Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells
ABSTRACT: Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalently marked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking at bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation. ChIP-Seq in mouse embryonic stem (mES) cells for MLL2. ChIP-seq of H3K4me1, H3K4me3 and H3K27me3 for mES cells with RNAi against MLL2(shMLL2) and control (shGFP). ChIP-seq of H3K4me3 in mES cells with RNAi against MLL3 (shMLL3). RNA-seq of mES cells with RNAi against MLL2 and control (shGFP). RNA-seq of control mES cells (shGFP) or MLL2 RNAi mES cells (shMLL2) induced with RA for 6h and 12h.
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. 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
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.
Project description:An immortalized multipotent otic progenitor (iMOP) cell was used to determine H3K4me3 and H3K27me3 histone marks. Promoters containing enriched with both histone marks were considered bivalent promoters. Neurog1 dependent transription was determined in genes with bivalently marked promoters. Overall design: ChIP-Seq was accomplished by immunoprecipitating endogenous H3K4me3 and H3K27me3
Project description:The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and the epigenetic states at cis-regulatory elements. Previously, we reported that Mll2 (KMT2B)/COMPASS is responsible for the implementation of H3K4me3 at promoters of bivalent genes. Here, we show that Mll2/COMPASS can also implements H3K4me3 at some of the non-TSS regulatory elements, a subset of which share epigenetic signatures of active enhancers. Our mechanistic studies reveal that the association of Mll2’s CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of sites, it appears to be essential for the expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development, indicating that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent. Overall design: Characterization of H3K4me3 and Mll2 occupancy by ChIP-seq in mouse embryonic stem cells and identifying their role in gene expression and during differentiation by RNA-seq studies. A high resolution 4C-seq experiments involving two restriction digests (HindIII and NlaIII) were performed to investigate the interaction bewteen promoters of Prdm1 and Prdm14 (viewponts) and cis-regulatory elements whose H3K4me3 is catalyzed by Mll2 in mouse embryonic stem cells.
Project description:Trimethylation of histone 3 lysine 4 (H3K4me3) is classically thought of as a mark of active promoters and yet it occurs at untranscribed domains. Partial redundancy of H3K4 methyltransferases has made it difficult to delineate the mechanisms underlying genomic targeting of H3K4me3. The oocyte provides an attractive system to investigate this, because extensive acquisition of H3K4me3 occurs in a non-dividing cell and ablation of a single H3K4 methyltransferase, Mll2, prevents most H3K4me3. We developed low-input chromatin immunoprecipitation to interrogate promoter associated histone modifications H3K4me3, H3K27ac and H3K27me3 throughout oogenesis. In non-growing oocytes, H3K4me3 was restricted to transcriptionally active promoters, but as oogenesis progresses, H3K4me3 accumulates in a transcription-independent manner: targeted to broad inter-genic regions, putative enhancers, and transcriptionally silent H3K27me3-marked promoters. Consequently, thousands of bivalent domains are established during oogenesis. Ablation of Mll2 resulted in loss of transcription-independent H3K4me3, with limited effects on transcription-coupled H3K4me3 or gene expression. Deletion of Dnmt3a/b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings show that there are two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription. Overall design: We first characterised histone modifications associated with transcriptional regulation (H3K4me3, H3K27ac, and H3K27me3) in oocytes by adapting an ultra-low input native ChIP (ULI-nChIP) sequencing method. This allowed us to obtain high-resolution chromatin maps throughout oogenesis, from non-growing oocytes (NGOs) to fully-grown germinal vesicle (GV) oocytes, and to compare these profiles to gene expression and DNA methylation. Furthermore, this has enabled the first molecular interrogation of H3K4me3 in the conditional Dnmt3a/b and Mll2 KO oocytes.
