Project description:We have developed a method for mapping unmethylated sites in human genome based on the resistant of TspR1 digested ends to exoIII nuclease degradation. Digestion with TspR1 and methylation-sensitive restriction endonuclease, HpaII, followed by exoIII and single strand DNA nuclease allows the removal of DNA fragments containing unmethylated HpaII sites. We then use array CGH to map the sequences depleted by this procedures in human genomes derived from five human tissues, a primary breast tumor and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5’ end of widely expressed genes including promoter, first exon and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas primary tumor exhibits intermediate pattern between normal tissue and cell lines. A striking characteristic of tumor genomes is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor supperessor genes, and with regions containing tissue-specific gene clusters or with gene poor region containing novel tissue-specific genes. Bisulfite sequencing analysis shows a novel mosaic methylation pattern with alternative methylated and unmethylated zones was found in human histone gene clusters in chromosome 6. Correlation with microarray analysis show that genes with hypomethylated sequence 2kb up- or down-stream of transcription start site are highly expressed whereas genes with extensive intragenic and 3’ UTR hypomethylation are silenced. The method described herein can be used for large scale screening of changes in methylation pattern in the genome of interest. Keywords: Genome-Wide Mapping of Hypomethylated Sites in Human Genomes
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.
Project description:We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40 % were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied functional p53 binding sites and to date not observed by previous genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely the different chromatin landscape in normal compared to cancer-derived cells influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIP-seq peaks to the recently published IMR90 methylome1, and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells. Identification of genomic p53 binding sites in normal human cells by ChIP-seq.
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.