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Laurie A Boyer,
Kathrin Plath,
Julia Zeitlinger,
Tobias Brambrink,
Lea A Medeiros, Tong Ihn Lee,
Stuart S Levine,
Marius Wernig,
Adriana Tajonar,
Mridula K Ray,
George W Bell,
Arie P Otte,
Miguel Vidal,
David K Gifford,
Richard A Young,
Rudolf Jaenisch
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ABSTRACT: The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.
Nature 06/2006; 441(7091):349-53. · 36.28 Impact Factor
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Tong Ihn Lee,
Richard G Jenner,
Laurie A Boyer,
Matthew G Guenther,
Stuart S Levine,
Roshan M Kumar,
Brett Chevalier,
Sarah E Johnstone,
Megan F Cole,
Kyo-ichi Isono, [......],
Nancy M Hannett,
Kaiming Sun,
Duncan T Odom,
Arie P Otte,
Thomas L Volkert,
David P Bartel,
Douglas A Melton,
David K Gifford,
Rudolf Jaenisch,
Richard A Young
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ABSTRACT: Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
Cell 05/2006; 125(2):301-13. · 32.40 Impact Factor
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ABSTRACT: Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.
Nature Protocol 02/2006; 1(2):729-48. · 8.36 Impact Factor
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ABSTRACT: The stepwise loss of cohesins, the complexes that hold sister chromatids together, is required for faithful meiotic chromosome segregation. Cohesins are removed from chromosome arms during meiosis I but are maintained around centromeres until meiosis II. Here we show that Sgo1, a protein required for protecting centromeric cohesins from removal during meiosis I, localizes to cohesin-associated regions (CARs) at the centromere and the 50-kb region surrounding it. Establishment of this Sgo1-binding domain requires the 120-base-pair (bp) core centromere, the kinetochore component Bub1, and the meiosis-specific factor Spo13. Interestingly, cohesins and the kinetochore proteins Iml3 and Chl4 are necessary for Sgo1 to associate with pericentric regions but less so for Sgo1 to associate with the core centromeric regions. Finally, we show that the 50-kb Sgo1-binding domain is the chromosomal region where cohesins are protected from removal during meiosis I. Our results identify the portions of chromosomes where cohesins are protected from removal during meiosis I and show that kinetochore components and cohesins themselves are required to establish this cohesin protective domain.
Genes & Development 01/2006; 19(24):3017-30. · 11.66 Impact Factor
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Laurie A Boyer, Tong Ihn Lee,
Megan F Cole,
Sarah E Johnstone,
Stuart S Levine,
Jacob P Zucker,
Matthew G Guenther,
Roshan M Kumar,
Heather L Murray,
Richard G Jenner,
David K Gifford,
Douglas A Melton,
Rudolf Jaenisch,
Richard A Young
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ABSTRACT: The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.
Cell 10/2005; 122(6):947-56. · 32.40 Impact Factor
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Dmitry K Pokholok,
Christopher T Harbison,
Stuart Levine,
Megan Cole,
Nancy M Hannett, Tong Ihn Lee,
George W Bell,
Kimberly Walker,
P Alex Rolfe,
Elizabeth Herbolsheimer,
Julia Zeitlinger,
Fran Lewitter,
David K Gifford,
Richard A Young
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ABSTRACT: Eukaryotic genomes are packaged into nucleosomes whose position and chemical modification state can profoundly influence regulation of gene expression. We profiled nucleosome modifications across the yeast genome using chromatin immunoprecipitation coupled with DNA microarrays to produce high-resolution genome-wide maps of histone acetylation and methylation. These maps take into account changes in nucleosome occupancy at actively transcribed genes and, in doing so, revise previous assessments of the modifications associated with gene expression. Both acetylation and methylation of histones are associated with transcriptional activity, but the former occurs predominantly at the beginning of genes, whereas the latter can occur throughout transcribed regions. Most notably, specific methylation events are associated with the beginning, middle, and end of actively transcribed genes. These maps provide the foundation for further understanding the roles of chromatin in gene expression and genome maintenance.
Cell 09/2005; 122(4):517-27. · 32.40 Impact Factor
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Christopher T Harbison,
D Benjamin Gordon, Tong Ihn Lee,
Nicola J Rinaldi,
Kenzie D Macisaac,
Timothy W Danford,
Nancy M Hannett,
Jean-Bosco Tagne,
David B Reynolds,
Jane Yoo,
Ezra G Jennings,
Julia Zeitlinger,
Dmitry K Pokholok,
Manolis Kellis,
P Alex Rolfe,
Ken T Takusagawa,
Eric S Lander,
David K Gifford,
Ernest Fraenkel,
Richard A Young
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ABSTRACT: DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.
Nature 10/2004; 431(7004):99-104. · 36.28 Impact Factor
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Ziv Bar-Joseph,
Georg K Gerber, Tong Ihn Lee,
Nicola J Rinaldi,
Jane Y Yoo,
François Robert,
D Benjamin Gordon,
Ernest Fraenkel,
Tommi S Jaakkola,
Richard A Young,
David K Gifford
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ABSTRACT: We describe an algorithm for discovering regulatory networks of gene modules, GRAM (Genetic Regulatory Modules), that combines information from genome-wide location and expression data sets. A gene module is defined as a set of coexpressed genes to which the same set of transcription factors binds. Unlike previous approaches that relied primarily on functional information from expression data, the GRAM algorithm explicitly links genes to the factors that regulate them by incorporating DNA binding data, which provide direct physical evidence of regulatory interactions. We use the GRAM algorithm to describe a genome-wide regulatory network in Saccharomyces cerevisiae using binding information for 106 transcription factors profiled in rich medium conditions data from over 500 expression experiments. We also present a genome-wide location analysis data set for regulators in yeast cells treated with rapamycin, and use the GRAM algorithm to provide biological insights into this regulatory network
Nature Biotechnology 12/2003; 21(11):1337-42. · 23.27 Impact Factor
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Ziv Bar-Joseph,
Georg K Gerber, Tong Ihn Lee,
Nicola J Rinaldi,
Jane Y Yoo,
Fran|[ccedil]|ois Robert,
D Benjamin Gordon,
Ernest Fraenkel,
Tommi S Jaakkola,
Richard A Young,
David K Gifford
[show abstract]
[hide abstract]
ABSTRACT: We describe an algorithm for discovering regulatory networks of gene modules, GRAM (Genetic Regulatory Modules), that combines information from genome-wide location and expression data sets. A gene module is defined as a set of coexpressed genes to which the same set of transcription factors binds. Unlike previous approaches
Nature Biotechnology 10/2003; 21(11):1337-1342. · 23.27 Impact Factor
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Tong Ihn Lee,
Nicola J Rinaldi,
François Robert,
Duncan T Odom,
Ziv Bar-Joseph,
Georg K Gerber,
Nancy M Hannett,
Christopher T Harbison,
Craig M Thompson,
Itamar Simon, [......],
Ezra G Jennings,
Heather L Murray,
D Benjamin Gordon,
Bing Ren,
John J Wyrick,
Jean-Bosco Tagne,
Thomas L Volkert,
Ernest Fraenkel,
David K Gifford,
Richard A Young
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ABSTRACT: We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.
Science 11/2002; 298(5594):799-804. · 31.20 Impact Factor
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[show abstract]
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ABSTRACT: The transcription factors TFIID and SAGA are multi-subunit complexes involved
in transcription by RNA polymerase II
Nature 06/2000; 405(6787):701-704. · 36.28 Impact Factor
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