Project description:Loss of INO80 from INO80 canonical bound regions decreases open chromatin. Loss of INO80 did not affect chromatin accessibility at autonomous sites.
Project description:In order to detect the effect of INO80 on chromatin accessibility, we will utilize the Assay for Transposase Accessible Chromatin with highthroughput sequencing (ATAC-Seq).
Project description:<p>In this study we profile the epigenomic enhancer landscapes of CLL B cells (CD19+/CD5+) harvested from peripheral blood of patients from our Center. Included are results of ChIPseq profiling using chromatin immunoprecipitation of the enhancer histone mark H3K27ac (acetylated lysine 27 on histone H3), and open chromatin profiles using ATAC-seq (assay for transposase accessible chromatin). These profiles are used to define the global enhancer and super enhancer landscape of CLL B cells, and to derive active transcription factor networks associated with this disease. Also included are H3K27ac ChIP-seq and ATAC-seq datasets for non-CLL B cells obtained from the peripheral blood of normal adult donors.</p>
Project description:We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500–50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with the nucleosome. Using ATAC-seq maps of human CD4+ T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual’s epigenome on a timescale compatible with clinical decision-making. We examined chromatin structure using ATAC-seq in 2 cell types (GM12878 cell line, purified CD4+ T cells).
Project description:We mapped 4 subunits of the INO80 complex using ChIP-seq in HepG2 and Huh7 liver cancer cell lines. We found a subclass of sites occupied by the INO80 ATPase subunit, but not by any accessory subunits that we call 'Autonomous' INO80 sites. These sites are present in both HepG2 and Huh7 cells and are characterized by repressed chromatin. Relief of reprissive histone modifications thorugh EZH2 inhbiition led to the increase in H3K27ac at INO80 targets.
Project description:We mapped 4 subunits of the INO80 complex using ChIP-seq in HepG2 liver cancer cells. We found a subclass of sites occupied by the INO80 ATPase subunit, but not by any accessory subunits that we call 'Non-canonical' INO80 sites. These sites are characterized by repressed chromatin.
Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs
Project description:Assay for Transposable Accessible Chromatin (ATAC) reveals a genome wide view of areas of open chromatin at very high resolution, which are often associated with regulatory activity. The ATAC-seq technology uses a Tn5 transposase loaded with nex-generation sequencing primers in order to simultaneously fragment areas of open chromatin and ligate adapters.