Project description:Bone marrow-derived multipotent stromal cells (BM-MSCs) exhibit therapuetically valuable properties, including the capacity to differentiate into skeletal tissues and modulate immune system activity. These properties depend on proper regulation of dynamic gene expression in response to environmental and developmental stimuli. This study used chromatin immunoprecipitation (ChIP) coupled with human promoter tiling microarray analysis (ChIP-on-chip) to profile histones H3K4me3 and H3K27me3 at promoters genome-wide. The goal of the study was to identify gene promoters marked by H3K27me3 and H3K4me3 in BM-MSCs. ChIP-on-chip performed with antibodies to H3K4me3 and H3K27me3 on BM-MSCs from 3 different donors (labeled 1632, 167696, and 8F3560) and with technical replicates.
Project description:To understand the epigenomic foundation of naive pluripotency, we implement a quantitative multiplexed chromatin immunoprecipitation sequencing (ChIP-seq) method comparing mouse embryonic stem cells (ESCs) grown in 2i versus 2i/serum and serum conditions. MINUTE-ChIP has a large linear dynamic range for accurately quantifying relative differences in genome-wide histone modification patterns across multiple pooled samples. We find compelling evidence for a broad H3 lysine 27 trimethylation (H3K27me3) hypermethylation of the genome, while bivalent promoters stably retain high H3K27me3 levels in 2i. We show that DNA hypomethylation, as observed in 2i, is a contributor to genome-wide gain of H3K27me3, while active demethylation by JMJD3/UTX counteracts further accumulation of H3K27me3. In parallel, we find hypomethylation of H3 lysine 4 trimethylation (H3K4me3), particularly at bivalent promoters, to be a characteristic of the 2i ground state. Serum stimulates H3K4me3 independent of GSK-3b and ERK signaling, suggesting that low H3K4me3 and high H3K27me3 levels at bivalent promoters are a product of two independent mechanisms that safeguard naive pluripotency.
Project description:BACKGROUND: Increasing evidence implicates the critical roles of epigenetic regulation in cancer. Very recent reports indicate that global gene silencing in cancer is associated with specific epigenetic modifications. However, the relationship between epigenetic switches and more dynamic patterns of gene activation and repression has remained largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide profiling of the trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) was performed using chromatin immunoprecipitation coupled with whole genome promoter microarray (ChIP-chip) techniques. Comparison of the ChIP-chip data and microarray gene expression data revealed that loss and/or gain of H3K4me3 and/or H3K27me3 were strongly associated with differential gene expression, including microRNA expression, between prostate cancer and primary cells. The most common switches were gain or loss of H3K27me3 coupled with low effect on gene expression. The least prevalent switches were between H3K4me3 and H3K27me3 coupled with much higher fractions of activated and silenced genes. Promoter patterns of H3K4me3 and H3K27me3 corresponded strongly with coordinated expression changes of regulatory gene modules, such as HOX and microRNA genes, and structural gene modules, such as desmosome and gap junction genes. A number of epigenetically switched oncogenes and tumor suppressor genes were found overexpressed and underexpressed accordingly in prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This work offers a dynamic picture of epigenetic switches in carcinogenesis and contributes to an overall understanding of coordinated regulation of gene expression in cancer. Our data indicate an H3K4me3/H3K27me3 epigenetic signature of prostate carcinogenesis.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. One-condition experment, gene expression of 3A6
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. Overall design: One-condition experment, gene expression of 3A6
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Overall design: Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:BACKGROUND:Key regulators of developmental processes can be prioritized through integrated analysis of ChIP-Seq data of master transcriptional factors (TFs) such as Nanog and Oct4, active histone modifications (HMs) such as H3K4me3 and H3K27ac, and repressive HMs such as H3K27me3. Recent studies show that broad enrichment signals such as super-enhancers and broad H3K4me3 enrichment signals play more dominant roles than short enrichment signals of the master TFs and H3K4me3 in epigenetic regulatory mechanism. Besides the broad enrichment signals, up to ten thousands of short enrichment signals of these TFs and HMs exist in genome. Prioritization of these broad enrichment signals from ChIP-Seq data is a prerequisite for such integrated analysis. RESULTS:Here, we present a method named Clustering-Local-Unique-Enriched-Signals (CLUES), which uses an adaptive-size-windows strategy to identify enriched regions (ERs) and cluster them into broad enrichment signals. Tested on 62 ENCODE ChIP-Seq datasets of Ctcf and Nrsf, CLUES performs equally well as MACS2 regarding prioritization of ERs with the TF's motif. Tested on 165 ENCODE ChIP-Seq datasets of H3K4me3, H3K27me3, and H3K36me3, CLUES performs better than existing algorithms on prioritizing broad enrichment signals implicating cell functions influenced by epigenetic regulatory mechanism in cells. Most importantly, CLUES helps to confirm several novel regulators of mouse ES cell self-renewal and pluripotency through integrated analysis of prioritized broad enrichment signals of H3K4me3, H3K27me3, Nanog and Oct4 with the support of a CRISPR/Cas9 negative selection genetic screen. CONCLUSIONS:CLUES holds promise for prioritizing broad enrichment signals from ChIP-Seq data. The download site for CLUES is https://github.com/Wuchao1984/CLUESv1.
Project description:Understanding how chromatin modification regulates development and disease can be limited by available material. Despite recent progress, balancing high-quality and reliable mapping using chromatin-immunoprecipitation-based deep sequencing (ChIP-seq) remains a challenge. We report two techniques, recovery via protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq), that provide reproducible mapping in as few as 500 cells. RP-ChIP-seq allows detection of age-associated epigenetic changes in a single mouse lens, whereas FARP-ChIP-seq accurately maps histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and multi-potent progenitors (MPPs) from one mouse. These datasets not only highlight genes that may be involved in lens aging but also indicate a lack of H3K4me3/H3K27me3 bivalency on hematopoietic genes in HSCs.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs. Two-condition experiment, KP MSCs vs. 3A6 MSCs.