessed smaller epididymal fat pads and showed reduced body weight compared with WT mice. When fed a high-fat diet, adiponectin KO mice showed significantly reduced lipid accumulation in the liver. These lipogenic defects are consistent with the down-regulation of lipogenic genes in the KO mice. PDF
Liu2, Q.S., Ren, T., Fresques, T., Oppliger, W., Niles, B.J., Hur, W., Sabatini, D.M., Hall, M.N., Powers, T., and Gray, N.S. (2012). Selective ATP-Competitive Inhibitors of TOR Suppress Rapamycin-Insensitive Function of TORC2 in Saccharomyces cerevisiae. ACS Chemical Biology 7, 982-987.The target of rapamycin (TOR) is a critical regulator of growth, survival, and energy metabolism. The allosteric TORC1 inhibitor rapamycin has been used extensively to elucidate the TOR related signal pathway but is limited by its inability to inhibit TORC2. We used an unbiased cell proliferation assay of a kinase inhibitor library to discover QL-IX-55 as a potent inhibitor of S. cerevisiae growth. The functional target of QL-IX-55 is the ATP-binding site of TOR2 as evidenced by the discovery of resistant alleles of TOR2 through rational design and unbiased selection strategies. QL-IX-55 is capable of potently inhibiting both TOR complex 1 and 2 (TORC1 and TORC2) as demonstrated by biochemical IP kinase assays (IC50 < 50 nM) and cellular assays for inhibition of substrate YPK1 phosphorylation. In contrast to rapamycin, QL-IX-55 is capable of inhibiting TORC2-dependent transcription, which suggests that this compound will be a powerful probe to dissect the Tor2/TORC2-related signaling pathway in yeast. Full Text
Liu, Q.S., Kirubakaran, S., Hur, W., Niepel, M., Westover, K., Thoreen, C.C., Wang, J.H., Ni, J., Patricelli, M.P., Vogel, K., Steve Riddle, David L. Waller, Ryan Traynor, Takaomi Sanda ,Zheng Zhao, Seong A. Kang ,Jean Zhao, A. Thomas Look, Peter K. Sorger ,David M. Sabatini, and Nathanael S. Gray (2012). Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics. Journal of Biological Chemistry 287, 9742-9752.An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting similar to 20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 mu M revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 mu M but did show that PP242 efficiently inhibited the RET receptor (EC50, 42 nM) and JAK1/2/3 kinases (EC50, 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 mu M and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases. Full Text.
Lodish2, H.F. (2012). Translational Control of Protein Synthesis: The Early Years. Journal of Biological Chemistry 287, 36528-36535.For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of alpha- and beta-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments. Full Text
Lodish, H., and Fedoroff, N. (2012). Retrospective. Norton Zinder (1928-2012). Science 335, 1316. Norton Zinder, a founder and leader of the then nascent field of molecular biology half a century ago, passed away on the 3rd of February in the Bronx, New York. He was a Professor at the Rockefeller University, where we were privileged to have been his graduate students and experienced how insightful mentorship and a fertile environment can inspire young students to do great science. Full Text.
Lorestani, A., Ivey, F.D., Thirugnanam, S., Busby, M.A., Marth, G.T., Cheeseman, I.M., and Gubbels, M.J. (2012). Targeted Proteomic Dissection of Toxoplasma Cytoskeleton Sub-Compartments using MORN1. Cytoskeleton 69, 1069-1085.The basal complex in Toxoplasma functions as the contractile ring in the cell division process. Basal complex contraction tapers the daughter cytoskeleton toward the basal end and is required for daughter segregation. We have previously shown that the protein MORN1 is essential for basal complex assembly and likely acts as a scaffolding protein. To further our understanding of the basal complex, we combined subcellular fractionation with an affinity purification of the MORN1 complex and identified its protein composition. We identified two new components of the basal complex, one of which uniquely associated with the basal complex in mature parasites, the first of its kind. In addition, we identified several other novel cytoskeleton proteins with different spatiotemporal dynamics throughout cell division. Since many of these proteins are unique to Apicomplexa this study significantly contributes to the annotation of their unique cytoskeleton. Furthermore, we show that G-actin binding protein TgCAP is localized at the apical cap region in intracellular parasites, but quickly redistributes to a cytoplasmic localization pattern upon egress. PDF
Loven, J., Orlando, D.A., Sigova, A.A., Lin, C.Y., Rahl, P.B., Burge, C.B., Levens, D.L., Lee, T.I., and Young, R.A. (2012). Revisiting global gene expression analysis. Cell 151, 476-482.Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms. Full Text
Maia, A.R.R., Garcia, Z., Kabeche, L., Barisic, M., Maffini, S., Macedo-Ribeiro, S., Cheeseman, I.M., Compton, D.A., Kaverina, I., and Maiato, H. (2012). Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore-microtubule attachments. Journal of Cell Biology 199, 285-301.Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT-MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 "phospho-switch" that temporally regulates KT-MT attachment stability. Full Text
Marjanovic, N.D., Weinberg, R.A., and Chaffer, C.L. (2012). Cell Plasticity and Heterogeneity in Cancer. Clinical Chemistry 59 (1) .BACKGROUND: Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models.Content:The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. Evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity.Summary:Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies. PDF
McGovern, A.P., Powell, B.E., and Chevassut, T.J.T. (2012). A dynamic multi-compartmental model of DNA methylation with demonstrable predictive value in hematological malignancies. Journal of Theoretical Biology 310, 14-20.Recent advances have highlighted the central role of DNA methylation in leukemogenesis and have led to clinical trials of epigenetic therapy, notably hypomethylating agents, in myelodysplasia and acute myeloid leukemia. However, despite these advances, our understanding of the dynamic regulation of the methylome remains poor. We have attempted to address this shortcoming by producing a dynamic, six-compartmental model of DNA methylation levels based on the activity of the Dnmt methyltransferase proteins. In addition, the model incorporates the recently discovered Tet family proteins which enzymatically convert methylcytosine to hydroxymethylcytosine. A set of first order, partial differential equations comprise the model and were solved via numerical integration. The model is able to predict the relative abundances of unmethylated, hemimethylated, fully methylated, and hydroxymethylated CpG dyads in the DNA of cells with fully functional Dnmt and Tet proteins. In addition, the model accurately predicts the experimentally measured changes in these abundances with disruption of Dnmt function. Furthermore, the model reveals the mechanism whereby CpG islands are maintained in a hypomethylated state via local modulation of Dnmt and Tet activities without any requirement for active demethylation. We conclude that this model provides an accurate depiction of the major epigenetic processes involving modification of DNA. (C) 2012 Elsevier Ltd. All rights reserved. Full Text
McLellan, C.A., Whitesell, L., King, O.D., Lancaster, A.K., Mazitschek, R., and Lindquist, S. (2012). Inhibiting GPI Anchor Biosynthesis in Fungi Stresses the Endoplasmic Reticulum and Enhances Immunogenicity. ACS Chem Bio Jun 22. [Epub ahead of print].In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall beta-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi. Full Text
