H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction (II) [ChIP-Seq]
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ABSTRACT: We report that acetylation of H4K16 is a new marker of active enhancers and that some enhancers are marked by H3K4me1, MOF and H4K16ac but not by acetylated H3K27 or p300, suggesting that they are novel p300-independent regulatory elements. ChIP-seq for H4K16 acetylation in undifferentiated 46c(sox1-gfp) ES cells, and FACS sorted day 5 Neural Progenitor Cells (differentiated with NB27 and Neuro2 medium supplements) , along with MNase digested input for both samples
Project description:Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. We found that Spt6 is required for proper engagement and function of the H3K27 demethylase KDM6A (UTX) on muscle genes and regulates muscle gene expression and cell differentiation. ChIP-Seq experiments revealed an extensive genome-wide overlap of Spt6, PolII and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus effectively controlling developmental gene expression and cell differentiation. Examination of Spt6 and KDM6A levels in a skeletal muscle cells at various developmental stages
Project description:Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in satellite cells To establish the role of the deacetylase SIRT1 in skeletal muscle we examined the genome wide distribution of H4K16ac in quiescent (FI) and proliferating (Cul) satellite cells isolated from WT mice (C57Bl/6 background) and SIRT1mKO (generated via breeding of Pax7cre/+ knock-in mice with mice containing the floxed exon 4 SIRT1 allele). We also analyzed the distribution of SIRT1 in quiescent and proliferating FACS isolated WT satellite cells (two replicates). We generated the mRNA profiles (at least two replicate for each experiment) of FACS isolated quiescent, proliferating and differentiating (1 day in differentiation medium) satellite cells of WT mice and SIRT1mKO. The selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program.
Project description:Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network (GRN). Here, by using genome-wide occupancy profiling of master regulators of MyoGenesis (MyoD and MyoGenin), we show their extensive occupancy in the extragenic enhancer regions coinciding with RNA synthesis (i.e. eRNA). In particular, multiple regions coding for eRNAs were observed within regulatory region of MYOD1, including previously characterized Distal Regulatory Regions (DRR) and Core Enhancer (CE). While CERNA enhanced RNA polymerase II (PolII) occupancy and transcription at MYOD1, DRRRNA acted in trans to activate the downstream MyoGenic GRN. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding PolII assembly. By nuclease sensitivity assay, we show that eRNAs increase genomic access to the transcriptional complex at defined regulatory regions. In conclusion, our data suggest eRNAs establish a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events. Examination of MyoD and MyoG binding events and production of RNA at the enhancer sites during myogenic differentiation. We performed RNAi against enhancer-derived RNA (DRRi) which resulted in reduction of chromatin accessiblity and RNA polymerase II occupancy at defined regulatory elements. In complementary experiments, overexpression of DRR-RNA (pHAN_DRR1.2) resulted in early activation of MyoG. GFPi and GFP overexpression (pHAN_GFP) were used as control in these experiments, respectively.
Project description:We report the acetylation of lysine residues in the globular domain of H3 (H3K64ac and H3K122ac) marks active gene promoters and also a subset of active enhancers in mouse embryonic stem cells (mESCs), human erythroleukemic cell line (K562). Moreover, we find a novel class of active functional enhancers in ESCs that are marked by H3K122ac but which lack H3K27ac. This work suggests that a more complex analysis of histone acetylation is required to identify enhancers than was previously considered. Examination of histone modifications in mouse ESCs (2 biological replicates) and K562 cells
Project description:Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and it is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells is sufficient to induce luminal-to-basal phenotypic switch implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells. Examination of histone H3K27me3 modifications in various breast cancer cell lines.
Project description:We report that acetylation of H4K16 is a new marker of active enhancers and that some enhancers are marked by H3K4me1, MOF and H4K16ac but not by acetylated H3K27 or p300, suggesting that they are novel p300-independent regulatory elements. ChIP-seq for H4K16 acetylation in undifferentiated ES cells, and cells after 3 days of retinoic acid differentiation, along with MNase digested input for both samples
Project description:Copy number variants (CNV) influence the expression of genes that map not only within, but also on their flanks. To assess the possible mechanism(s) underlying this “neighboring effect”, we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. We detected alteration of intrachromosomal interactions (chromosomal looping) between the loci of affected genes and the rearranged interval using chromosome conformation capture (4C-seq). These results are consistent with the observed gene expression alterations. We also pinpointed concomitant changes in histone modifications between samples. Modified genes were often looping together, possibly forming an interacting cluster. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thus possibly modulating expression globally and modifying the phenotype. For example, we observe that the chromatin conformation, histone marks and relative expression levels of the AUTS2 gene, mutations of which are associated with autism and intellectual disabilities, are modified in Williams-Beuren syndrome patients cell lines. Examination of 2 different histone modifications in genomic disorders patients' cell lines.
Project description:Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential meiotic genes and repressing a M-bM-^@M-^\progenitor cellsM-bM-^@M-^] gene expression program, while M-bM-^@M-^\primingM-bM-^@M-^] a post-meiotic gene group for further activation. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for BrdtM-bM-^@M-^Ys first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome. Examination of Brdt binding on chromatin in meiotic (spermatocytes) and post-meiotic (round spermatids) male germ cells from adult wild type mice
Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Neural stem cells derived from mouse embryonic stem cells were differentiated into neurons and FACS purified based on RedStar fluorescence driven by the Tau promoter. Chromatin was prepared from NSCs and neurons (n=1), sonicated to roughly 300bp and immunoprecipitated with antibodies against H3K4me3, H3K27me3, total Histone H3 and total IgG, alongside a 5% input sample. K4/K27 and corresponding input samples were analysed by ChIPSeq
Project description:This SuperSeries is composed of the following subset Series: GSE39908: Bromodomain-dependent stage-specific male genome programming by Brdt [ChIP-Seq] GSE39909: Bromodomain-dependent stage-specific male genome programming by Brdt [Illumina BeadArray] Refer to individual Series