Project description:PcG protein complex PRC2 is a methyltransferase specific for histone H3 lysine27, and H3K27me3 is essential for stable transcription silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates ~70% of total H3K27. Here we show that H3K27me2 occurs in inverse proportion to transcriptional activity in genes and intergenic regions and its loss results in global transcriptional derepression proportionally greatest in previously silent or weakly transcribed regions. H3K27me2 levels are controlled by opposing roaming activities of PRC2 and the H3K27 demethylase dUTX. Unexpectedly, we find an equally pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub), attributed to the RING1 subunit of PRC1-type complexes. Examination of global changes in five epigenetic marks when E(z) is inactivated in E(z) temperature-sensitive cells at 25°C and 31°C.
Project description:We generated maps of H3K4me1, H3K27ac (enhancers), H3K4me3, Pol II (promoters) and H3K27me3 (repressed chromatin) in the genome of human iPSC-derived cardiomyocytes Differentiation of cardiomyocytes from iPSC followed by ChIP-seq of H3K27ac, H34me1, H327me3, H3K4me3 and PolII
Project description:During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation. Examination of TF binding by ChIP-seq in a CRC cell-line treated with non-targeting siRNA, or siRNA specific to the cohesin subunit RAD21.
Project description:We report how CSB affects globally the density of RNA Pol II at TSS and how this effect correlates with the gene expression alterations observed from microarray analysis. We also show globally the distribution of CSB. RNA Pol II and CSB ChIP-Seq in CS1AN cells and CSB reconstituted wild type cells, in duplicate, using Illumina GAIIx
Project description:Nucleosomal DNA was prepared using Simple ChIP Enzymatic Chromatin IP Kit according to manufacturer’s instruction. Briefly, Nuclei were isolated from purified Ter119 negative or in vitro cultured erythroblasts. Cross-linked native chromatin was then digested with MNase into mononucleosomal DNA. Sequencing libraries were generated from nucleosomal DNA, and sequencing was carried out using the Illumina system according to the manufacturer’s specification. In this study, we purified chromatin from in vitro cultured mouse fetal liver erythroblasts on day 0, day 1, and day 2. The chromatins were digested by micrococcal nuclease to make mononucleosomal products, which were further analyzed by next generation sequencing analysis. We aim to determine the dynamic changes of nucleosome during terminal erythropoiesis.
Project description:In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. Plants homozygous for null mutations in the KRYPTONITE H3K9 methyltransferase were stably transformed with transgenes encoding the wildtype KYP protein or transgenes carrying induced point mutations in the KYP active site. The resulting lines were assayed for DNA methylation by whole-genome bisulfite sequencing to learn the efficiency with which wildtype and mutant versions of the KYP protein could restore DNA methylation lost in a kyp mutant. Samples 7 and 8 were run as single Illumina lanes and as such were compared to a previous Col sample (GSM881756), this Col sample was realigned to the TAIR10 genome for this study and as such updated processed files are available with this submission. These samples were used to define kyp mutant CHG context DMRs that were complemented upon introduction of the wildtype KYP protein. Samples 1-6 were run as multiplexed samples and were used to assay the degree of complementation for various point mutants. All plants are in the Col ecotype background.
Project description:DNA methylation plays critical roles in gene regulation and cellular specification without altering DNA sequences. The wide application of reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (bis-seq) opens the door to study DNA methylation at single CpG site resolution. One challenging question is how best to test for significant methylation differences between groups of biological samples in order to minimize false positive findings. Current methods to analyze genome-wide bisulfite sequencing data use a smoothing approach or a simple statistical test based on the binomial distribution. Comparative DNA methylation profiling in AML blasts and normal CD34(+) control cells
Project description:Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia. We performed DNA methylation (HELP array) and gene expression profiling in 215 samples of adult B-lineage acute lymphoblastic leukemia (ALL) and 12 normal preB samples. Adult B-lineage acute lymphoblastic leukemia (B-ALL) is an aggressive disease with <40% long-term survival. Genetic alterations such as BCR/ABL, E2A/PBX1 and MLL rearrangement (tMLL) define distinct B-ALL subtypes, which are associated with poor clinical outcome. It has been shown that these B-ALL subtypes have distinct expression profiles. However, the role of the epigenome in shaping these expression profiles and how the aberrant epigenetic gene regulation contributes to the biological and clinical features of those ALL subtypes is largely unknown. To address this question, we performed genome-wide DNA methylation and gene expression profiling on a large cohort of 215 well-characterized adult B-ALL specimens from the ECOG E2993 phase III clinical trial and a cohort of normal precursor B (preB) cells from 12 healthy bone marrows. The integrative analysis of these profiles led to the identification of key gene networks deregulated at the epigenetic and transcriptional levels within each subtype. In BCR/ABL, we identified a network centered on IL2RA(CD25), which is itself hypomethylated and overexpressed in most BCR/ABL B-ALL and confers poor clinical outcomes. In the tMLL subtype, we uncovered aberrant epigenetic and transcriptional activities that include hypomethylation and upregulation of FLT3 and BCL6. After showing that MLL/AF4 fusion protein binds to these genes as well as other hypomethylated and overexpressed genes in tMLL ALL cells, we showed that a specific BCL6 inhibitor, RI-BPI, kills tumor cells in both tMLL ALL cell lines and patient samples. BCL6 inhibition may therefore represent a novel therapeutic strategy for B-ALL patients with MLL translocations. RUNX1 is a key target gene in MLL-AF4 leukemias and contributes to gene activation by interacting with the AF4-MLL complex. The Mixed Lineage Leukemia 1 protein (MLL1) is an important epigenetic protein that is required for the maintenance of gene activation during development, but is also mutated in a subset of aggressive human leukemias. The most common leukemogenic MLL1 mutations are chromosome translocations that fuse MLL1 in-frame to produce novel fusion proteins. Different MLL1 fusion proteins cause unique leukemias even when they are expressed in the same cell type, suggesting that they function through unique molecular mechanisms. We used ChIP-seq in MLL-AF4 patient cell lines to identify target genes that are involved in leukemogenesis. ChIP-seq using MLLN, AF4, H3K4me3 and H3K79me2 antibodies in RS4;11 cells.
Project description:We utilized FAIRE-seq to identify accesible chromatin in mouse embryonic-, epiblast-, and neural- stem cells in addition to mouse embryonic fibroblasts. Analysis of these data sets reveal cell type specific chromatin signatures that differentiate naïve and primed pluripotency. Functional analysis of type-specific peaks revealed cell-type specific enhancers. FAIRE-seq of mESC, EpiSC, NSC and MEF