Mendillo, M.L., Santagata, S., Koeva, M., Bell, G.W., Hu, R., Tamimi, R.M., Fraenkel, E., Ince, T.A., Whitesell, L., and Lindquist, S. (2012). HSF1 Drives a Transcriptional Program Distinct from Heat Shock to Support Highly Malignant Human Cancers. Cell 150, 549-562.Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications. Full Text
MeyerHermann, M., Mohr, E., Pelletier, N., Zhang, Y., Victora, G.D., and Toellner, K.M. (2012). A Theory of Germinal Center B Cell Selection, Division, and Exit. Cell Reports 2, 162-174.High-affinity antibodies are generated in germinal centers in a process involving mutation and selection of B cells. Information processing in germinal center reactions has been investigated in a number of recent experiments. These have revealed cell migration patterns, asymmetric cell divisions, and cell-cell interaction characteristics, used here to develop a theory of germinal center B cell selection, division, and exit (the LEDA model). According to this model, B cells selected by T follicular helper cells on the basis of successful antigen processing always return to the dark zone for asymmetric division, and acquired antigen is inherited by one daughter cell only. Antigen-retaining B cells differentiate to plasma cells and leave the germinal center through the dark zone. This theory has implications for the functioning of germinal centers because compared to previous models, high-affinity antibodies appear one day earlier and the amount of derived plasma cells is considerably larger. Full Text
Muralidharan, V., Oksman, A., Pal, P., Lindquist, S., and Goldberg, D.E. (2012). Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat Commun 3,# 1310 .One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes. Full Text
NadlerHolly, M., Breker, M., Gruber, R., Azia, A., Gymrek, M., Eisenstein, M., Willison, K.R., Schuldiner, M., and Horovitz, A. (2012). Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis. Proceedings of the National Academy of Sciences of the United States of America 109, 18833-18838.The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems. Full Text
Nakanishi, K., Weinberg, D.E., Bartel, D.P., and Patel, D.J. (2012). Structure of yeast Argonaute with guide RNA. Nature 486, 368-374.The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 A crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage. Full Text
Nam, J.W., and Bartel, D. (2012). Long non-coding RNAs in C. elegans. Genome Research Jun 15. [Epub ahead of print] .Thousands of long non-coding RNAs (lncRNAs) have been found in vertebrate animals, a few of which have known biological roles. To better understand the genomics and features of lncRNAs in invertebrates, we used available RNA-seq, poly(A)-site, and ribosome-mapping data to identify lncRNAs of C. elegans. We found hundreds of long intervening ncRNAs (lincRNAs), which had single- or multi-exonic structures that did not overlap protein-coding transcripts, and about seventy antisense lncRNAs (ancRNAs), which were complementary to protein-coding transcripts. Compared to protein-coding genes, the lncRNA genes tended to be expressed in stage-dependent manner. Approximately 30% of the newly identified lincRNAs showed little signal for sequence conservation and mapped antisense to clusters of 22G or 26G endogenous siRNAs, as would be expected if they serve as templates and targets for these siRNAs. The other 70% tended to be more conserved and included lincRNAs with intriguing expression and sequence features associating them with processes such as dauer formation, male identity, sperm formation, and interaction with sperm-specific mRNAs. Our study provides a glimpse into the lncRNA content of a non-vertebrate animal and a resource for future studies of lncRNA function. PDF
Nishino, T., Takeuchi, K., Gascoigne, K.E., Suzuki, A., Hori, T., Oyama, T., Morikawa, K., Cheeseman, I.M., and Fukagawa, T. (2012). CENP-T-W-S-X Forms a Unique Centromeric Chromatin Structure with a Histone-like Fold. Cell 148, 487-501.The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code'' beyond canonical nucleosome proteins. Full Text.
Nodine, M.D., and Bartel, D.P. (2012). Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature. Jan 22. [Epub ahead of print] In animals, maternal gene products deposited into eggs regulate embryonic development before activation of the zygotic genome. In plants, an analogous period of prolonged maternal control over embryogenesis is thought to occur based on some gene-expression studies. However, other gene-expression studies and genetic analyses show that some transcripts must derive from the early zygotic genome, implying that the prevailing model does not fully explain the nature of zygotic genome activation in plants. To determine the maternal, paternal and zygotic contributions to the early embryonic transcriptome, we sequenced the transcripts of hybrid embryos from crosses between two polymorphic inbred lines of Arabidopsis thaliana and used single-nucleotide polymorphisms diagnostic of each parental line to quantify parental contributions. Although some transcripts seemed to be either inherited from primarily one parent or transcribed from imprinted loci, the vast majority of transcripts were produced in near-equal amounts from both maternal and paternal alleles, even during the initial stages of embryogenesis. Results of reporter experiments and analyses of transcripts from genes that are not expressed in sperm and egg indicate early and widespread zygotic transcription. Thus, in contrast to early animal embryogenesis, early plant embryogenesis is mostly under zygotic control. Full Text.
Nordman, J., and Orr-Weaver, T.L. (2012). Regulation of DNA replication during development. Development 139, 455-464.As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication. Full Text.
OlveraGomez, I., Hamilton, S.E., Xiao, Z., Guimaraes, C.P., Ploegh, H.L., Hogquist, K.A., Wang, L., and Jameson, S.C. (2012). Cholera toxin activates nonconventional adjuvant pathways that induce protective CD8 T-cell responses after epicutaneous vaccination. Proceedings of the National Academy of Sciences of the United States of America 109, 2072-2077.The ability to induce humoral and cellular immunity via antigen delivery through the unbroken skin (epicutaneous immunization, EPI) has immediate relevance for vaccine development. However, it is unclear which adjuvants induce protective memory CD8 T-cell responses by this route, and the molecular and cellular requirements for priming through intact skin are not defined. We report that cholera toxin (CT) is superior to other adjuvants in its ability to prime memory CD8 T cells that control bacterial and viral challenges. Epicutaneous immunization with CT does not require engagement of classic toll-like receptor (TLR) and inflammasome pathways and, surprisingly, is independent of skin langerin-expressing cells (including Langerhans cells). However, CT adjuvanticity required type-I IFN sensitivity, participation of a Batf3-dependent dendritic cell (DC) population and engagement of CT with suitable gangliosides. Chemoenzymatic generation of CT-antigen fusion proteins led to efficient priming of the CD8 T-cell responses, paving the way for development of this immunization strategy as a therapeutic option. Full Text.
Onder, T.T., Kara, N., Cherry, A., Sinha, A.U., Zhu, N., Bernt, K.M., Cahan, P., Mancarci, B.O., Unternaehrer, J., Gupta, P.B ,Lander ES, Armstrong SA, and Daley GQ. (2012). Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598-602 .Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling(1). Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming(2,3), the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatinmodifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. Full Text.
