Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases

Hypermethylation associated silencing of the CpG islands of tumor suppressor genes is a common hallmark of human cancer. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using two molecular screenings for differentially methylated loci [differential methylation hybridization (DMH) and amplification of inter-methylated sites (AIMS)], we found that DKO cells, but not the single DNMT1 or DNMT3b knockouts, have a massive loss of hypermethylated CpG islands that induces the re-activation of the contiguous genes. We have characterized a substantial number of these CpG island associated genes with potentially important roles in tumorigenesis, such as the cadherin member FAT, or the homeobox genes LMX-1 and DUX-4. For other genes whose role in transformation has not been characterized, such as the calcium channel alpha1I or the thromboxane A2 receptor, their re-introduction in DKO cells inhibited colony formation. Thus, our results demonstrate the role of DNMT1 and DNMT3b in CpG island methylation associated silencing and the usefulness of genetic disruption strategies in searching for new hypermethylated loci.