We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

It is increasingly apparent that cancer development not only depends on genetic alterations but on an abnormal cellular memory, or epigenetic changes, which convey heritable gene expression patterns critical for neoplastic initiation and progression. These aberrant epigenetic mechanisms are manifest in both global changes in chromatin packaging and in localized gene promoter changes that influence the transcription of genes important to the cancer process. An exciting emerging theme is that an understanding of stem cell chromatin control of gene expression, including relationships between histone modifications and DNA methylation, may hold a key to understanding the origins of cancer epigenetic changes. This possibility, coupled with the reversible nature of epigenetics, has enormous significance for the prevention and control of cancer.

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.

Double stranded RNAs (dsRNA) molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation dependent manner in plants (RNAdependent DNA methylation or RdDM). 1-3 Whether a similar mechanism exists in mammalian systems is a vital and currently controversial issue. 4-6 DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and x-chromosome inactivation, 7-9 and aberrant CpG island hypermethylation at tumor suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer. 10 Hence, we investigated whether RdDM may operate in human cancers to mediate epigenetic silencing using the endogenous CDH1 gene as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers. 11-14 We find that, although small dsRNAs targete exclusively to the CDH1 promoter can effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this is entirely independent of DNA methylation. Moreover, we can accomplish such silencing in a cancer cell line genetically modified such that it lacks virtually any capacity to methylate DNA.To test whether dsRNAs could induce transcriptional gene silencing at the endogenous CDH1 promoter, we transfected HCT116 human colorectal cancer cells with two 21-nucleotide long dsRNA oligos (dsCDH1-1 and dsCDH1-2). These sequences are homologous to the CpG island of the CDH1 promoter but do not overlap any known transcribed sequences ( Fig. 1a and Supplementary Note). When both oligos were administered simultaneously, CDH1 protein was decreased to barely detectable levels by day 7 of treatment as compared to treatments with either the mock or dsCtrl, a scrambled control dsRNA oligo (Fig. 1b). We verified this decrement of CDH1 expression by real time RT-PCR (qRT-PCR) and found that there was a corresponding decline in CDH1 mRNA level in the dsCDH1s treated cells when compared to the untreated cells (Fig. 1c). When used alone, the dsCDH1-1 treatment resulted in a greater down-regulation of CDH1 expression than dsCDH1-2 alone but not greater than the two oligos combined (Fig. 1d). To establish that loss of the CDH1 protein was due to transcriptional silencing, we performed nuclear run-on assays. Analysis on the dsCDH1s treated cells indicated that CDH1 transcription on day 7 was decreased by 61% as detected by a 3′ cDNA probe when compared to the dsCtrl treated cells (Fig. 1e). Thus, promoter-targeting dsRNAs could effectively silence CDH1 transcription resulting in a net decrease in protein production. Competing Interest Statement:The authors declare that they have no competing financial interests regarding the particular studies in this manuscript. Drs. Baylin and Herman are paid consultants to OncoMethylome Sciences. Under licensing agreement between the Johns Hopkins University and this company, M.S.P. ...