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Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

Accumulation of tau and amyloid-β are two pathologic hallmarks of Alzheimer's disease. We conducted an epigenome-wide association study using the histone 3 lysine 9 acetylation (H3K9ac) mark in 669 aged human prefrontal cortices; in contrast with amyloid-β, tau protein burden had a broad effect on the epigenome, affecting 5,990 of 26,384 H3K9ac domains. Tau-related alterations aggregated in large genomic segments reflecting spatial chromatin organization, and the magnitude of these effects correlated with the segment's nuclear lamina association. Functional relevance of these chromatin changes was demonstrated by (1) consistent transcriptional changes in three independent datasets and (2) similar findings in two mouse models of Alzheimer's disease. Finally, we found that tau overexpression in induced pluripotent stem cell-derived neurons altered chromatin structure and that these effects could be blocked by a small molecule predicted to reverse the tau effect. Thus, we report broad tau-driven chromatin rearrangements in the aging human brain that may be reversible with heat-shock protein 90 (Hsp90) inhibitors.

Different combinations of histone modifications have been proposed to signal distinct gene regulatory functions, but this area is poorly addressed by existing technologies. We applied high-throughput single-molecule imaging to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. We identified definitively bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. We showed that genetic and chemical perturbations of chromatin enzymes preferentially affect nucleosomes harboring specific modification states. Last, we combined this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This single-molecule technology has the potential to address fundamental questions in chromatin biology and epigenetic regulation., (Copyright © 2016, American Association for the Advancement of Science.)

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type-specific chromatin signatures and striking enrichments for disease-associated sequence variants. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and did not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator-like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes downregulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of epigenome editing tools to characterize an important class of functional genomic elements.

Background: The histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure., Results: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin., Conclusions: Our findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles.

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation., Competing Interests: The authors have declared that no competing interests exist.

Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A succession of technological advances over the past decade have enabled researchers to chart maps of histone modifications and related chromatin structures with increasing accuracy, comprehensiveness and throughput. The resulting data sets highlight the interplay between chromatin and genome function, dynamic variations in chromatin structure across cellular conditions, and emerging roles for large-scale domains and higher-ordered chromatin organization. Here we review a selection of recent studies that have probed histone modifications and successive layers of chromatin structure in mammalian genomes, the patterns that have been identified and future directions for research.

Post-translational modifications to histone proteins regulate the packaging of genomic DNA into chromatin, gene activity and other functions of the genome. They are understood to play key roles in embryonic development and disease pathogenesis. Recent advances in technology have made it possible to analyze chromatin structure genome-wide in mammalian cells. Global patterns of histone modifications can be observed using a technique called ChIP-on-chip, which combines the specificity of chromatin immunoprecipitation with the unbiased, high-throughput capabilities of microarrays. The resulting maps provide insight into the functions of, and relationships between, different modifications. Here, we provide validated ChIP-on-chip methods for analyzing histone modification patterns at genome-scale in mammalian cells.

MLL complexes are homologs of yeast COMPASS capable of methylating histone H3 Lys4 (H3K4). ASH2L, RbBP5 and WDR5 are conserved subunits of MLL complexes with homology to the Cps40/Cps60, Cps50 and Cps30 subunits of COMPASS, respectively. We report that ASH2L differentially regulates MLL's catalysis of H3K4 trimethylation similarly to Cps40 and Cps60. Furthermore, WDR5 is required to maintain MLL complex integrity, including the stability of ASH2L within the complex. These findings offer insight into the molecular role of ASH2L, and by extension that of WDR5, in proper H3K4 trimethylation.

The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.

We mapped histone H3 lysine 4 di- and trimethylation and lysine 9/14 acetylation across the nonrepetitive portions of human chromosomes 21 and 22 and compared patterns of lysine 4 dimethylation for several orthologous human and mouse loci. Both chromosomes show punctate sites enriched for modified histones. Sites showing trimethylation correlate with transcription starts, while those showing mainly dimethylation occur elsewhere in the vicinity of active genes. Punctate methylation patterns are also evident at the cytokine and IL-4 receptor loci. The Hox clusters present a strikingly different picture, with broad lysine 4-methylated regions that overlay multiple active genes. We suggest these regions represent active chromatin domains required for the maintenance of Hox gene expression. Methylation patterns at orthologous loci are strongly conserved between human and mouse even though many methylated sites do not show sequence conservation notably higher than background. This suggests that the DNA elements that direct the methylation represent only a small fraction of the region or lie at some distance from the site.

Set1p methylates lysine 4 (K4) of histone H3 and regulates the expression of many genes in yeast. Here we use a biochemical approach to identify a protein, Isw1p, which recognizes chromatin preferentially when it is di- and trimethylated at K4 H3. We show that on certain actively transcribed genes, the Isw1p chromatin remodeling ATPase requires K4 H3 methylation to associate with chromatin in vivo. Analysis of one such gene, MET16, shows that the enzymatic activities of Set1p and Isw1p are functionally connected: Set1p methylation and Isw1p ATPase generate specific chromatin changes at the 5' end of the gene, are necessary for the correct distribution of RNA polymerase II over the coding region, and are required for the recruitment of the cleavage and polyadenylation factor Rna15p. These results indicate that K4 H3 methylation and Isw1p ATPase activity are intimately linked in regulating transcription of certain genes in yeast.

Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.

Posttranslational modifications of histone tails regulate chromatin structure and transcription. Here we present global analyses of histone acetylation and histone H3 Lys 4 methylation patterns in yeast. We observe a significant correlation between acetylation of histones H3 and H4 in promoter regions and transcriptional activity. In contrast, we find that dimethylation of histone H3 Lys 4 in coding regions correlates with transcriptional activity. The histone methyltransferase Set1 is required to maintain expression of these active, promoter-acetylated, and coding region-methylated genes. Global comparisons reveal that genomic regions deacetylated by the yeast enzymes Rpd3 and Hda1 overlap extensively with Lys 4 hypo- but not hypermethylated regions. In the context of recent studies showing that Lys 4 methylation precludes histone deacetylase recruitment, we conclude that Set1 facilitates transcription, in part, by protecting active coding regions from deacetylation.

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML. Chromatin accessibility is a key mediator of gene expression and mutations in chromatin modifiers are frequently seen in cancers. Here, the authors show that the chromatin accessibility regulator HMGN1 - which is frequently mutated by amplification in leukemias - acts by blocking myeloid differentiation. [ABSTRACT FROM AUTHOR]

Current methods for whole-genome mapping of protein-DNA interactions, performed by coupling chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), require large amounts of starting materials, which precludes their application to rare cell types. Here we combine a high-sensitivity ChIP assay with a new library preparation procedure to map histone modifications in as few as 10,000 cells. We used the technique to characterize mouse hematopoietic progenitors and thereby gain insight into their developmental program. [ABSTRACT FROM AUTHOR]

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that ‘weak’ CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine. [ABSTRACT FROM AUTHOR]

Chemical modifications to DNA and histone proteins form a complex regulatory network that modulates chromatin structure and genome function. The epigenome refers to the complete description of these potentially heritable changes across the genome. The composition of the epigenome within a given cell is a function of genetic determinants, lineage, and environment. With the sequencing of the human genome completed, investigators now seek a comprehensive view of the epigenetic changes that determine how genetic information is made manifest across an incredibly varied background of developmental stages, tissue types, and disease states. Here we review current research efforts, with an emphasis on large-scale studies, emerging technologies, and challenges ahead. [Copyright &y& Elsevier]

Background: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. Results: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. Conclusion: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments. [ABSTRACT FROM AUTHOR]

Discusses the combination of reverse genetic and reverse chemical genetic experiments involving transcription profiling to study the function of histone deacetylases (HDAC) in Saccharomyces cerevisiae. Importance of the small molecules trapoxin and trichostatin A in the initial characterization of an HDAC.

The Polycomb group of proteins forms at least two distinct complexes designated the Polycomb repressive complex-1 (PRC1) and PRC2. These complexes cooperate to mediate transcriptional repression of their target genes, including the Hox gene cluster and the Cdkn2a genes. Mammalian Polycomb-like gene Pcl2/Mtf2 is expressed as four different isoforms, and the longest one contains a Tudor domain and two plant homeodomain (PHD) fingers. Pcl2 forms a complex with PRC2 and binds to Hox genes in a PRC2-dependent manner. We show that Pcl2 is a functional component of PRC2 and is required for PRC2-mediated Hox repression. Pcl2, however, exhibits a profound synergistic effect on PRC1-mediated Hox repression, which is not accompanied by major alterations in the local trimethylation of histone H3 at lysine 27 (H3K27me3) or PRC1 deposition. Pcl2 therefore functions in collaboration with both PRC2 and PRC1 to repress Hox gene expression during axial development. Paradoxically, in embryonic fibroblasts, Pcl2 is shown to activate the expression of Cdkn2a and promote cellular senescence, presumably by suppressing the catalytic activity of PRC2 locally. Taken together, we show that Pcl2 differentially regulates Polycomb-mediated repression of Hox and Cdkn2a genes. We therefore propose a novel role for Pcl2 to modify functional engagement of PRC2 and PRC1, which could be modulated by sensing cellular circumstances. [ABSTRACT FROM AUTHOR]

Recent genomic analyses of pediatric glioblastoma, a poorly understood tumor with dismal outcome, have identified mutations in histone H3 variants that affect critical amino acids in the tail. The findings extend discoveries of chromatin regulator inactivation and gain-of-function mutations by documenting alteration of a modifiable histone residue in human cancer. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode. org/) portals. [ABSTRACT FROM AUTHOR]

Summary: Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis. [ABSTRACT FROM AUTHOR]

Summary: Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function. [ABSTRACT FROM AUTHOR]

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing. [ABSTRACT FROM AUTHOR]