RNA-seq analysis of neuronal differentiation of KAT6A/B knockout mouse embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE250570 | GEO | 2024/12/27
REPOSITORIES: GEO
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Project description:Promoters of developmental genes in embryonic stem cells (ESCs) are marked by histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in an asymmetric nucleosomal conformation, with each sister histone H3 carrying one of the two marks. These so-called bivalent domains are thought to poise genes for timely activation upon differentiation. Here we show that asymmetric bivalent nucleosomes recruit repressive H3K27me3 binders but fail to enrich activating H3K4me3 binders, despite presence of H3K4me3, thereby promoting a poised state. Strikingly, the bivalent mark combination further promotes binding of chromatin proteins that are not recruited by each mark individually, including the histone acetyltransferase complex KAT6B (MORF). Knockout of KAT6B blocks neuronal differentiation, demonstrating that bivalency-specific readers are critical for proper ESC differentiation. These findings reveal how histone mark bivalency directly promotes establishment of a poised state at developmental genes, while highlighting how nucleosomal asymmetry is critical for histone mark readout and function.
2024-12-27 | GSE229987 | GEO
Project description:Bivalency, the paradoxical juxtaposition of transcriptionally activating trimethylation of histone H3 lysine 4 (H3K4me3) and the repressive trimethylation of histone H3 lysine 27 (H3K27me3), has been proposed to decorate developmental genes poised for gene expression regulation. Here, we report development of sequential internally calibrated chromatin immunoprecipitation (Re-ICeChIP-seq), capable of measuring absolute quantities of nucleosomal patterns of histone marks in a genome-wide fashion, combined with in situ control of antibody specificity. Re-ICeChIP-seq of H3K4me3/H3K27me3 in mESC reveals that bivalent genes can be delineated into two classes, distinguished by the nucleosomal ratio of H3K4me3 to H3K27me3. Consistent with the canonical role of bivalency, H3K27me3-rich bivalent nucleosomes demarcate promoters of poorly expressed developmental genes that may be poised for activation or repression. Yet our measurements reveal surprisingly widespread presence of bivalency at promoters of highly-expressed housekeeping genes, characterized by H3K4me3-rich bivalent nucleosomes. Moreover, the ratio of H3K4me3 to H3K27me3 at transcription start sites better correlates with gene expression than H3K4me3 or H3K27me3 alone, suggesting cooperation between opposing marks to fine-tune gene expression. Finally, we report that major H3K4 methyltransferases exhibit wide acceptance of various H3K27me3 substrates.
2021-01-04 | GSE108747 | GEO
Project description:Epigenetic priming factors establish a permissive epigenetic landscape which is not required until a later developmental or physiological time point, temporally uncoupling the presence of these factors with their phenotypic effects. One classic example of epigenetic priming is in the context of bivalent chromatin, found in pluripotent stem cells and early embryos at key developmental gene promoters marked by both activating-associated H3K4me3 and repressive-associated H3K27me3 histone modifications. It is currently unknown how these bivalent domains are targeted, or precisely how they impact on lineage commitment. Here we show that the small heterodimerising non-enzymatic DNA binding proteins Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) act as epigenetic priming factors to establish bivalency at a subset of developmental genes. Dppa2/4 localise to the +1 nucleosome position of bivalent genes and while they are not required for pluripotency in embryonic stem cells (ESCs), double knockout cells fail to exit pluripotency and to differentiate efficiently, with delays in upregulating bivalently marked lineage genes. Proteomics reveal that Dppa2/4 interact on chromatin with members of the COMPASS and Polycomb complexes important for H3K4me3 and H3K27me3 deposition, respectively. Epigenetic profiling reveals a striking loss of H3K4me3, H3K27me3, and their associated enzymatic machinery at a significant subset of bivalent promoters in Dppa2/4 mutants, in addition to loss of H2A.Z and chromatin accessibility. In wild-type ESCs, these “Dppa2/4-dependent” bivalent promoters are characterised by low H3K4me3 enrichment and breadth, near-absent expression levels and initiating but not elongating RNA polymerase. Notably, Dppa2/4-dependent promoters are less evolutionarily conserved suggesting that they lack additional safeguard measures to maintain bivalency at these genes in the absence of Dppa2/4. Concomitantly with the loss of bivalency, Dppa2/4-dependent bivalent promoters gain DNA methylation and consequently are no longer able to be effectively activated upon ESC differentiation, leading to defects in cell fate acquisition. Our findings reveal a targeting principle for bivalency to developmental gene promoters poising them for future lineage specific gene activation.
2020-05-26 | PXD014981 | Pride
Project description:Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity. Traditionally, trimethylation of histone H3K4 (H3K4me3) is associated with transcriptional initiation, whereas trimethylation of H3K27 (H3K27me3) is transcriptionally repressive. The apparent juxtaposition of these opposing marks, termed “bivalent domains”, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.
2021-09-10 | GSE183155 | GEO
Project description:Bivalent chromatin refers to the simultaneous occurrence of transcription activation (H3K4me3)- and repression (H3K27me3)-associated histone modifications at gene promoters. This mark was first identified in ES cells and proposed to maintain genes in a poised state for future resolution to fully-active (H3K4me3-only) or fully-repressed (H3K27me3-only) states. In this report we rigorously test the poising hypothesis using a well-established developmental paradigm of hematopoietic stem cell differentiation to T lymphoid lineage committed cells. We show that bivalent chromatin is generated and resolved at specific stages of hematopoiesis. The epigenetic states from which it is generated and the states to which it is resolved vary with developmental stage, suggesting that bivalency serves different functions at each stage. Moreover, singly-marked genes do not transition to the opposing univalent state via a bivalent intermediate.
