Project description:The control of cell identity is orchestrated by transcriptional and chromatin regulators in the context of specific chromosome structures. With the recent isolation of human naive embryonic stem cells (ESCs) representative of the ground state of pluripotency, it is possible to deduce this regulatory landscape in one of the earliest stages of human development. Here we generate cohesin ChIA-PET chromatin interaction data in naive and primed human ESCs and use it to reconstruct and compare the 3D regulatory landscapes of these two stages of early human development. The results reveal shared and stage-specific regulatory landscapes of topological domains and their subdomains, which consist of CTCF-CTCF/cohesin loops and enhancer-promoter/cohesin loops. The enhancer-promoter loop data reveal that genes with key roles in pluripotency are nearly always regulated by one or more super-enhancers, and show that these genes tend to occur in insulated neighborhoods. Our results reveal the key features of the 3D regulatory landscape of early human cells that form the foundation for embryonic development. ChIA_PET data against SMC1 from naive and primed human embroynic stem cells.
Project description:The control of cell identity is orchestrated by transcriptional and chromatin regulators in the context of specific chromosome structures. With the recent isolation of human naive embryonic stem cells (ESCs) representative of the ground state of pluripotency, it is possible to deduce this regulatory landscape in one of the earliest stages of human development. Here we generate cohesin ChIA-PET chromatin interaction data in naive and primed human ESCs and use it to reconstruct and compare the 3D regulatory landscapes of these two stages of early human development. The results reveal shared and stage-specific regulatory landscapes of topological domains and their subdomains, which consist of CTCF-CTCF/cohesin loops and enhancer-promoter/cohesin loops. The enhancer-promoter loop data reveal that genes with key roles in pluripotency are nearly always regulated by one or more super-enhancers, and show that these genes tend to occur in insulated neighborhoods. Our results reveal the key features of the 3D regulatory landscape of early human cells that form the foundation for embryonic development. ChIP-seq data from naive and primed human embroynic stem cells.
Project description:The control of cell identity is orchestrated by transcriptional and chromatin regulators in the context of specific chromosome structures. With the recent isolation of human naive embryonic stem cells (ESCs) representative of the ground state of pluripotency, it is possible to deduce this regulatory landscape in one of the earliest stages of human development. Here we generate cohesin ChIA-PET chromatin interaction data in naive and primed human ESCs and use it to reconstruct and compare the 3D regulatory landscapes of these two stages of early human development. The results reveal shared and stage-specific regulatory landscapes of topological domains and their subdomains, which consist of CTCF-CTCF/cohesin loops and enhancer-promoter/cohesin loops. The enhancer-promoter loop data reveal that genes with key roles in pluripotency are nearly always regulated by one or more super-enhancers, and show that these genes tend to occur in insulated neighborhoods. Our results reveal the key features of the 3D regulatory landscape of early human cells that form the foundation for embryonic development. Polyadenylated RNA-seq from naive and primed human embroynic stem cells.
Project description:Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing, we mapped long-range chromatin interactions associated with RNA polymerase II in three different mouse cell lines and uncovered widespread promoter-centered interactions. These interactions further aggregated into higher-order clusters, in which proximal and distant genes are engaged through enhancer-promoter interactions. Comparative analyses of different cell lines imply that cell specific enhancer interactions are dynamic among different cell specific transcription, and suggest significant enrichment of enhancer-promoter interactions for cell specific manner. Overall, our study provides novel insights into the three-dimensional basis of transcription activity in mouse cells. RNA polymerase II (RNAPII) guided chromatin interactions were discovered by Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study genome-wise the enhancer-promoter interactions. Three cell lines, namely mouse embryonic stem cell E14, Neural stem cell NS5 and neuroshpere cells were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al, 2009. The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al, 2010)
Project description:Recently, various groups managed to isolate naïve human embryonic stem cell (hESC) state in vitro has come into acceptance. However, a thorough epigenetic characterization of this human ground state, defined as a state without any epigenetic restrictions, and how that compares to mouse is currently lacking. Also, the epigenomic remodeling required to obtain the ground state, and the important transient processes occurring during the remodeling, have remained elusive in human. Here, we address these issues by using an untargeted mass spectrometry-based (MS) approach to profile the histone epigenome in a time-resolved experimental design. Special care was given to defining the naïve hESC state that was reached over 12 passages (P12, 37 days) in feeder-free conditions in this study. We found that conversion is a multi-staged process with a prominent cellular disturbance after stimulation (P3), an increase in cell proliferation between P3 and P6 and a naïve cell state stabilizing between P9 and P12. In total, 20 different histone post-translational modifications (hPTMs) changed significantly over time from primed to naïve hESCs. Most notably, H3K27me3 is the most prominently increasing hPTM in naïve hESCs, in line with what we recently described in mouse. Essentially, we present a first roadmap of the changing human histone epigenome from primed to naïve state and emphasize that the overlap with mouse hints at a conserved Mammalian epigenetic signature of the ground state.
