Project description:The rapidly expanding information on the structural and functional characteristics of the human genome allows the development of genome-wide approaches to investigate the molecular circuitry wiring the genetic and epigenetic programs of clinically relevant stem/progenitor cells. Here, we define the transcriptional and epigenetic profile of human hematopoietic stem/progenitor cells (HSPC) and their committed, early myeloid and erythroid progeny. Cap Analysis of Gene Expression showed that the transcriptional state is largely maintained in the transition from early hematopoietic stem/progenitors to committed progenitor/precursors. A large fraction of differentially expressed active promoters in each cell type were classified as novel, possibly driving the expression of transcripts involved in HSPC commitment. Unannotated promoters harbored an enhancer chromatin signature, were enriched in cell-specific transcription factor binding sites, and were close to protein-coding cell-specific genes. To obtain a genome-wide description of regulatory regions, we performed ChIP-seq for histone modifications typical of promoters, enhancers and super-enhancers. ChIP-defined promoters were shared by HSPC and committed cells, whereas enhancer usage consistently changed upon commitment, thus indicating that the fine regulation of commitment is carried out by enhancer elements. The expression level of genes targeted by enhancers and super-enhancers was moderately higher in cells actively using these regulatory elements. However, functional annotation of targeted genes showed a low enrichment for cell-specific gene ontology categories. Finally, we used Moloney leukemia virus (MLV) to map functional regions of chromatin. Genomic regions targeted by MLV co-mapped with a fraction of epigenetically defined active regulatory elements, with two-third of MLV integrations falling in enhancers, and one-third in promoters. Most of MLV-targeted regulatory regions were differentially used between HSPC and their committed progeny. Analysis of the expression levels of promoters close to cell-specific MLV-targeted enhancers and functional assays indicated that MLV-defined regulatory regions are bona fide cell-specific enhancers. Finally, gene ontology analyses showed that MLV-targeted genes related to the peculiar cell identity. Overall, this study provided an overview of the differential transcriptional and epigenetic programs of HSPC and committed progenitors and represents a unique source of regulatory regions involved in HSPC commitment.

Project description:Genome-wide mapping of transcriptional regulatory elements are essential tools for the understanding of the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of nascent, Pol-II-transcribed RNAs by Cap Analysis of Gene Expression (CAGE-Seq) with genome-wide profiling of histones modifications by chromatin immunoprecipitation (ChIP-seq) to map active promoters and enhancers in a model of human neural commitment, represented by embryonic stem cells (ESCs) induced to differentiate into self-renewing neuroepithelial-like stem cells (NESC). We integrated CAGE-seq, ChIP-seq and gene expression profiles to discover shared or cell-specific regulatory elements, transcription start sites and transcripts associated to the transition from pluripotent to neural-restricted stem cell. Our analysis showed that >90% of the promoters are in common between the two cell types, while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a “bivalent” histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provide a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and gene expression programs occurring in the transition from a pluripotent to a neural-restricted cell fate. Investiagtion of promoters usage changes during ESCs neural induction ESCs and NESCs promoter usage profiling by CAGE-seq

Project description:Genome-wide mapping of transcriptional regulatory elements are essential tools for the understanding of the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of nascent, Pol-II-transcribed RNAs by Cap Analysis of Gene Expression (CAGE-Seq) with genome-wide profiling of histones modifications by chromatin immunoprecipitation (ChIP-seq) to map active promoters and enhancers in a model of human neural commitment, represented by embryonic stem cells (ESCs) induced to differentiate into self-renewing neuroepithelial-like stem cells (NESC). We integrated CAGE-seq, ChIP-seq and gene expression profiles to discover shared or cell-specific regulatory elements, transcription start sites and transcripts associated to the transition from pluripotent to neural-restricted stem cell. Our analysis showed that >90% of the promoters are in common between the two cell types, while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a “bivalent” histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provide a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and gene expression programs occurring in the transition from a pluripotent to a neural-restricted cell fate. Genome-wide mapping of H3K4me1 and H3K4me3 in NESCs ChIP-seq for H3K4me1 and H3K4me3 in NESCs

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Genome-wide definition of regulatory elements in hematopoietic stem cell differentiation [RNA-seq (CAGE)]

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