Orlando, D.A., Guenther, M.G., Frampton, G.M., and Young, R.A. (2012). CpG Island Structure and Trithorax/Polycomb Chromatin Domains in Human Cells. Genomics Jul 17. [Epub ahead of print] .TrxG and PcG complexes play key roles in the epigenetic regulation of development through H3K4me3 and H3K27me3 modification at specific sites throughout the human genome, but how these sites are selected is poorly understood. We find that in pluripotent cells, clustered CpG-islands at genes predict occupancy of H3K4me3 and H3K27me3, and these "bivalent" chromatin domains precisely span the boundaries of CpG-island clusters. These relationships are specific to pluripotent stem cells and are not retained at H3K4me3 and H3K27me3 sites unique to differentiated cells. We show that putative transcripts from clustered CpG-islands predict stem-loop structures characteristic of those bound by PcG complexes, consistent with the possibility that RNA facilitates PcG recruitment or maintenance at these sites. These studies suggest that CpG-island structure plays a fundamental role in establishing developmentally important chromatin structures in the pluripotent genome, and a subordinate role in establishing TrxG/PcG chromatin structure at sites unique to differentiated cells. Full Text
Pignatta, D., and Gehring, M. (2012). Imprinting meets genomics: new insights and new challenges. Curr Opin Plant Biol.Available online 20 September 2012 Until recently, only a handful of imprinted genes, or genes with parent-of-origin dependent expression patterns, were known in plants. Study of these genes yielded key insights into mechanisms of monoallelic expression and imprinted gene function. The recent application of high throughput sequencing to the study of imprinting has confirmed that many previous findings are relevant on a genome-wide scale. The catalogue of imprinted genes in monocots and dicots now includes a large number of transcription factors, chromatin related genes, and metabolic or hormone biosynthesis enzymes. Interpretation of allele specific expression data remains a challenge, with careful validation of candidate imprinted genes necessary. Full Text
Ploegh, H., and Meyers, M. (2012). Prize Fight: The Race and the Rivalry to be the First in Science. Nature 486, 318-319. An examination of the battles behind the prestige of top awards grips Hidde Ploegh. Full Text
Popp, M.W., Karssemeijer, R.A., and Ploegh, H.L. (2012). Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time. PLoS Pathogens 8, e1002604.The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging. Full Text.
Qin, J.Z., Whyte, W.A., Anderssen, E., Apostolou, E., Chen, H.H., Akbarian, S., Bronson, R.T., Hochedlinger, K., Ramaswamy, S., Young, R.A. ,and Hanno Hock. (2012). The Polycomb Group Protein L3mbtl2 Assembles an Atypical PRC1-Family Complex that Is Essential in Pluripotent Stem Cells and Early Development. Cell Stem Cell 11, 319-332 .L3mbtl2 has been implicated in transcriptional repression and chromatin compaction but its biological function has not been defined. Here we show that disruption of L3mbtl2 results in embryonic lethality with failure of gastrulation. This correlates with compromised proliferation and abnormal differentiation of L3mbtl2(-/-) embryonic stem (ES) cells. L3mbtl2 regulates genes by recruiting a Polycomb Repressive Complex1 (PRC1)-related complex, resembling the previously described E2F6-complex, and including G9A, Hdac1, and Ring1b. The presence of L3mbtl2 at target genes is associated with H3K9 dimethylation, low histone acetylation, and H2AK119 ubiquitination, but the latter is neither dependent on L3mbtl2 nor sufficient for repression. Genomewide studies revealed that the L3mbtl2-dependent complex predominantly regulates genes not bound by canonical PRC1 and PRC2. However, some developmental regulators are repressed by the combined activity of all three complexes. Together, we have uncovered a highly selective, essential role for an atypical PRC1-family complex in ES cells and early development. Full Text
Raddatz, G., Gao, Q., Bender, S., Jaenisch, R., and Lyko, F. (2012). Dnmt3a Protects Active Chromosome Domains against Cancer-Associated Hypomethylation. Plos Genetics 8(12):e1003146 .Changes in genomic DNA methylation patterns are generally assumed to play an important role in the etiology of human cancers. The Dnmt3a enzyme is required for the establishment of normal methylation patterns, and mutations in Dnmt3a have been described in leukemias. Deletion of Dnmt3a in a K-ras-dependent mouse lung cancer model has been shown to promote tumor progression, which suggested that the enzyme might suppress tumor development by stabilizing DNA methylation patterns. We have used whole-genome bisulfite sequencing to comprehensively characterize the methylomes from Dnmt3a wildtype and Dnmt3a-deficient mouse lung tumors. Our results show that profound global methylation changes can occur in K-ras-induced lung cancer. Dnmt3a wild-type tumors were characterized by large hypomethylated domains that correspond to nuclear lamina-associated domains. In contrast, Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for efficient maintenance methylation of active chromosome domains and that Dnmt3a-deficient tumors show moderate levels of gene deregulation in these domains. In summary, our results uncover conserved features of cancer methylomes and define the role of Dnmt3a in maintaining DNA methylation patterns in cancer. Full Text
Ray, A., Amato, A.A., Bradshaw, E.M., Felice, K.J., DiCapua, D.B., Goldstein, J.M., Lundberg, I.E., Nowak, R.J., Ploegh, H.L., Spooner, E. ,Qian Wu, Simon N. Willis, and Kevin C. O’Connor. (2012). Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis. PLOS One 7(10): e46709.Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens. Full Text
RobidaStubbs, S., Glover-Cutter, K., Lamming, D.W., Mizunuma, M., Narasimhan, S.D., Neumann-Haefelin, E., Sabatini, D.M., and Blackwell, T.K. (2012). TOR Signaling and Rapamycin Influence Longevity by Regulating SKN-1/Nrf and DAF-16/FoxO. Cell Metabolism 15, 713-724.The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity. Full Text
Rolfe, P.A., Bernstein, D.A., Grisafi, P., Fink, G.R., and Gifford, D.K. (2012). Ruler Arrays Reveal Haploid Genomic Structural Variation. Plos One 7(8): e43210 .Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase's processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence. The method combines a sample preparation protocol, tiling genomic microarrays, and a new computational analysis. The analysis of ruler array data from two genomic samples enables the identification of structural variation between the samples. In an empirical test between two closely related haploid strains of yeast ruler arrays detected 78% of the structural variants larger than 100 bp. Full Text
Rosmarin, D.M., Carette, J.E., Olive, A.J., Starnbach, M.N., Brummelkamp, T.R., and Ploegh, H.L. (2012). Attachment of Chlamydia trachomatis L2 to host cells requires sulfation. Proc Natl Acad Sci U S A Jun 6. [Epub ahead of print].Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3, B4GALT7, and SLC35B2, which encode glucuronosyltransferase I, galactosyltransferase I, and the 3'-phosphoadenosine 5'-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.PDF
Rozen, S.G., Marszalek, J.D., Irenze, K., Skaletsky, H., Brown, L.G., Oates, R.D., Silber, S.J., Ardlie, K., and Page, D.C. (2012). AZFc Deletions and Spermatogenic Failure: A Population-Based Survey of 20,000 Y Chromosomes. Am J Hum Genet. Oct 24. [Epub ahead of print] Deletions involving the Y chromosome's AZFc region are the most common known genetic cause of severe spermatogenic failure (SSF). Six recurrent interstitial deletions affecting the region have been reported, but their population genetics are largely unexplored. We assessed the deletions' prevalence in 20,884 men in five populations and found four of the six deletions (presented here in descending order of prevalence): gr/gr, b2/b3, b1/b3, and b2/b4. One of every 27 men carried one of these four deletions. The 1.6 Mb gr/gr deletion, found in one of every 41 men, almost doubles the risk of SSF and accounts for approximately 2% of SSF, although <2% of men with the deletion are affected. The 1.8 Mb b2/b3 deletion, found in one of every 90 men, does not appear to be a risk factor for SSF. The 1.6 Mb b1/b3 deletion, found in one of every 994 men, appears to increase the risk of SSF by a factor of 2.5, although <2% of men with the deletion are affected, and it accounts for only 0.15% of SSF. The 3.5 Mb b2/b4 deletion, found in one of every 2,320 men, increases the risk of SSF 145 times and accounts for approximately 6% of SSF; the observed prevalence should approximate the rate at which the deletion arises anew in each generation. We conclude that a single rare variant of major effect (the b2/b4 deletion) and a single common variant of modest effect (the gr/gr deletion) are largely responsible for the AZFc region's contribution to SSF in the population. Full Text
