Project description:Our understanding of the functions of DNA elements is limited by the paucity of information about the spectrum of proteins that occupy these genomic regions. Here we describe an approach to identify proteins associated with genomic regions whose chromatin is marked by specific modified histones, which we term chromatin profiling. We used chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) to identify proteins associated with genomic regions marked by histone H3K27Ac, H3K4me3, H3K79me2 and H3K36me3 in mouse embryonic stem (mES) cells. We identified 385 known and 224 novel candidate proteins associated with these histone-marked genomic segments and confirmed that several of the novel candidates are indeed associated with histone-marked segments of the genome. Future study of the novel candidates, many of which have been implicated in various diseases, should lead to an improved understanding of gene control and its dysregulation in disease. ChIP-seq for nucleosomes with modified histones and DNA-binding proteins in mouse embryonic stem cells, and DNA-binding proteins in Jurkat cells
Project description:Our understanding of the functions of DNA elements is limited by the paucity of information about the spectrum of proteins that occupy these genomic regions. Here we describe an approach to identify proteins associated with genomic regions whose chromatin is marked by specific modified histones, which we term chromatin profiling. We used chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) to identify proteins associated with genomic regions marked by histone H3K27Ac, H3K4me3, H3K79me2 and H3K36me3 in mouse embryonic stem (mES) cells. We identified 385 known and 224 novel candidate proteins associated with these histone-marked genomic segments and confirmed that several of the novel candidates are indeed associated with histone-marked segments of the genome. Future study of the novel candidates, many of which have been implicated in various diseases, should lead to an improved understanding of gene control and its dysregulation in disease.
Project description:We employed the DamID technique to systematically map the genomic distribution of all canonical somatic H1 subtypes (H1.1-H1.5) in human IMR90 cells. Human cells contain up to eleven histone H1 proteins, with different spatial and temporal expression patterns. These include five canonical, replication-dependent somatic H1 subtypes (H1.1, H1.2, H1.3, H1.4 and H1.5). Despite being a key chromatin component, the genomic distribution of the somatic canonical H1 subtypes is still unknown and their role in chromatin related processes has so far remained elusive. Here we employed a DamID approach to map for the first time the genomic localization of all somatic canonical H1 subtypes in human cells. Our integrative analysis reveals novel insights into H1 subtype distribution and uncovers functional chromatin features potentially regulating the H1 genomic landscape. In general H1.2 to H1.5 are depleted from GC-rich regions and regulatory regions associated with active transcription. H1.1 shows a binding profile distinct from the other subtypes, suggesting a unique function for H1.1 in chromatin-regulated processes. Interestingly, our data indicate a novel role for somatic H1 subtypes in the three-dimensional organization of the genome by marking repressive regions within topological domains such as LADs. Our work integrates the five somatic linker histone H1 subtypes into the epigenome maps of human cells and provides a resource to refine our understanding of the significance of H1 and its heterogeneity in the control of genome function. DamID profiling of somatic linker histone variants H1.1, H1.2, H1.3, H1.4 and H1.5 in human fibroblasts. Two biological replicate samples of all H1 variants were hybridized on NimbleGen Human ChIP-chip 2.1M Economy Whole-Genome Tiling - Array GPL16055 covering small human chromosomes.
Project description:The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type–specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K36me2/K4me1 marks transcribed enhancers, while H3K36me3/K4me1 and H3K79me2/K4me1 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics.
Project description:Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1, in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. However, the ratio of HMGD1 to H1 is better correlated with chromatin accessibility, gene expression and nucleosome spacing variation than either protein alone suggesting a competitive mechanism between these proteins. Indeed, we show that HMGD1 and H1 compensate each other’s absence by binding reciprocally to chromatin resulting in changes to nucleosome repeat length and distinct gene expression patterns. Collectively our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study thus provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation. ChIP-seq of HMGD1 and Histone H1 bound nucleosomes as well as MNase-seq of total nucleosome in Drosophila S2 cells
Project description:BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite previous structural studies and recent advance of SWI/SNF family complexes, it remains incompletely understood how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions. PBAF-nucleosome structure reveals six histone-binding domains and four DNA-binding domains/modules, the majority of which directly bind histone/DNA. This multivalent nucleosome-binding pattern, not observed in previous studies, suggests that PBAF may integrate comprehensive chromatin information to target genomic loci for function. Our study reveals molecular organization of subunits and histone/DNA-binding domains/modules in PBAF-nucleosome complex and provides structural insights into PBAF-mediated nucleosome association complimentary to the recently reported PBAF-nucleosome structure.
Project description:To identify chromatin alterations in primary gastric adenocarcionomas, we performed nano-scale chromatin immunoprecipitation-sequencing (Nano-CHiPseq) of histone modifications in 5 gastric cancers and matched normal tissues, We identified hundreds of somatically-altered promoters (marked by H3K4me3) and enhancers (H3K4me1). The majority of cancer-associated promoters localized to genomic sites lacking previously-annotated transcription start sites (“cryptic promoters”), driving high expression of nearby genes implicated in gastrointestinal cancers, embryonic development, and tissue specification. Our findings demonstrate the feasibility of performing chromatin profiling on solid tumors where tissue is limiting, to identify in non-coding regions regulatory elements, transcriptional patterns and genetic variants associated with cancer. We propose a pervasive role for cryptic promoters in the reactivation of early developmental programs in gastric cancer, and the potential utility of cryptic promoters as biomarkers of malignancy. Five gastric cancer tumor normal pairs are profiling in multiple number of chromatin marks
Project description:We employed the DamID technique to systematically map the genomic distribution of all canonical somatic H1 subtypes (H1.1-H1.5) in human IMR90 cells. Human cells contain up to eleven histone H1 proteins, with different spatial and temporal expression patterns. These include five canonical, replication-dependent somatic H1 subtypes (H1.1, H1.2, H1.3, H1.4 and H1.5). Despite being a key chromatin component, the genomic distribution of the somatic canonical H1 subtypes is still unknown and their role in chromatin related processes has so far remained elusive. Here we employed a DamID approach to map for the first time the genomic localization of all somatic canonical H1 subtypes in human cells. Our integrative analysis reveals novel insights into H1 subtype distribution and uncovers functional chromatin features potentially regulating the H1 genomic landscape. In general H1.2 to H1.5 are depleted from GC-rich regions and regulatory regions associated with active transcription. H1.1 shows a binding profile distinct from the other subtypes, suggesting a unique function for H1.1 in chromatin-regulated processes. Interestingly, our data indicate a novel role for somatic H1 subtypes in the three-dimensional organization of the genome by marking repressive regions within topological domains such as LADs. Our work integrates the five somatic linker histone H1 subtypes into the epigenome maps of human cells and provides a resource to refine our understanding of the significance of H1 and its heterogeneity in the control of genome function.
Project description:Post-translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We discovered a novel bivalent combination, a dually-marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent genome-wide location analysis (ChIP-Seq) identified 1032 and 668 bivalent regions in young and old livers, respectively, with 280 in common. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually-marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.
Project description:The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae . Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest.