Project description:We quantified the targets and kinetics of DNA methylation acquisition in mouse embryos, and determined the contribution of the de novo methyltransferases DNMT3A and DNMT3B to this process. We provide single-base maps of cytosine methylation by RRBS from the blastocysts to post-implantation stages and in embryos lacking DNMT3A or DNMT3B activity, and performed RNA-Seq in embryos lacking DNMT3B activity. We sequenced RRBS libraries prepared from genomic DNA isolated from embryos at consecutive stages of development between E3.5 and E11.5,and adult differentiated cells (sperm, liver). We performed RRBS on blastocysts at E3.5/E4.5, dissected epiblasts at E5.5/E6.5/E7/5, whole embryos at E8.5/E10.5 and limbs at E11.5. RRBS experiments in Dnmt3a-/- and Dnmt3b-/- embryos were performed in biological duplicates on individual embryos. We sequenced RNA-Seq libraries prepared from total RNAs of three WT and Dnmt3b-/- littermate embryos collected at E8.5.
Project description:Histone modifications and DNA methylation represent two distinct modes of varying epigenetic landscapes, but whose exact interrelationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation (H3K4me3) inhibits the binding of de novo DNA methyltransferases (Dnmt) through the ATRX-DNMT3-DNMTL (ADD) domain, thus protecting H3K4me3 marked CpG islands (CGI) from DNA methylation. In addition to H3K4me3, we identified antagonistic relationship between H3T3 phosphorylation and the binding of the ADD domain to the unmodified H3 N-terminus. To assess the physiological relevance of these restrictions, we engineered the wild-type ADD domain of Dnmt3a (WT) to permit additional binding to either H3K4me3 (WWD) or H3T3ph (R) and stably introduced FLAG-tagged, full-length normal or mutant Dnmt3a2 into ESCs lacking all Dnmts (TKO; triple knock-out of Dnmt1, Dnmt3a, and Dnmt3b) using the PiggyBac transposon system. For each WT-, WWD-, and R-Dnmt3a2, we generated bulk and clonally-derived ESC lines. We then employed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify the genomic distribution of full-length WT-, WWD-, R-Dnmt3a2, and the H3K4me3 distribution. In parallel, we quantitatively measured genome-wide CpG (cytosine) methylation at base-pair resolution using an enhanced form of reduced representation bisulfite sequencing (RRBS), and performed RNA-seq to assess transcription in matched ESC lines. Examination of DNA methylation levels in Dnmt TKO-ESCs expressing wild-type/mutant Dnmt3a2.
Project description:This data includes regulatory factor profiling using DNase and ChIP-seq and methylation profiling using bisulfite-seq. We investigated CTCF occupancy in the context of reduced methylation by performing genome-wide profiling with chromatin immunoprecipitation (ChIP-seq) in HCT116 cells and DNMT1 and DNMT3B double knockout (DKO) HCT116 cells. We also profiled HCT116 and DKO using DNaseI-seq and ChIP-seq for trimethylation of histone 3 lysine 4 (H3K4me3) and acetylation of histone 3 lysine 27 (H3K27ac), Finally, we performed ChIP-seq on 3 replicates of mock-treated and 2 replicates of 5-aza-CdR-treated K562 cells.
Project description:Maternal and fetal monocytes and tissue macrophages (decidual macrophages, Hofbauer cells) at the feto-maternal interface have different methylome. Paired and balanced design. We compared maternal blood monocytes (MB) vs. cord blood monocytes (CB), maternal blood monocytes (MB) vs. decidual macrophages (Deci), cord blood monocytes (CB) vs placental macrophages (villi) and decidual macrophages (Deci) vs. placental macrophages (villi).
Project description:The methylation status of colon epithelial cells was profiled in wild type mice and mice expressing a Dnmt3b transgene. Genome-scale methylation profiles were generated using reduced representation bisulfite sequencing (RRBS), with CpG methylation scored by promoter and also summarized by gene. Dnmt3b expression is associated with a strong increase in de novo methylation of a discrete subset of "methylation sensitive" genes which show a strong concordance with genes methylated in human colon cancer. These results, together with further analysis, indicate that colon epithelial cell methylation in the Dnmt3b mouse model predicts DNA methylation of human colon cancer with high confidence. Three each of Dnmt3b-induced and control mice, each split into two fractions.
Project description:Arabidopsis shoot apical meristem (SAM) stem cells were labelled with a CLV3:H2B-mCherry reporter. This reporter allowed fluorescence activated nuclear sorting (FANS) of nuclei of stem cells at 2 different developmental stages: 14d old seedling and 5 weeks old seedlings. DNA was extracted, bisulfite-converted and sequenced on an illumina HiSeq 2500 platform.