Ryan, O., Shapiro, R.S., Kurat, C.F., Mayhew, D., Baryshnikova, A., Chin, B., Lin, Z.Y., Cox, M.J., Vizeacoumar, F., Cheung, D .David K. Gifford , Gerald R. Fink, et al. (2012). Global Gene Deletion Analysis Exploring Yeast Filamentous Growth. Science 337, 1353-1356.The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Sigma 1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans. Full Text
Ryu, S., Lang, M.J., and Matsudaira, P. (2012). Maximal Force Characteristics of the Ca(2+)-Powered Actuator of Vorticella convallaria. Biophys J 103, 860-867.The millisecond stalk contraction of the sessile ciliate Vorticella convallaria is powered by energy from Ca(2+) binding to generate contractile forces of approximately 10 nN. Its contractile organelle, the spasmoneme, generates higher contractile force under increased stall resistances. By applying viscous drag force to contracting V. convallaria in a microfluidic channel, we observed that the mechanical force and work of the spasmoneme depended on the stalk length, i.e., the maximum tension (150-350 nN) and work linearly depended on the stalk length ( approximately 2.5 nN and approximately 30 fJ per 1 mum of the stalk). This stalk-length dependency suggests that motor units of the spasmoneme may be organized in such a way that the mechanical force and work of each unit cumulate in series along the spasmoneme. Full Text
Saibil, H.R., Seybert, A., Habermann, A., Winkler, J., Eltsov, M., Perkovic, M., Castano-Diez, D., Scheffer, M.P., Haselmann, U., Chlanda, P, .Lindquist S, Tyedmers J, Frangakis AS.l. (2012). Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures. Proceedings of the National Academy of Sciences of the United States of America 109, 14906-14911.Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI+], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI+] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance. Full Text
Sanda, T., Lawton, L.N., Barrasa, M.I., Fan, Z.P., Kohlhammer, H., Gutierrez, A., Ma, W., Tatarek, J., Ahn, Y., Kelliher, M.A,.Richard A. Young, et al. (2012). Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia. Cancer Cell 22, 209-221.The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells. Full Text
Sankaran, V.G., Ludwig, L.S., Sicinska, E., Xu, J., Bauer, D.E., Eng, J.C., Patterson, H.C., Metcalf, R.A., Natkunam, Y., Orkin, S.H., Sicinski P, Lander ES ,and Lodish HF (2012). Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes & Development 26, 2075-2087.Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies. Full Text
Santagata, S., Xu, Y.-m., Wijeratne, E.M.K., Kontnik, R., Rooney, C., Perley, C.C., Kwon, H., Clardy, J., Kesari, S., Whitesell, L., Susan Lindquist, and A. A. Leslie Gunatilaka(2012). Using the Heat-Shock Response To Discover Anticancer Compounds that Target Protein Homeostasis. ACS Chemical Biology 7, 339-348.Unlike normal tissues, cancers experience profound alterations in protein homeostasis. Powerful innate adaptive mechanisms, especially the transcriptional response regulated by Heat Shock Factor 1 (HSF1), are activated in cancers to enable survival under these stressful conditions. Natural products that further tax these stress responses can overwhelm the ability to cope and could provide leads for the development of new, broadly effective anticancer drugs. To identify compounds that drive the HSF1-dependent stress response, we evaluated over 80,000 natural and synthetic compounds as well as partially purified natural product extracts using a reporter cell line optimized for high-throughput screening. Surprisingly, many of the strongly active compounds identified were natural products representing five diverse chemical classes (limonoids, curvularins, withanolides, celastraloids, and colletofragarones). All of these compounds share the same chemical motif, an alpha,beta-unsaturated carbonyl functionality, with strong potential for thiol-reactivity. Despite the lack of a priori mechanistic requirements in our primary phenotypic screen, this motif was found to be necessary albeit not sufficient, for both heat-shock activation and inhibition of glioma tumor cell growth. Within the withanolide class, a promising therapeutic index for the compound withaferin A was demonstrated in vivo using a stringent orthotopic human glioma xenograft model in mice. Our findings reveal that diverse organisms elaborate structurally complex thiol-reactive metabolites that at on the stress responses of heterologous organisms including humans. From a chemical biology perspective, they define a robust approach for discovering candidate compounds that target the malignant phenotype by disrupting protein homeostasis. Full Text.
Sanyal, S., Claessen, J.H., and Ploegh, H.L. (2012). A viral deubiquitylating enzyme restores dislocation of substrates from the ER in semi-intact cells. J Biol Chem. May 22. [Epub ahead of print] Terminally misfolded glycoproteins are ejected from the endoplasmic reticulum (ER) to the cytosol and destroyed by the ubiquitin proteasome system (UPS). A dominant negative version of the deubiquitylating enzyme Yod1 (Yod1C160S) causes accumulation of dislocation substrates in the ER. Failure to remove ubiquitin from the dislocation substrate might therefore stall the reaction at the exit site from the ER. We hypothesized that addition of a promiscuous deubiquitylase (DUB) should overcome this blockade and restore dislocation. We monitored ER-to-cytosol transport of misfolded proteins in cells permeabilized at high cell density by perfringolysin O, a pore-forming cytolysin. This method allows ready access of otherwise impermeant reagents to the intracellular milieu with minimal dilution of cytoplasmic components. We show that addition of the purified Epstein-Barr Virus (EBV) DUB to semi-intact cells indeed initiates dislocation of a stalled substrate intermediate, resulting in stabilization of substrates in the cytosol. Our data provide new mechanistic insight in the dislocation reaction and support a model where failure to deubiquitylate an ER-resident protein occludes the dislocon and causes upstream misfolded intermediates to accumulate. PDF
Scheel, C., and Weinberg, R.A. (2012). Cancer stem cells and epithelial-mesenchymal transition: Concepts and molecular links. Semin Cancer Biol. Apr 23. [Epub ahead of print] The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by carcinoma cells. EMT programs are orchestrated by a set of pleiotropically acting transcription factors (TFs). The actions of these EMT-TFs enable the early steps of metastasis: local invasion and subsequent dissemination of carcinoma cells to distant sites. However, in most malignancies, the subsequent outgrowth of micrometastatic deposits into macroscopic metastases has the greatest impact on clinical progression. Such metastatic "colonization" reflects the ability of disseminated tumor cells to adapt to a foreign tissue microenvironment. The outgrowth of a metastasis is also thought to be associated with self-renewal, the defining cellular trait of cancer stem cells (CSCs), also termed tumor-initiating cells. Importantly, molecular links between EMT-TFs and self-renewal have emerged, suggesting that EMT programs play critical roles both early and late in the metastatic cascade. The genetic and epigenetic mechanisms that regulate the activation of EMT-TFs and the traits they induce are areas under intensive investigation. Such studies may provide new opportunities for therapeutic intervention and help to overcome tumor heterogeneity and therapeutic resistance. Full Text.
