Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. DamID experiment was performed in human breast cancer cell lines (MCF-7 and MDA-MB-231) in triplicate. Samples were hybridized to NimbleGen Human Whole-Genome Tiling Arrays (0701_HG18_TILE_05_HX1 and 100718_HG18_TILE_05_HX1). MBD3-Dam material was hybridized over Dam-only control.
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. DamID experiment was performed in human breast cancer cell lines (MCF-7 and MDA-MB-231) in duplicate. Samples were hybridized to Nimblegen 2.1M Deluxe promoter array. MBD3-Dam material was hybridized over Dam-only control.
Project description:A large body of literature suggests that local features of chromatin are crucial in determining functional properties of underlying DNA. The extent to which DNA sequence plays an active role in the establishment and maintenance of patterns of histone and DNA modification at promoters in cells and tissues in an adult animal remains poorly understood. Likewise, whether passage through development is required for refinement of transcriptional states and chromatin marks characteristic of fully differentiated adult cells remains unclear. Here we undertook analysis of gene expression along with the genomic distribution of DNA methylation and a set of histone marks in primary tissues derived from different primordial germ layers in adult mice. We find that promoter CpG content correlated strongly with specific repressive mechanisms. Likewise, RNA polymerase II occupancy and active histone marks were omnipresent on CpG rich promoters regardless of transcriptional output and of cell type. However, these same chromatin features cleanly demarcate transcriptional activity on promoters with low CpG content. While these observations imply an instructive role for DNA sequence in establishment and maintenance of epigenetic states, experimental evidence presented here also suggests an additional role for development in refining these features at individual promoters. DNA methylation and a set of histone marks (H3K4me3, H3K9me2 and H3K27me3) were examined in B cells and liver using Nimblegen mouse promoter arrays.
Project description:DNA methylation profiling of human B cell populations representing a developmental series before and after immune activation. The B cells are derived from inflamed tonsils. The B cells were already activated in vivo in patients, therefore there was no need to stimulate the B cells after isolation. Gene expression profiling was also performed from the same samples. Four B cell subsets: Naïve, germinal center B cells (GC), memory B cells, and plasma cells (PC) were purified by FACS ex vivo from tonsils of 8 human subjects. Methylated CpG island Recovery assay (MIRA) was utilized to enrich for methylated DNA fragments from each sample for microarray analysis. *** This Series reports DNA methylation profiling data. ***
Project description:Similar to other genotoxic polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) is known to produce stable DNA adducts and other DNA damage. BaP is a powerful carcinogen and can induce tumors in the accessory sex organs (including prostate) of rodents. In the present study, we have investigated the potential role of BaP as an epimutagen in vivo during chemical carcinogenesis. We have analyzed the DNA methylation profile, genome-wide, of seminal vesicles of Big Blue® mice exposed to chronic doses of BaP over a period of 6 weeks, both immediately after the termination of exposure and several weeks after-treatment in mice that have developed tumors on the accessory sex organs. Mice treated with chronic doses of DMSO (sham) were used as control. We have used a genome-wide microarray-based approach to catalogue the DNA methylation profile in the seminal vesicles of mice injected intraperitoneally with chronic doses of BaP (experimental) or DMSO (control).
Project description:Elevated DNA methylation in the first intronic region of the BCL6 locus in B cell lymphomas enforces transcription of the BCL6 gene Promoter tiling arrays were used to investigate the distribution of DNA methylation near the promoter region of BCL6 locus Comparison of DNA methylation at promoter regions of lymphoma (Raji) and myeloma (H929) cell lines by methylated CpG island recovery assay (MIRA-chip)
Project description:Epithelial to mesenchymal transition (EMT) is an extreme example of cell plasticity, important for normal development, injury repair, and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during TGF-β-mediated EMT. While DNA methylation was unchanged during EMT, we found global reduction of the heterochromatin mark H3-lys9 dimethylation (H3K9Me2), increase of the euchromatin mark H3-lys4 trimethylation (H3K4Me3), and increase of the transcriptional mark H3-lys36 trimethylation (H3K36Me3). These changes were largely dependent on lysine-specific deaminase-1 (LSD1), and LSD1 loss-of-function experiments showed marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping revealed that chromatin changes were largely specific to large organized heterochromatin K9-modifications (LOCKs), suggesting that EMT is characterized by reprogramming of specific chromatin domains across the genome. Chromatin immunoprecipitation (ChIP) was performed with antibodies against H3K9Me2 (Abcam, ab1220), H3K36Me3 (ab9050), and H3K4Me3 (ab8580) on native (unfixed) chromatin isolated from fully differentiated mouse AML12 cells (confluent, serum starved for 48hrs) either treated with TGF-β for 36hrs to induce EMT or not treated with TGF-β (0hrs, differentiated AML12 cells). DNA purified from these samples was then either whole-genome amplified (H3K36Me3 and H3K4Me3) and hybridized or directly (H3K9Me2) hybridized to NimbleGen 2.1M economy whole-genome tiling arrays #2 (listed below as array 1) and #3 (listed below as array 2), which cover mouse chromosomes 4-14. For each sample, the immunoprecipitated DNA (IP) and the input (control) DNA were hybridized to the arrays, and the IP is normalized to the input. There are 16 total samples listed below. There are 8 sample for H3K9Me2 (TGF-β and no TGF-β for arrays 1 and 2, done in replicate for a total of 8). There are 4 samples for H3K36Me3 (TGF-β and no TGF-β done on array 1 and array 2). There are 4 total samples for H3K4Me3 (TGF-β and no TGF-β done on array 1 and array 2).
Project description:Higher eukaryotes must adapt a totipotent genome to specialized cell types with a stable but limited repertoire of functions. One potential mechanism for lineage restriction is changes in chromatin, and differentiation-related chromatin changes have been observed for individual genes. We have taken a genome-wide view of histone H3 lysine-9 dimethylation (H3K9Me2). We find that differentiated tissues exhibit surprisingly large K9-modified regions (up to 4.9 Mb), that are highly conserved between human and mouse, and differentiation-specific, covering only ~4% of the genome in undifferentiated mouse embryonic stem (ES) cells, compared to 31% in differentiated ES cells, ~46% in liver and ~10% in brain. They require histone methyltransferase G9a, and are inversely related to expression of genes within them, and we term them Large Organized Chromatin K9-modifications (LOCKs). LOCKs are are substantially lost in cancer cell lines, and they may provide a cell type-heritable mechanism for phenotypic plasticity in development and disease. Chromatin was isolated without sonication or formalin cross linking, by digested with micrococcal nuclease, and then chromatin immunoprecipitation was performed with an antibody specific to H3K9Me2. ChIP and input DNA were amplified, labeled by Cy5 and Cy3 respectively, and hybridized to NimbleGen tilling arrays.