Project description:Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2,264 SMC1A interactions, of which 1,491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue- specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues, but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin- associated interactions in the genome and highlight their role in establishing the regulatory architecture of development. Smc1a ChIA-PET, RNA-seq, chromatin state maps (H3K27ac, H3K27me3, H3K4m2), and CTCF and Smc1a binding in mouse embryonic limb bud (E11.5)
Project description:The antibody gene mutator AID promiscuously damages oncogenes and B cell identity genes leading to chromosomal translocations and tumorigenesis. Why non-immunoglobulin loci are susceptible to AID activity is unknown. Here we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome, but are predominantly clustered within super-enhancers. Unexpectedly, in these domains AID deaminates highly active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment. Examination of AID activity in human cells via Single Nucleotide Variant discovery in H3K4me3 ChIP-seq data from 26 MSH2-/-; AIDtg; UGItg, 18 AICDA-/- and 2 unmodified RAMOS clonal populations. Examination of PolII mediated long-range interactions via Chia-PET of RAMOS cells (2 sample). Identification of super-enhancers from H3K27Ac ChIP-Seq data from activated B cells (3 replicates and 1 input control) and RAMOS cells (1 sample and 1 input control), 2 preparations of naive and 2 of germinal center (GC) B cells from human tonsilectomy samples. Mapping of regulatory elements in RAMOS based on H3K4me1 (1 sample) and Nipbl (2 replicates) ChIP-Seq. RNA expression analyses of activated B cells from 3 WT and 3 Il4raU/U mice and RAMOS cells (3 replicates). Mapping of long-range interactions by 4C in activated B cells from a WT and an Il4raU/U mouse with the IL4ra and Il21r locus, respectively, as a viewpoint. Mapping of Super-Enhancers in activated B cells from Il4raU/U and WT control mice (2 samples).
Project description:Tumor suppressor p53 promotes differentiation of human embryonic stem cells (hESCs), but an in-depth understanding of mechanism is lacking. Here, we define p53 functions in hESCs by genome wide profiling of p53 chromatin interactions and intersection with gene expression during early differentiation and in response to DNA damage. During differentiation, p53 targets and regulates a unique collection of genes, many of which encode transcription factors and developmental regulators with chromatin structure poised by OCT4 and NANOG and marked by repressive H3K27me3 in pluripotent hESCs. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in regulation of cell death and cell cycle genes are conserved during both DNA damage and differentiation. Our findings expand the registry of p53 -regulated genes in hESCs and define specific functions of p53 in opposing pluripotency, which are highly distinct from stress-induced p53 response in stem cells. Identification of p53 binding sites in hESC under three conditions : Pluripotent, DNA damaged, Differentiating
Project description:Recent epigenomic studies have predicted thousands of potential enhancers in the human genome. However, there has not been systematic characterization of target promoters for these potential enhancers. Using H3K4me2 as a mark for active enhancers, we identified genome-wide enhancer-promoter interactions in human CD4+ T cells. Among the 6,520 long-distance chromatin interactions, we identify 2,067 enhancers that interact with 1,619 promoters and enhance their expression. These enhancers exist in accessible chromatin regions and are associated with various histone modifications and Pol II binding. The promoters with interacting enhancers are expressed at higher levels than those without interacting enhancers and their expression levels are positively correlated with the number of interacting enhancers. Interestingly, interacting promoters are co-expressed in a tissue-specific manner. We also find that chromosomes are organized into multiple levels of interacting domains. Our results define a global view of enhancer-promoter interactions and provide a dataset to further understand mechanisms of enhancer targeting and long-range chromatin organization. Two biological replicates of ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) experiment in CD4+ T cells