Project description:The ground state of pluripotency is defined as a basal proliferative state free of epigenetic restriction, represented by mouse embryonic stem cells (ESCs) cultured with two kinase inhibitors (so-called “2i”). Through comparison with serum-grown ESCs, we identify epigenetic features characterizing 2i ESCs by proteome profiling of chromatin including post-translational histone modifications. The most prominent difference is H3K27me3 and its enzymatic writer complex PRC2 that are highly abundant on eu- and heterochromatin in 2i ESCs, with H3K27me3 redistributing outside canonical PRC2 targets in a CpG-dependent fashion. Using PRC2-deficient 2i ESCs, we identify epigenetic crosstalk with H3K27me3, including significant increases in H4 acetylation and DNA methylation. This suggests that the unique H3K27me3 configuration protects 2i ESCs from preparation to lineage priming. Interestingly, removal of DNA methylation in PRC2-deficient 2i ESCs lacking H3K27me3 using 5-azacytidine hardly affected ESC viability and transcriptome, showing that ESCs are independent of both major repressive epigenetic marks.
Project description:Genome-wide analysis of histone modification (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3), protein-DNA binding (TAF1, P300, Pou5f1 and Nanog), cytosine methylation and transcriptome data in mouse and human ES cells and pig iPS cells We generated histone modification data (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3) and protein-DNA binding data (TAF1, P300, Pou5f1 and Nanog) using Chromatin Immunoprecipitation followed by short sequencing (ChIP-seq), cytosine methylation data using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq), transcriptome data with RNA short sequencing (RNA-seq) in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation. Examination of 8 histone modifications, 4 protein-DNA binding, cytosine methylation and transcriptome in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation.
Project description:The conversion of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPS) by forced expression of Oct4, Sox2 and Klf4 is among the earliest demonstrations of reprogramming to a pluripotent state by forced expression of transcription factors. To gain insights into the chromatin state of genes required for reprogramming, we profiled H3K4me3, H3K27me3 and H3K9me3. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. ChIP was performed using an antibody against H3K4me3, H3K27me3 and H3K9me3.
Project description:DNA methylation and the Polycomb Repression System are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. By genome-wide mapping of the Polycomb Repressive Complex 2 (PRC2)-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and PRC2 from Polycomb-target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining PRC2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease. comparison of Dnmt1+/+ vs Dnmt1-/- mouse embryonic fibroblasts by ChIP-seq for H3K27me3 and H3K4me3
Project description:Genome-wide analysis of histone modification (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3), protein-DNA binding (TAF1, P300, Pou5f1 and Nanog), cytosine methylation and transcriptome data in mouse and human ES cells and pig iPS cells We generated histone modification data (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3) and protein-DNA binding data (TAF1, P300, Pou5f1 and Nanog) using Chromatin Immunoprecipitation followed by short sequencing (ChIP-seq), cytosine methylation data using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq), transcriptome data with RNA short sequencing (RNA-seq) in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation.
Project description:Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.
Project description:MBD-affinity purification (MAP) was employed to investigate the DNA methylation status at promoters of mouse embryonic fibroblasts (MEFs), both wild type and Lsh knock-out cells.<br>MAP is conceptually identical to ChIP, using an affinity column rather than an antibody.