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ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS

ABSTRACT: Many DNA-hypermethylated cancer genes are occupied by the polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that approximately 75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESC and adult stem/progenitor cells. A large number of these genes are key developmental regulators and a subset, which we call the "DNA hypermethylation module", comprise a portion of the PcG target genes that are downregulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA-hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE35573 | GEO | 2012/03/02

SECONDARY ACCESSION(S): PRJNA156085

REPOSITORIES: GEO

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Project description:Many DNA-hypermethylated cancer genes are occupied by the polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that approximately 75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESC and adult stem/progenitor cells. A large number of these genes are key developmental regulators and a subset, which we call the "DNA hypermethylation module", comprise a portion of the PcG target genes that are downregulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA-hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions. The goal was to analyze the chromatin in MSCs, osteoblasts and U2OS cells of genes that are methylated in the osteosarcoma cell line U2OS. Cell culture: MSCs derived from adult bone marrow were cultured and differentiated to osteoblasts as described by Jaiswal et al., 1997 (J Cell Biochem 64, 295-312). U2OS cells were cultured in McCoy's 5A medium supplemented with 10% FBS. ChIP-seq: Cells were crosslinked in 3.7% formaldehyde, lysed and sonicated to obtain chromatin fragments in the range of 200 to 600 base pairs. Sonicated chromatin was incubated with primary antibody, H3K4Me3 (Millipore, 07-473) or H3K27Me3 (kind gift from Thomas Jenuwein/ Nicholas Shukeir), overnight at 4°C, followed by capturing primary antibodies by adding ProteinA/G magnetic beads (DynaBeads) and incubating at room temperature for 3-4 hrs. Captured chromatin was washed in low- and high-salt buffers, as well as TE. Chromatin fragments were subjected to simultaneous elution, de-crosslinking and Proteinase-K treatment at 65°C in elution buffer followed by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Sequencing and alignment: Purified DNA was used to prepare the sequencing library and sequenced on Applied Biosystems SOLiD (V3). Sequencing reads were aligned to hg18 (NCBI 36) using Bioscope 1.2.1. The software guarantees finding all alignments between the first 25 base pairs of the read (seed) and the reference sequence with up to 2 mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and -2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported. Peak identification: Aligned reads were analyzed using Model-based Analysis of ChIP-Seq (MACS) to detect regions enriched for the histone marks (called peaks) with default settings. Input was used as background for peak identification.

Project description:Aberrant DNA hypermethylation of CpG island (CGI) promoters are associated with transcriptional repression of many tumor suppressor genes and lead to tumor progression in many cancers. Most recently, one research group observed that aberrantly hypermethylated genes in multiple cancers are already repressed, but their promoters are maintained in a hypomethylated state in pre-cancerous tissues.Their studies didn't provide a clue to explain by what mechanisms those genes were repressed in pre-cancerous tissues. Another research group found that many genes with de novo promoter hypermethylation in colon cancer were among the subset of genes "bivalently" marked in embryonic stem cells and adult stem/progenitor cells by repressive Polycomb group proteins (PcG), which are known for maintaining low, but poised, transcription.These observations provide a clue that CGI promoter hypermethylation in cancers is associated with PcG target genes in pre-cancerous tissues.we took advantage of ChIP-BS-seq technology and applied it to examine H3K27me3 and H3K4me3 profiles for one normal lymphoblastoid cell line (YH) and three cancer cell lines including one cervical cancer cell line (Hela) and two gastric cancer (GC) cell lines (BGC-823 and AGS). We aplied ChIP-BS technology to examine H3K27me3 marks, which are catalyzed by the SET domain histone methyltransferase EZH2 and have a repressive function with 50bp pair-end sequencing. found H3K27me3 marks were enriched preferentially at CpG islands, (+/-500) transcription start sites (TSSs) and exons in two GC cell lines (BGC-823 and AGS). In YH cells, H3K27me3 marks were only preferentially enriched at CpG islands. In contrast, Hela cells presented a reverse pattern with highest H3K27me3 enrichment in intergenic regions. To confirm this result in Hela cells, we performed two independent replicates of ChIP-Seq and ChIP-BS-seq. Cause of useful was relative small. we still sequenced one 100bp pe reads replicate for H3K4me3 and two replicate for H3K27me3 ChIP-BS-seq.

Dataset Information

ChIP-seq analysis of H3K4Me3- and H3K27Me3-marked chromatin in mesenchymal stem cells (MSCs), osteoblasts derived from MSCs and the osteosarcoma cell line U2OS

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