Project description:TET protein-catalyzed 5mC oxidation not only creates novel DNA modifications such as 5hmC, but also initiates active or passive DNA demethylation. However, the TETs’ function in crosstalk with specific histone modifications is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domain underlying the methylated DNA CpG islands (CGIs). Overexpression of wild type (WT) but not catalytic inactive mutant (Mut) TET2 in TET-low-expressing cells results in increase of 5hmC level and accompanying DNA demethylation at a subset of CGIs. Importantly, this is sufficient to create de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of key developmental gene promoters, which are often located within CpG islands that are previously hyper-methylated and silenced. On the other hand, depletion of Tet1 and Tet2 in mouse ES cells results in an apparent loss of H3K27me3 at bivalent domains, which are located within CGIs and associated with a particular set of key developmental gene promoters. Collectively, these data suggest that TET proteins have a primary role in charge of regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at a subset of CpG islands associated with developmental genes. We examined H3K4me3,H3K27me3,5mC and 5hmC in 293T and mES cell types,using Illumina Hiseq2500.
Project description:Epigenetic regulation of transcription plays a crucial role in lineage commitment of embryonic stem cells. Promoters of key lineage-specific differentiation genes are found in a repressed bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3 in response to environmental cues. Whether the tumour-initiating, self-renewing, cancer-initiating cells (C-ICs) have similar epigenetic regulatory mechanism that prevent lineage commitment is unknown. In order to investigate bivalently marked and repressed promoters, we used a patient-derived CC-IC enriched model to identify the changes in transcriptome following inhibition of EZH2, the H3K27 methyltransferase. We also performed ChIP-seq for H3K27me3 and H3K4me3 at baseline in order to identify repressed and bivalently marked promoters.
Project description:Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes in self-renewal of ESCs. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a 'general facilitator' that generates access for a variety of complexes both activating and repressive. ChIP-Seq in murine embryonic stem (mES) cells for H2A.Z and acetylated H2A.Z. ChIP-Seq of H3K4me3, H3K27me3, RbBP5, SUZ12 and OCT4 for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of RARalpha in H2A.Z knockdown (withdraw of LIF and exposure to RA for 3h) and control cells. MNase-Seq and chromatin accessibility assay using Benzonase digestion followed by next-generation sequencing for mES cells of both H2A.Z RNAi knockdown and shLuc control. ChIP-Seq of H2A.Z and H3K4me3 for mES cells of both MLL4 RNAi knockdown and shLuc control. RNA-Seq for mES cells of H2A.Z knockdown and shluc control. RNA-Seq for embryonic bodies derived from mES cells (H2A.Z knockdown and shLuc control) at day 3 and day 7.
Project description:In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes: the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes. Keywords: cell type comparison Suz12, Ezh2, Ring1b ChIP-Seq in singlicate from mouse embryonic stem (mES) cells. H3K4me3, H3K27me3, H3K36me3, Ezh2 and Ring1b ChIP-Seq in singlicate from human embyonic stem cells (hES; H9).
Project description:Background: Previously we reported extensive gene expression reprogramming during epithelial to mesenchymal transition (EMT) of primary prostate cells. Here we investigated the hypothesis that specific histone and DNA methylations are involved in coordination of gene expression during EMT and early stages of transformation. Results: Genome-wide profiling of histone methylations (H3K4me3 and H3K27me3) and DNA methylation (DNAMe) was applied on a prostate cell model during EMT and malignant transformation. Integrated analyses of promoter epigenetic modifications and gene expression changes revealed strong correlations between the dynamic changes of histone methylations and gene expression. DNA methylation was weakly associated with global gene repression, but strongly correlated to gene silencing when genes co-modified by H3K4me3 were excluded. In genes labeled with multiple epigenetic marks in their promoters, the level of transcription was associated with the net signal intensity of the activating mark H3K4me3 minus the repressive mark H3K27me3 or DNAMe, indicating that the effect on gene expression of bivalent marks (H3K4/K27me3 or H3K4me3/DNAMe) depends on relative modification intensities. Sets of genes, including epithelial cell junction and EMT associated fibroblast growth factor receptor genes, showed corresponding changes concerning epigenetic modifications and gene expression during EMT. Conclusions: This work presents the first blueprint of epigenetic modifications during EMT in prostate cells and shows that specific histone methylations are extensively involved in gene expression reprogramming during EMT and carcinogenesis. The observation that transcription activity of bivalently marked genes depends on the relative labeling intensity of individual marks provides a new view of quantitative regulation of epigenetic modification.