Chromatin States in Human ES Cells Reveal Key Regulatory Sequences and Genes Involved in Pluripotency and Self-renewal
ABSTRACT: Human embryonic stem cells (hESCs) are offering a new therapeutic approach because of their unique ability to proliferate indefinitely in vitro and differentiate into multiple cell types. However, our understanding of the molecular mechanisms of pluripotency and self-renewal remain incomplete. To elucidate the key regulatory sequences and genes responsible for these cellular properties, we have determined potential enhancers and insulators in the genome of human ES cells and examined the dynamics of four key chromatin modifications (H3K4me1, H3K4me3, H3K27ac and H3K27me3) at both promoters and enhancers during the differentiation of these cells. We observe that most enhancers gain or lose H3K4me1 and H3K27ac during differentiation in a manner that correlates with expression of their potential target genes. By contrast, chromatin modifications at promoters remain stable and largely invariant during hESC differentiation, with the exception of a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression. Our results reveal more than 50,000 potential enhancers for early human development, and identify key genes that are involved in differentiation and maintenance of pluripotency in human ES cells. Overall design: ChIP-Seq Analysis of SOX2 and NANOG in hESC H1 cells. 36 cycles of sequencing was done on the Illumina Genome Analyzer II platform.
INSTRUMENT(S): Illumina Genome Analyzer II (Homo sapiens)
Project description:Human embryonic stem cells (hESCs) are offering a new therapeutic approach because of their unique ability to proliferate indefinitely in vitro and differentiate into multiple cell types. However, our understanding of the molecular mechanisms of pluripotency and self-renewal remain incomplete. To elucidate the key regulatory sequences and genes responsible for these cellular properties, we have determined potential enhancers and insulators in the genome of human ES cells and examined the dynamics of four key chromatin modifications (H3K4me1, H3K4me3, H3K27ac and H3K27me3) at both promoters and enhancers during the differentiation of these cells. We observe that most enhancers gain or lose H3K4me1 and H3K27ac during differentiation in a manner that correlates with expression of their potential target genes. By contrast, chromatin modifications at promoters remain stable and largely invariant during hESC differentiation, with the exception of a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression. Our results reveal more than 50,000 potential enhancers for early human development, and identify key genes that are involved in differentiation and maintenance of pluripotency in human ES cells. ChIP-Seq Analysis of SOX2 and NANOG in hESC H1 cells. 36 cycles of sequencing was done on the Illumina Genome Analyzer II platform.
Project description:Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC. Genome wide enrichment profile of 5-hmC in H1 human embryonic stem cells
Project description:We generated maps of H3K4me1, H3K27ac (enhancers), H3K4me3, Pol II (promoters) and H3K27me3 (repressed chromatin) in the genome of human iPSC-derived cardiomyocytes Differentiation of cardiomyocytes from iPSC followed by ChIP-seq of H3K27ac, H34me1, H327me3, H3K4me3 and PolII
Project description:Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function. Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes. Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation. ChIP-Seq for KDM5B, H3K4 methylation and H3K27ac in murine shLuc, shKdm5b, shLuc-LSD1i, shKdm5b-LSD1i ES cells, and during differentiation of shLuc and shKdm5b ES cells for 2 days, 3 days, and 4 days without LIF.
Project description:ChIP-seq was performed using Drosophila Kc167 cells using antibodies against H3K4me3 to identify active promoters and H3K4me1 to identify active enhancers. H3K27ac ChIPseq was performed to identify active promoters and enhancers. Once enhancers and promoters were identified, JIL-1 and histone phosphorylation, H3K9acS10ph and H3K27acS28ph, ChIP-seq was performed to look at binding trends. JIL-1 and phosphoacetlation is found at low levels at inactive enhancers and shows increase at active enhancers and promoters. Here we examine histone phosphorylation by JIL-1 and acetylation of H3K27ac by CBP at transcriptionally active vs. inactive promoters and enhancers. ChIP-seq is performed in Kc167 Drosophila cells using antibodies against JIL-1, H3K27acS28ph, H3K9acS10ph, H3K4me3, H3K4me1, and H3K27ac.