Schmidt, J.C., Arthanari, H., Boeszoermenyi, A., Dashkevich, N.M., Wilson-Kubalek, E.M., Monnier, N., Markus, M., Oberer, M., Milligan, R.A.,Bathe M, Wagner G, Grishchuk EL, Cheeseman IM. (2012). The Kinetochore-Bound Ska1 Complex Tracks Depolymerizing Microtubules and Binds to Curved Protofilaments. Developmental Cell.Available online 18 October 2012. To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule-binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments. Full Text
Sebastian, C., Zwaans, B.M.M., Silberman, D.M., Gymrek, M., Goren, A., Zhong, L., Ram, O., Truelove, J., Guimaraes, A.R., Toiber, D., et al. (2012). The Histone Deacetylase SIRT6 Is a Tumor Suppressor that Controls Cancer Metabolism. Cell 151, 1185-1199.Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism. Full Text
Sehrawat2, S., Koenig, P.A., Kirak, O., Schlieker, C., Fankhauser, M., and Ploegh, H.L. (2012). A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation. Blood Dec 13. [Epub ahead of print].Antigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8(+) T cells, both in vitro and in vivo. Compared to controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8(+) T cells and showed improved control of infection with a recombinant gamma herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8(+) T cells was likewise observed upon infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced cross-presentation by YOD1-C160S APCs was TAP-independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigen-specific CD8(+) T cell responses during immunization. Full Text
Sehrawat, S., Kirak, O., Koenig, P.A., Isaacson, M.K., Marques, S., Bozkurt, G., Simas, J.P., Jaenisch, R., and Ploegh, H.L. (2012). CD8(+) T Cells from Mice Transnuclear for a TCR that Recognizes a Single H-2K(b)-Restricted MHV68 Epitope Derived from gB-ORF8 Help Control Infection. Cell Reports 1, 461-471.To study the CD8(+) T cell response against a mouse gamma-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to gamma-herpes viruses. Full Text
Shah, D.I., Takahashi-Makise, N., Cooney, J.D., Li, L.T., Schultz, I.J., Pierce, E.L., Narla, A., Seguin, A., Hattangadi, S.M., Medlock, A.E., et al. (2012). Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts. Nature 491, 608-612.Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias(1,2). The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies. Full Text
Sheikh, M.A., and Erlich, Y. (2012). Base-Calling for Bioinformaticians. In Bioinformatics for High Throughput Sequencing, pp. 67-83. Full Text
Sher, N., Bell, G.W., Li, S., Nordman, J., Eng, T., Eaton, M.L., MacAlpine, D.M., and Orr-Weaver, T.L. (2012). Developmental control of gene copy number by repression of replication initiation and fork progression. Genome Research 22, 64-75.Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation. Full Text.
Shibue, T., Brooks, M.W., Inan, M.F., Reinhardt, F., and Weinberg, R.A. (2012). The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions.Cancer Discovery Published Online on May 18 .Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix (ECM) components in order to survive, proliferate and form macroscopic metastases. The biochemical and cell-biological mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated, integrin beta1-containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase (FAK). These plaques originate in and appear only after the formation of filopodium-like protrusions (FLPs) that harbor integrin beta1 along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases. PDF
Sinisi, A., Popp, M.W., Antos, J.M., Pansegrau, W., Savino, S., Nissum, M., Rappuoli, R., Ploegh, H.L., and Buti, L. (2012). Development of an Influenza virus Protein Array Using Sortagging Technology. Bioconjugate Chemistry Article ASAP Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly(3) linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening. Full Text
Smith, L.M., Hensley, L.E., Geisbert, T.W., Johnson, J., Stossel, A., Honko, A., Yen, J.Y., Geisbert, J., Paragas, J., Fritz, E.,Olinger G, Young HA, Rubins KH, Karp CLl. (2012). Interferon-beta Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever. J Infect Dis.Dec 18. [Epub ahead of print] There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust IFN-alpha production, with plasma concentrations of IFN-alpha that greatly (60-100-fold) exceed those seen in other viral infections, but little IFN-beta production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-beta therapy in filovirus infection. Here we show that early postexposure treatment with IFN-beta significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-beta also significantly increased survival time after Marburg virus infection. IFN-beta may have promise as an adjunctive postexposure therapy in filovirus infection. Full Text
Soldner, F., and Jaenisch, R. (2012). Medicine. iPSC disease modeling. Science 338, 1155-1156 .Induced pluripotent stem cell (iPSC) technology has provided previously unanticipated possibilities to model human disease in the culture dish. Reprogramming somatic cells from patients into an embryonic stem cell–like state followed by differentiation into disease-relevant cell types can generate an unlimited source of human tissue carrying the genetic variations that caused or facilitated disease development . Yet, despite the excitement over this “disease-in-a-dish” approach, studying genetic disorders in patient-derived cells faces more challenges than studies using genetically well-defined model systems. Here we describe some of these limitations, and also present some solutions for ensuring that iPSC technology lives up to at least some of its promise. Full Text
Strijbis, K., Spooner, E., and Ploegh, H.L. (2012). Protein ligation in living cells using sortase. Traffic Feb 20. [Epub ahead of print].Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging we employ the Ca(2+) -independent SrtA from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); as nucleophiles we used proteins with a N-terminal (oligo)glycine. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER-luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 minutes after translation. The versatility of the method is shown by protein ligation of multiple substrates with GFP-based nucleophiles in different intracellular compartments.PDF
Taipale, M., Krykbaeva, I., Koeva, M., Kayatekin, C., Westover, K.D., Karras, G.I., and Lindquist, S. (2012). Quantitative analysis of hsp90-client interactions reveals principles of substrate recognition. Cell 150, 987-1001.HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family. Full Text
Tam, J.M., Mansour, M.K., Khan, N.S., Yoder, N.C., and Vyas, J.M. (2012). Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity. Integrative Biology 4, 220-227.The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of beta-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of "fungal like particles" was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified beta-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the beta-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the beta-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating beta-1,3-glucan beads with laminarinase, a specific beta-1,3-gluconase, the reactivity of the anti-beta-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly beta-1,3-glucan. TNF-alpha was also measured by stimulating bone-marrow derived macrophages with the beta-1,3-glucan beads, and showed a dose dependent response compared to soluble beta-glucan, insoluble beta-1,3-glucan, uncoated beads, and soluble beta-1,3-glucan mixed with uncoated beads. Finally, beta-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. beta-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells. PDF
Tardiff, D.F., Tucci, M.L., Caldwell, K.A., Caldwell, G.A., and Lindquist, S. (2012). Different 8-Hydroxyquinolines Protect Models of TDP-43 Protein, alpha-Synuclein, and Polyglutamine Proteotoxicity through Distinct Mechanisms. Journal of biological chemistry 287, 4107-4120.No current therapies target the underlying cellular pathologies of age-related neurodegenerative diseases. Model organisms provide a platform for discovering compounds that protect against the toxic, misfolded proteins that initiate these diseases. One such protein, TDP-43, is implicated in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In yeast, TDP-43 expression is toxic, and genetic modifiers first discovered in yeast have proven to modulate TDP-43 toxicity in both neurons and humans. Here, we describe a phenotypic screen for small molecules that reverse TDP-43 toxicity in yeast. One group of hit compounds was 8-hydroxyquinolines (8-OHQ), a class of clinically relevant bioactive metal chelators related to clioquinol. Surprisingly, in otherwise wild-type yeast cells, different 8-OHQs had selectivity for rescuing the distinct toxicities caused by the expression of TDP-43, alpha-synuclein, or polyglutamine proteins. In fact, each 8-OHQ synergized with the other, clearly establishing that they function in different ways. Comparative growth and molecular analyses also revealed that 8-OHQs have distinct metal chelation and ionophore activities. The diverse bioactivity of 8-OHQs indicates that altering different aspects of metal homeostasis and/or metalloprotein activity elicits distinct protective mechanisms against several neurotoxic proteins. Indeed, phase II clinical trials of an 8-OHQ has produced encouraging results in modifying Alzheimer disease. Our unbiased identification of 8-OHQs in a yeast TDP-43 toxicity model suggests that tailoring 8-OHQ activity to a particular neurodegenerative disease may be a viable therapeutic strategy. Full Text.
