Project description:This experiment aimed at investigating how O-GlcNac occupancy sites are impacted by RNA Polymerase II removal upon doxycycline stimulation. These human colon adenocarcinoma DLD-1 cells express OsTIR and a cassette encoding mini-AID (mAID) and fluorescent protein mClover (mAID+mClover) at the initiation site of the endogenous Rpb1 gene locus (POLR2A) (Nagashima 2019).
Project description:This experiment uses a transgenic cell line expressing bacterial OGA BtGH84 fused to a localization peptide (NLS) and regulated by Tet-On system. OGA is a glycosidase that removes O-GlcNAc modifications. We evaluated the changes in chromatin openness before and after O-GlcNac removal by OGA.
Project description:This experiment uses a transgenic cell line expressing bacterial OGA BtGH84 fused to a localization peptide (NLS) and regulated by Tet-On system. OGA is a glycosidase that removes O-GlcNAc modifications. We evaluated the changes in gene expression before and after O-GlcNac removal by OGA.
Project description:The established hierarchical model explaining co-occupancy of Polycomb repressor complexes 1 and 2 (PRC 1 and 2) at target loci proposes that the chromodomain of the polycomb protein, a core PRC1 subunit, recognises the H3K27me3 histone modification catalysed by PRC2. We used chromatin immunoprecipitation to analyse PRC1 occupancy at target loci in Eed-/- mouse embryonic stem cells (ESCs) that lack H3K27me3. Occupancy of the core PRC1 proteins Ring1B and Mel18 was strongly reduced, consistent with the hierarchical model. However, levels of H2A ubiquitylation (H2AK119u1), the histone modification catalysed by PRC1, were similar to wild-type cells, suggesting PRC1 recruitment is independent of H3K27me3. ChIP-sequencing analysis of Ring1B occupancy genome wide substantiated this conclusion, demonstrating significant Ring1B levels at polycomb target loci in Eed-/- ESCs. Thus PRC1 and PRC2 are recruited independently to sites that they co-occupy. We conclude that the primary function of H3K27me3 is to increase the residency of PRC1 at target loci and thereby to contribute to the stability of PRC1 mediated silencing. Examination of Ring1B binding in WT, Eed ko and Input of ESCs Examination of CBX7 in WT and Eed ko of ESCs
Project description:We performed ChIP-Seq analysis of SOX10, histone H3 lysine 27 acetylation (H3K27ac) and H3K27 trimethylation (H3K27me3) in melanocytes to profile the genomic binding sites of SOX10 and the chromatin landscape. In parallel, we generated Sox10 haploinsufficient cell lines using gene knockout approaches and conducted microarray gene expression analysis to identify functional gene targets of SOX10 transcriptional regulation in melanocytes. We demonstrate that SOX10 predominantly engages “open” chromatin, binds to melanocyte enhancer elements and plays a central role in transcriptional activation and repression of functionally distinct classes of genes. Furthermore, we identified cis-regulatory sequence motifs of putative co-regulatory transcription factors that define SOX10-activated and SOX10-repressed target genes. Our results uncover novel mechanisms and roles of SOX10 in global transcriptional regulation of diverse regulatory pathways in the melanocyte lineage. ChIP-seq profiling of SOX10, H3K27ac, and H3K27me3 in the mouse melanocyte cell line melan-Ink4a-Arf-1 (melan-a).
Project description:To identify the sequences responsible for recruitment of Glucocorticoid receptor (GR) to individual loci, we performed ChIP-seq and ChIP-exo that combines chromatin immunoprecipitation with an exonuclease digestion step. We performed these experiments in three cell lines : IMR90 (ATTC:CCL-186), U2OS osteosarcoma cell lines, K562 (ATCC:CCL243), upon glucocorticoid treatment. The U2OS assays are the same as those in E-MTAB-2731.
Project description:In this experiment, we sought to identify how the distribution of CTCF, H3K27me3, H3K4me3, Pol II, H2AK119Ub1, EZH2 3 lines (WT, Δ5|6, Δ5|6:7|9)