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Methods and Resources

Genome-Wide Mapping and Characterization of Hypomethylated Sites in Human Tissues and Breast Cancer Cell Lines

Biochemistry and Molecular Biology

We have developed a method for mapping unmethylated sites in human genome based on the resistant of TspRI digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease, HpaII, followed by ExoIII and single strand DNA nuclease allowed the removal of DNA fragments containing unmethylated HpaII sites. We then used 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 cell lines is the presence of megabase-size hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene poor region containing novel tissue-specific genes. Correlation with microarray analysis shows 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.

E-mail mth{at}ym.edu.tw

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