Thoreen, C.C., Chantranupong, L., Keys, H.R., Wang, T., Gray, N.S., and Sabatini, D.M. (2012). A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109-113.The mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses(1). mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1 (ref. 2). Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5' terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5' untranslated region length or complexity(3). mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown(4). Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors. Full Text
Treusch, S., and Lindquist, S. (2012). An intrinsically disordered yeast prion arrests the cell cycle by sequestering a spindle pole body component. J Cell Biol. 2012 Apr 23. [Epub ahead of print] .Intrinsically disordered proteins play causative roles in many human diseases. Their overexpression is toxic in many organisms, but the causes of toxicity are opaque. In this paper, we exploit yeast technologies to determine the root of toxicity for one such protein, the yeast prion Rnq1. This protein is profoundly toxic when overexpressed but only in cells carrying the endogenous Rnq1 protein in its [RNQ(+)] prion (amyloid) conformation. Surprisingly, toxicity was not caused by general proteotoxic stress. Rather, it involved a highly specific mitotic arrest mediated by the Mad2 cell cycle checkpoint. Monopolar spindles accumulated as a result of defective duplication of the yeast centrosome (spindle pole body [SPB]). This arose from selective Rnq1-mediated sequestration of the core SPB component Spc42 in the insoluble protein deposit (IPOD). Rnq1 does not normally participate in spindle pole dynamics, but it does assemble at the IPOD when aggregated. Our work illustrates how the promiscuous interactions of an intrinsically disordered protein can produce highly specific cellular toxicities through illicit, yet highly specific, interactions with the proteome. Full Text.
Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal Regulation of Epithelial-Mesenchymal Transition Is Essential for Squamous Cell Carcinoma Metastasis. Cancer Cell 22, 725-736.Epithelial-mesenchymal transition (EMT) is implicated in converting stationary epithelial tumor cells into motile mesenchymal cells during metastasis. However, the involvement of EMT in metastasis is still controversial, due to the lack of a mesenchymal phenotype in human carcinoma metastases. Using a spontaneous squamous cell carcinoma mouse model, we show that activation of the EMT-inducing transcription factor Twist1 is sufficient to promote carcinoma cells to undergo EMT and disseminate into blood circulation. Importantly, in distant sites, turning off Twist1 to allow reversion of EMT is essential for disseminated tumor cells to proliferate and form metastases. Our study demonstrates in vivo the requirement of "reversible EMT" in tumor metastasis and may resolve the controversy on the importance of EMT in carcinoma metastasis. Full Text
Tuncbag, N., McCallum, S., Huang, S.S., and Fraenkel, E. (2012). SteinerNet: a web server for integrating 'omic' data to discover hidden components of response pathways. Nucleic Acids Research Published online: May 25, 2012.High-throughput technologies including transcriptional profiling, proteomics and reverse genetics screens provide detailed molecular descriptions of cellular responses to perturbations. However, it is difficult to integrate these diverse data to reconstruct biologically meaningful signaling networks. Previously, we have established a framework for integrating transcriptional, proteomic and interactome data by searching for the solution to the prize-collecting Steiner tree problem. Here, we present a web server, SteinerNet, to make this method available in a user-friendly format for a broad range of users with data from any species. At a minimum, a user only needs to provide a set of experimentally detected proteins and/or genes and the server will search for connections among these data from the provided interactomes for yeast, human, mouse, Drosophila melanogaster and Caenorhabditis elegans. More advanced users can upload their own interactome data as well. The server provides interactive visualization of the resulting optimal network and downloadable files detailing the analysis and results. We believe that SteinerNet will be useful for researchers who would like to integrate their high-throughput data for a specific condition or cellular response and to find biologically meaningful pathways. SteinerNet is accessible at http://fraenkel.mit.edu/steinernet. Full Text
Ulitsky, I., Shkumatava, A., Jan, C., Subtelny, A.O., Koppstein, D., Bell, G., Sive, H., and Bartel, D. (2012). Extensive alternative polyadenylation during zebrafish development. Genome Res.Jun 21. [Epub ahead of print] The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3'UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially increased and improved existing 3'UTR annotations, resulting in confidently identified 3'UTRs for more than 79% of the annotated protein-coding genes in zebrafish. mRNAs from most zebrafish genes undergo alternative CPA, with those from more than a thousand genes using different dominant 3'UTRs at different stages. These included one of the poly(A) polymerase genes, for which alternative CPA reinforces its repression in the ovary. 3'UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3'UTRs are highly expressed in the ovary yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3'UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At two hours post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3'UTRs provide a resource for studying gene regulation during vertebrate development. PDF
Unhavaithaya, Y., and Orr-Weaver, T.L. (2012). Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes Dev 26, 31-36.Proper development requires coordination in growth of the cell types composing an organ. Many plant and animal cells are polyploid, but how these polyploid tissues contribute to organ growth is not well understood. We found the Drosophila melanogaster subperineurial glia (SPG) to be polyploid, and ploidy is coordinated with brain mass. Inhibition of SPG polyploidy caused rupture of the septate junctions necessary for the blood-brain barrier. Thus, the increased SPG cell size resulting from polyploidization is required to maintain the SPG envelope surrounding the growing brain. Polyploidization likely is a conserved strategy to coordinate tissue growth during organogenesis, with potential vertebrate examples. Full Text.
vanderVeen, A.G., and Ploegh, H.L. (2012). Ubiquitin-Like Proteins. Annual Review of Biochemistry. 81 Reviews in Advance.The eukaryotic ubiquitin family encompasses nearly 20 proteins that are involved in the posttranslational modification of various macromolecules. The ubiquitin-like proteins (UBLs) that are part of this family adopt the beta-grasp fold that is characteristic of its founding member ubiquitin (Ub). Although structurally related, UBLs regulate a strikingly diverse set of cellular processes, including nuclear transport, proteolysis, translation, autophagy, and antiviral pathways. New UBL substrates continue to be identified and further expand the functional diversity of UBL pathways in cellular homeostasis and physiology. Here, we review recent findings on such novel substrates, mechanisms, and functions of UBLs. Full Text
Victora, G.D., and Nussenzweig, M.C. (2012). Germinal Centers. Annual Review of Immunology, Vol 30, pp. 429-45 Germinal centers (GCs) were described more than 125 years ago as compartments within secondary lymphoid organs that contained mitotic cells. Since then, it has become clear that this structure is the site of B cell clonal expansion, somatic hypermutation, and affinity-based selection, the combination of which results in the production of high-affinity antibodies. Decades of anatomical and functional studies have led to an overall model of how the GC reaction and affinity-based selection operate. More recently, the introduction of intravital imaging into the GC field has opened the door to direct investigation of certain key dynamic features of this microanatomic structure, sparking renewed interest in the relationship between cell movement and affinity maturation. We review these and other recent advances in our understanding of GCs, focusing on cellular dynamics and on the mechanism of selection of high-affinity B cells. .Full Text
Voeltz, G., and Cheeseman, I. (2012). Building a path in cell biology. Mol Biol Cell 23, 4145-4147.Setting up a new lab is an exciting but challenging prospect. We discuss our experiences in finding a path to tackle some of the key current questions in cell biology and the hurdles that we have encountered along the way. Full Text
Wagner, D.E., Ho, J.J., and Reddien, P.W. (2012). Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10, 299-311.Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types and identify genetic regulators of the planarian stem cell system. Full Text.
