Project description:We report a comprehensive analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line.
Project description:We report a comprehensive analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line.
Project description:We report an in-depth analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors, and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line.
Project description:We report a comprehensive analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line.
Project description:Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells (chip-seq gradient)
Project description:Heat shock rapidly induces expression of a small set of genes while globally repressing transcription, making it an attractive system for studying alterations in the chromatin landscape that accompany changes in gene regulation. We have characterized these changes using low-salt extraction of intact micrococcal nuclease (MNase)-treated Drosophila S2 cell nuclei to determine the active nucleosomal and subnucleosomal chromatin landscapes. The low-salt-soluble fraction corresponds to classical "active" chromatin and includes distinct size fractions of MNase-protected particles that can be precisely mapped by paired-end sequencing. After heat shock, the distribution of low-salt-soluble nucleosomes showed an overall reduction over gene bodies, consistent with down-regulation of transcription. No global changes were detected in the subnucleosomal landscape upstream of transcriptional start sites, however, we observed a genome-wide reduction of paused RNA Polymerase II from the active chromatin fraction. Furthermore, nucleosome turnover decreased within gene bodies in a pattern similar to that observed when transcription elongation was artificially inhibited. These observations suggest that reduced Pol II affinity and processivity is the dominant nuclear mechanism for genome-wide repression during heat shock. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from classical active chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization. High-throughput sequencing (Illumina HiSeq 2000) We have characterized changes to the active nucleosomal and subnucleosomal landscape during the heat shock response in Drosophila cells by genome-wide profiling of low-salt extracted micrococcal nuclease-treated nuclei, paused RNA Polymerase II and CATCH-IT nucleosome turnover.
