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Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.