Wang, H., Mayhew, D., Chen, X., Johnston, M., and Mitra, R.D. (2012). "Calling Cards" for DNA-binding Proteins in Mammalian Cells. Genetics.Published online before print January 3, 2012 The ability to chronicle transcription factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell fate decisions. We describe a method for permanently recording protein-DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a "Calling Card" the transcription factor leaves behind to record its visit to the genome. The locations of the Calling Cards can be determined by massively-parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible, and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription factor binding throughout cellular and organismal development in a way that has heretofore not been possible. Full Text
Wang, J., Ramirez, T., Ji, P., Jayapal, S.R., Lodish, H.F., and Murata-Hori, M. (2012). Mammalian erythroblast enucleation requires PI3K-dependent cell polarization. J Cell Sci 125, 340-349.Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion. Full Text.
Weinberg, R.A. (2012). Bengt Westermark and our current understanding of tumor pathogenesis. Upsala Journal of Medical Sciences 117, 81-82. Full Text.
Welburn, J.P., and Cheeseman, I.M. (2012). The microtubule-binding protein Cep170 promotes the targeting of the kinesin-13 depolymerase Kif2b to the mitotic spindle. Mol Biol Cell.Oct 19. [Epub ahead of print] Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. In humans, there are three closely related kinesin-13 isoforms (Kif2a, Kif2b, and Kif2c/MCAK) that are highly conserved in their primary sequences, but display distinct localization and non-overlapping functions. Here, we demonstrate that the N-terminus is a primary determinant of kinesin-13 localization. However, we also find that differences in the C-terminus alter the properties of kinesin-13, in part by facilitating unique protein-protein interactions. We identify the spindle-localized proteins Cep170 and Cep170R (KIAA0284) as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides Kif2b with a second microtubule binding site to target it to the spindle. Thus, the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family .PDF
Welstead, G.G., Creyghton, M.P., Bilodeau, S., Cheng, A.W., Markoulaki, S., Young, R.A., and Jaenisch, R. (2012). X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner. Proc Natl Acad Sci U S A. Jul 23. [Epub ahead of print] Embryogenesis requires the timely and coordinated activation of developmental regulators. It has been suggested that the recently discovered class of histone demethylases (UTX and JMJD3) that specifically target the repressive H3K27me3 modification play an important role in the activation of "bivalent" genes in response to specific developmental cues. To determine the requirements for UTX in pluripotency and development, we have generated Utx-null ES cells and mutant mice. The loss of UTX had a profound effect during embryogenesis. Utx-null embryos had reduced somite counts, neural tube closure defects and heart malformation that presented between E9.5 and E13.5. Unexpectedly, homozygous mutant female embryos were more severely affected than hemizygous mutant male embryos. In fact, we observed the survival of a subset of UTX-deficient males that were smaller in size and had reduced lifespan. Interestingly, these animals were fertile with normal spermatogenesis. Consistent with a midgestation lethality, UTX-null male and female ES cells gave rise to all three germ layers in teratoma assays, though sex-specific differences could be observed in the activation of developmental regulators in embryoid body assays. Lastly, ChIP-seq analysis revealed an increase in H3K27me3 in Utx-null male ES cells. In summary, our data demonstrate sex-specific requirements for this X-linked gene while suggesting a role for UTY during development.PDF
Wenemoser, D., Lapan, S.W., Wilkinson, A.W., Bell, G.W., and Reddien, P.W. (2012). A molecular wound response program associated with regeneration initiation in planarians. Genes Dev 26, 988-1002.Planarians are capable of regenerating any missing body part and present an attractive system for molecular investigation of regeneration initiation. The gene activation program that occurs at planarian wounds to coordinate regenerative responses remains unknown. We identified a large set of wound-induced genes during regeneration initiation in planarians. Two waves of wound-induced gene expression occurred in differentiated tissues. The first wave includes conserved immediate early genes. Many second-wave genes encode conserved patterning factors required for proper regeneration. Genes of both classes were generally induced by wounding, indicating that a common initial gene expression program is triggered regardless of missing tissue identity. Planarian regeneration uses a population of regenerative cells (neoblasts), including pluripotent stem cells. A class of wound-induced genes was activated directly within neoblasts, including the Runx transcription factor-encoding runt-1 gene. runt-1 was required for specifying different cell types during regeneration, promoting heterogeneity in neoblasts near wounds. Wound-induced gene expression in neoblasts, including that of runt-1, required SRF (serum response factor) and sos-1. Taken together, these data connect wound sensation to the activation of specific cell type regeneration programs in neoblasts. Most planarian wound-induced genes are conserved across metazoans, and identified genes and mechanisms should be important broadly for understanding wound signaling and regeneration initiation. Full Text.
Whitesell, L., Santagata, S., and Lin, N.U. (2012). Inhibiting HSP90 to treat cancer: a strategy in evolution. Current Molecular Medicine .Jul 17. [Epub ahead of print] Since discovery of the first HSP90 inhibitor almost two decades ago, tremendous progress has been made in developing potent and selective compounds with which to target this chaperone for the treatment of cancers. In turn, these compounds have been invaluable in probing how HSP90 supports the dramatic alterations in cellular physiology that constitute the malignant phenotype. Unfortunately, single agent activity for HSP90 inhibitors has proven disappointingly modest against advanced cancers in most of the clinical trials that have been reported to date. This problem could be due to the serious pharmacological liabilities of the first-generation inhibitors that have been most extensively studied. But, we suggest it could well be intrinsic to the target itself. Systemically tolerable exposure to HSP90 inhibitors may not be particularly cytotoxic for the majority of common clinical cancers. Instead, the utilization of HSP90 by cancer cells might better be targeted to enhance the activity of other anticancer drugs while at the same time limiting the ability of cancers to adapt and evolve drug resistance; the net result being more durable disease control. This review will focus on these fundamental issues with the goal of helping the clinical development of HSP90 inhibitors become less empiric, more efficient and hopefully more successful.
Whitesell, L., and Lin, N.U. (2012). HSP90 as a platform for the assembly of more effective cancer chemotherapy. Biochimica et biophysica acta 1823, 756-766.Since initial discovery of the first HSP90 inhibitor over a decade and a half ago, tremendous progress has been made in developing potent and selective compounds with which to target this chaperone in the treatment of cancers. These compounds have been invaluable in dissecting how HSP90 supports the dramatic alterations in cellular physiology that constitute the malignant phenotype and give rise to the clinical manifestations of diverse cancers. Unfortunately, single agent activity for HSP90 inhibitors has been disappointingly modest against recurrent, refractory cancers in most of the clinical trials that have been reported to date. This problem could be due to pharmacological limitations of the first-generation inhibitors that have been most extensively studied. But we suggest it may well be intrinsic to the target itself. This review will focus on how the utilization of HSP90 by cancer cells might be targeted to enhance the activity of other anticancer drugs while at the same time limiting the ability of advanced cancers to adapt and evolve drug resistance; the net result being more durable disease control. A better understanding of these fundamental issues will surely make the ongoing clinical development of HSP90 inhibitors as anticancer drugs less empiric, more efficient and hopefully more successful. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90). Full Text.