Project description:The Zinc finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Our molecular and biochemical studies have demonstrated that Zic2 to preferentially bind to transcriptional enhancers and functions as a cofactor that interacts with the NuRD complex in ES cells. Detailed genome-wide studies demonstrate that Zic2 function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is dispensable for the selfrenewal of ES cells but is required for proper differentiation, similar to what has been previously reported for Mbd3/NuRD. Our study identifies Zic2 as a key factor in the execution of the pluripotency program with Mbd3/NuRD in ES cells. ChIP-seq of Zic2, Chd4, Mbd3 and Zic3 in mES cells. ChIP-seq of H3K27me3, H3K27ac, H3K4me3, H3K4me1 and PolII in mES cells after Zic2 shRNA and non-targeting shRNA. RNA-seq of mES cells after Zic2, Zic3, Mbd3 and Mta2 shRNA and non-targeting shRNA.
Project description:To understand epigenetic mechanisms underlying CD8 T cell differentiation, we performed ChIP-seq of 4 histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) and ATAC-seq to probe chromatin states and accessibility. We identified subset-specific regulatory elements (enhancers and promoters) from chromatin states and predicted key transcription factors from accessible regulatory regions using ATAC-seq. Overall design: Examination of 4 different histone modifications and open chromatin in 4 cell types.
Project description:The emergence of distinct cell types during embryonic development relies on the ability of progenitor cells to properly interpret environmental cues; yet how this developmental competence is established is unknown. Here we show that epigenetic priming of enhancers signifies developmental competence. Chromatin mapping during endodermal lineage diversification of human pluripotent stem cells revealed en masse acquisition of a poised chromatin state at enhancers for multiple descendant lineages in developmental intermediates. The responsiveness of developmental intermediates to lineage-inductive signals is dependent on a poised enhancer state. We further find that lineage-specific enhancers are first recognized by transcription factors involved in chromatin priming, while subsequent recruitment of lineage-inductive transcription factors leads to enhancer and target gene activation. Together, our results identify acquisition of a poised chromatin state at enhancers as a general mechanism by which progenitor cells gain the competence to rapidly activate lineage-specific genes in response to inductive signals. Overall design: The overall goal of this study was the examine the dynamic changes in the enhancer landscape during endodermal and pancreatic differentiation of hESCs. Specifically we performed ChIP-seq of enhancer related histone modifications (H3K4me1 and H3K27ac) during a five stage differentiation (ES, DE, GT, FG and PE) of hESCs to the pancreatic lineage with two independent biological replicates. To further investigate enhancer activity we performed GRO-seq analysis during the same time course. Additionally, we performed ChIP-seq of the pancreatic regulator PDX1 in hESC derived pancreatic endoderm. To assess its effect on enhancers we also performed H3K27ac ChIP-seq on hESC derived PE transduced with shRNA targeting PDX1 as well as a scrambled control. H3K27ac ChIP-seq was also performed on hESC derived hepatic endoderm using two different protocols to examine enhancer activity during the liver/pancreas lineage decision. Lastly, to investigate the role of pioneer transcription factors at enhancers, we performed ChIP-seq of FOXA1 and FOXA2 during the same five stage pancreatic timecourse.
Project description:The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the non-coding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that: (i) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression; (ii) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog; (iii) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage. ChIP-seq analysis of H3K4me3, H3K4me1 and H3K27ac at four developmental time-points (dome, 80% epiboly, 24hpf and 48pf) of zebrafish embryogenesis.
Project description:Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for the deposition of H3K4me1/2 on enhancers remain elusive. Furthermore, the functions of these methyltransferases on enhancers and associated cell-type-specific gene expression are poorly understood. Here, we identify MLL4 (KMT2D) as a major H3K4 mono- and di-methyltransferase in mammalian cells. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 dramatically decreases H3K4me1/2 and H3K27ac on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Finally, we provide evidence that lineage-determining TFs recruit and require MLL4 to establish enhancers critical for cell-type-specific gene expression. Together, these results identify MLL4 as an H3K4 mono-/di-methyltransferase required for enhancer activation during cell differentiation. ChIP-Seq analyses of C/EBPbeta, MLL4 and histone modifications (H3K4me1, H3K27ac) in vec- or C/EBPbeta-overexpressing, adenoviral GFP- or Cre-infected, MLL3-/-MLL4-flox/flox brown preadipocytes without induction of differentiation.