2018-12-31 | GSE43428 | GEO
Project description:Trimethylation of histone 3 lysine 4 (H3K4me3) at promoters of actively transcribed genes is a universal epigenetic mark and a key product of Trithorax-Group action. Here we show that Mll2, one of the six Set1/Trithorax-type H3K4 methyltransferases in mammals, is required for trimethylation of bivalent promoters in mouse embryonic stem cells. Mll2 is bound to bivalent promoters but also to most active promoters, which do not require Mll2 for H3K4me3 or mRNA expression. In contrast, the Set1 complex (Set1C) subunit Cxxc1 is primarily bound to active but not bivalent promoters. This indicates that bivalent promoters rely on Mll2 for H3K4me3 whereas active promoters have more than one bound H3K4 methyltransferase including Set1C. Removal of Mll1, sister to Mll2, had almost no effect on any promoter unless Mll2 was also removed indicating functional back-up between these enzymes. Except for a subset, loss of H3K4me3 on bivalent promoters did not prevent responsiveness to retinoic acid thereby arguing against a priming model for bivalency. In contrast, we propose that Mll2 is the pioneer trimethyltransferase for promoter definition in the naM-CM-/ve epigenome and Polycomb-Group action on bivalent promoters blocks premature establishment of active, Set1C bound, promoters. ChIP-Seq to study MLL2 function using H3K4me3 (12 samples), H3K27me3 (4 samples), Pol2 (1 sample) or GFP (7 samples) antibody, and 6 RNA-Seq profiles
2014-01-20 | E-GEOD-52071 | biostudies-arrayexpress
Project description:Background: Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes. Results: Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status. Conclusions: We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells.
2016-09-13 | GSE86838 | GEO
Project description:Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1 which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis. H3K4me3, H3K27me3, H3K9me3, SETDB1, MBD1, and Pol II ChIP-seq. m5CpG pull-down using recombinant MBD domain of MBD1 followed by next-generation sequencing.
2015-11-19 | E-GEOD-73432 | biostudies-arrayexpress
Project description:Trimethylation of histone 3 lysine 4 (H3K4me3) at promoters of actively transcribed genes is a universal epigenetic mark and a key product of Trithorax-Group action. Here we show that Mll2, one of the six Set1/Trithorax-type H3K4 methyltransferases in mammals, is required for trimethylation of bivalent promoters in mouse embryonic stem cells. Mll2 is bound to bivalent promoters but also to most active promoters, which do not require Mll2 for H3K4me3 or mRNA expression. In contrast, the Set1 complex (Set1C) subunit Cxxc1 is primarily bound to active but not bivalent promoters. This indicates that bivalent promoters rely on Mll2 for H3K4me3 whereas active promoters have more than one bound H3K4 methyltransferase including Set1C. Removal of Mll1, sister to Mll2, had almost no effect on any promoter unless Mll2 was also removed indicating functional back-up between these enzymes. Except for a subset, loss of H3K4me3 on bivalent promoters did not prevent responsiveness to retinoic acid thereby arguing against a priming model for bivalency. In contrast, we propose that Mll2 is the pioneer trimethyltransferase for promoter definition in the naïve epigenome and Polycomb-Group action on bivalent promoters blocks premature establishment of active, Set1C bound, promoters.
2014-01-20 | GSE52071 | GEO
Project description:Ketone 3-hydroxybutyrate (3OHB) has been proved to be a neuroprotective endogenous molecule in multiple neurodegenerative diseases, but the detailed mechanisms of action has not been fully uncovered. Here, we employed a quantitative proteomics approach to assess the global protein expression pattern of neuron cells which were incorporated with 3OHB. While 3OHB did not derive dramatic proteome pattern fluctuation in HT22 cells, a big portion of the proteome alteration are found to be 3OHB specific responses (including: cell metabolism, protein acetylation, cognition, neurodegeneration diseases). Combining with disease related protein-protein interaction network analysis, we successfully pinpointed a hub marker, Histone lysine 27 trimethylation (H3K27me3), which has been widely studied in multiple neurodegenerative diseases, but still hasn’t been connected to 3OHB derived neuroprotective effects yet. In addition, our data suggested that the co-occurrence of H3K4me3 and H3K27me3 which was defined as chromatin bivalency referring “poised” transcriptional state could be perturbed by 3OHB, since both H3K4me3 and H3K27me3 were showing sensitive response to 3OHB. Integrative transcriptomics and epigenomics analysis highlighted bivalent transcription factors may play critical roles in 3OHB derived disease protection and alteration of neuronal development processes. To further validate and explore the possible scenario, we performed transcriptomics profiling of 3OHB perturbation upon neural differentiation process. The gene set enrichment analysis has shown that 3OHB could impair the fate decision of neural precursor cells by repressing and promoting their differentiation and proliferation related biological processes, respectively. Another interesting phenomenon is that two of relative high abundant Histone lysine hydroxybutyrylation sites (H2AK118bhb, H2BK34bhb) also responded to 3OHB fulguration. Those two sites are happen to be the most important monoubiquitylation sites that play critical role in regulating of H3K27 methylation and H3K4 (and K79) methylation, respectively. One may speculate that H2AK118bhb and H2BK34bhb involve in or even derive chromatin bivalency alteration upon 3OHB perturbation in neural systems.Taking together, our study provides a novel scenario for deciphering the 3OHB and its mechanisms of action in neural systems both in neurodegeneration disease pathogenesis and neural development process.
2023-03-10 | PXD008053 | Pride