Whyte, W.A., Bilodeau, S., Orlando, D.A., Hoke, H.A., Frampton, G.M., Foster, C.T., Cowley, S.M., and Young, R.A. (2012). Enhancer decommissioning by LSD1 during embryonic stem cell differentiation. Nature 482, 221-225.Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. Full Text.
Wijeratne, E.M.K., Bashyal, B.P., Liu, M.P.X., Rocha, D.D., Gunaherath, G., U'Ren, J.M., Gunatilaka, M.K., Arnold, A.E., Whitesell, L., and Gunatilaka, A.A.L. (2012). Geopyxins A-E, ent-Kaurane Diterpenoids from Endolichenic Fungal Strains Geopyxis aff. majalis and Geopyxis sp AZ0066: Structure-Activity Relationships of Geopyxins and Their Analogues. Journal of Natural Products 75, 361-369.Four new ent-kaurane diterpenoids, geopyxins A-D (1-4), were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6), together with 1 and 3. The structures of 1-6 were established on the basis of their spectroscopic data, while the absolute configurations were assigned using modified Mosher's ester method. Methylation of 1-3, 5, and 6 gave their corresponding methyl esters 7-11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7-10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7-9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic alpha,beta-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation. Full Text.
Witte, M.D., Cragnolini, J.J., Dougan, S.K., Yoder, N.C., Popp, M.W., and Ploegh, H.L. (2012). Preparation of unnatural N-to-N and C-to-C protein fusions. Proc Natl Acad Sci U S AJul 9. [Epub ahead of print] .Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest. PDF
Wood, K.C., Konieczkowski, D.J., Johannessen, C.M., Boehm, J.S., Tamayo, P., Botvinnik, O.B., Mesirov, J.P., Hahn, W.C., Root, D.E., Garraway, L.A.,and David M. Sabatini (2012). MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma. Science Signaling May 15;5(224):rs4. Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but owing to technical limitations they have been restricted for use with a narrow range of cell lines and short-term assays. Here, we describe MicroSCALE (Microarrays of Spatially Confined Adhesive Lentiviral Features), a cell microarray-based platform that enables application of this technology to a wide range of cell types and longer-term assays. We used MicroSCALE to uncover kinases that when overexpressed partially desensitized B-RAF(V600E)-mutant melanoma cells to inhibitors of the mitogen-activated protein kinase kinase kinase (MAPKKK) RAF, the MAPKKs MEK1 and 2 (MEK1/2, mitogen-activated protein kinase kinase 1 and 2), mTOR (mammalian target of rapamycin), or PI3K (phosphatidylinositol 3-kinase). These screens indicated that cells treated with inhibitors acting through common mechanisms were affected by a similar profile of overexpressed proteins. In contrast, screens involving inhibitors acting through distinct mechanisms yielded unique profiles, a finding that has potential relevance for small-molecule target identification and combination drugging studies. Further, by integrating large-scale functional screening results with cancer cell line gene expression and pharmacological sensitivity data, we validated the nuclear factor kappa B pathway as a potential mediator of resistance to MAPK pathway inhibitors. The MicroSCALE platform described here may enable new classes of large-scale, resource-efficient screens that were not previously feasible, including those involving combinations of cell lines, perturbations, and assay outputs or those involving limited numbers of cells and limited or expensive reagents. Full Text
Xu, C., Fan, Z.P., Muller, P., Fogley, R., DiBiase, A., Trompouki, E., Unternaehrer, J., Xiong, F.Z., Torregroza, I., Evans, T Sean G. Megason George Q. Daley Alexander F. Schier Richard A. Young and Leonard I. Zon. (2012). Nanog-like Regulates Endoderm Formation through the Mxbc2-Nodal Pathway. Developmental Cell 22, 625-638.In mammalian embryonic stem cells, the acquisition of pluripotency is dependent on Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extraembryonic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We examined the genes that were regulated by Nanog-like and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which, in turn, specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extraembryonic tissue required for endoderm induction. Full Text.
Yilmaz, O.H., Katajisto, P., Lamming, D.W., Gultekin, Y., Bauer-Rowe, K.E., Sengupta, S., Birsoy, K., Dursun, A., Yilmaz, V.O., Selig, M.Nielsen GP, Mino-Kenudson M, Zukerberg LR, Bhan AK, Deshpande V, Sabatini DM.l. (2012). mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake. Nature.Published online 20 May 2012 How adult tissue stem and niche cells respond to the nutritional state of an organism is not well understood. Here we find that Paneth cells, a key constituent of the mammalian intestinal stem-cell (ISC) niche, augment stem-cell function in response to calorie restriction. Calorie restriction acts by reducing mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells, and the ISC-enhancing effects of calorie restriction can be mimicked by rapamycin. Calorie intake regulates mTORC1 in Paneth cells, but not ISCs, and forced activation of mTORC1 in Paneth cells during calorie restriction abolishes the ISC-augmenting effects of the niche. Finally, increased expression of bone stromal antigen 1 (Bst1) in Paneth cells-an ectoenzyme that produces the paracrine factor cyclic ADP ribose-mediates the effects of calorie restriction and rapamycin on ISC function. Our findings establish that mTORC1 non-cell-autonomously regulates stem-cell self-renewal, and highlight a significant role of the mammalian intestinal niche in coupling stem-cell function to organismal physiology. Full Text
Youngsaye, W., Dockendorff, C., Vincent, B., Hartland, C.L., Bittker, J.A., Dandapani, S., Palmer, M., Whitesell, L., Lindquist, S. ,Schreiber SL, Munoz B. (2012). Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles. Bioorganic & Medicinal Chemistry Letters 22, 3362-3365.Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways. Full Text
Zielinski, D., Gymrek, M., and Erlich, Y. (2012). Back to the family: a renewed approach to rare variant studies. Genome Med 4, 97. A report on the 62nd Annual Meeting of the American Society of Human Genetics, San Francisco, California, USA, 6-10 November 2012.
Zhang, L., Sankaran, V.G., and Lodish, H.F. (2012). MicroRNAs in erythroid and megakaryocytic differentiation and megakaryocyte-erythroid progenitor lineage commitment. Leukemia. May 23. [Epub ahead of print] MicroRNAs are a class of small regulatory non-coding RNAs that modulate the expression of their target genes through either mRNA degradation or inhibition of protein translation. In recent years, microRNAs have been shown to be critical regulators of hematopoiesis and play important roles in the differentiation of specific lineages. Here, we summarize our current understanding of microRNAs involved in hematopoiesis with a focus on the role of microRNAs in regulating erythroid and megakaryocytic differentiation and megakaryocyte-erythroid progenitor (MEP) lineage commitment. Full Text.
Zhou, H., Cheng, J.S., Wang, B.L., Fink, G.R., and Stephanopoulos, G. (2012). Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metabolic Engineering 14, 611-622.Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL(CS) displayed significantly increased anaerobic growth rate (0.203 +/- 0.006 h(-1)) and xylose consumption rate (1.866 g g(-1) h(-1)) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain. Full Text
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