Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:Eukaryotic transcription factors (TFs) are key determinants of gene activity, yet they bind only a fraction of their corresponding DNA sequence motifs in any given cell type. Chromatin has the potential to restrict accessibility of binding sites; however, in which context chromatin states are instructive for TF binding remains mainly unknown. To explore the contribution of DNA methylation to constrained TF binding, we mapped DNase-I-hypersensitive sites in murine stem cells in the presence and absence of DNA methylation. Methylation-restricted sites are enriched for TF motifs containing CpGs, especially for those of NRF1. In fact, the TF NRF1 occupies several thousand additional sites in the unmethylated genome, resulting in increased transcription. Restoring de novo methyltransferase activity initiates remethylation at these sites and outcompetes NRF1 binding. This suggests that binding of DNA-methylationsensitive TFs relies on additional determinants to induce local hypomethylation. In support of this model, removal of neighbouring motifs in cis or of a TF in trans causes local hypermethylation and subsequent loss of NRF1 binding. This competition between DNA methylation and TFs in vivo reveals a case of cooperativity between TFs that acts indirectly via DNA methylation. Methylation removal by methylation-insensitive factors enables occupancy of methylation-sensitive factors, a principle that rationalizes hypomethylation of regulatory regions. DNase-seq (2 replicates) in mouse embryonic stem cells with (WT) and without DNA methylation (DNMT TKO). RNA-seq (3 replicates) in WT and DNMT TKO cells and in DNMT TKO cells after treatment with control siRNA or siRNA targeting Nrf1. H3K27ac ChIP-seq (2 replicates) in WT and DNMT TKO cells. NRF1 ChIP-seq (2 replicates) in WT and DNMT TKO cells, in WT upon culture in different conditions (adaptation to 2i and back to serum), upon transient overexpression of NRF1 and after differentiation into neuronal progenitor cells (NP). Whole-genome bisulfite sequencing in DNMT TKO cells and in WT upon culture in different conditions (adaptation to 2i and back to serum). NRF1 ChIP-seq (2 replicates) in human HMEC and HCC1954 cells.
Project description:ChIP-Seq experiments targeting H3K4me2, H3K4me3, H3K9ac, H3K27ac, H3K36me3 histone modifications have been performed in order to assess if SETBP1 binding to gDNA was associated with chromatin remodeling and to further characterize the mechanisms responsible for SETBP1-mediated transcriptional regulation
Project description:Global gene expression changes induced by Setbp1 and Setbp1(D/N) in purified mouse hematopoietic stem and progenitor cells were characterized by RNA-seq analysis; ChIP-seq studies to identify cooperation between SETBP1/SETBP1(D/N) and MLL1 in regulating gene transcription in hematopoietic stem and progenitor cells.