Abstract LB-84: DNA methylation centers in CpG-island promoters are context-specific

Repression of many TSGs (tumor suppressor genes) in cancer is mediated by aberrantly increased DNA methylation levels of CpG islands at promoters. Evidence from plants and mammalian cells indicate that sequences within or around target sites could influence DNA methylation patterns. However, it is still unknown whether aberrant methylation in cancer starts randomly or is triggered by specific DNA sequences. Here, we test the hypothesis that cisacting elements trigger de novo CpG-island promoter methylation. We first examined methylation patterns of the RIL promoter region (from −722 to +630 from transcription start) in several cell types and tissues. RIL is a candidate tumor suppressor gene that is frequently hypermethylated in cancer. Bisulfite-sequencing results identified 11 CpG sites upstream and 17 CpG sites downstream that are densely methylated both in normal blood (average methylation density, upstream 82.2± 11.6% and downstream 73.6± 17.7%) and colon tissues (upstream 80.5±14.6% and downstream 76.2±12.1%), compared with those within the CpG island (17.6± 10.0% in blood and 20.2±15.0% in colon tissues). This sharp transition of methylation surrounding the CpG island has also been seen in some genes such as p16, VHL and CDH1. We hypothesized that these highly methylated CpG sites in normal tissues serve as methylation centers that attract DNMT binding and trigger de-novo DNA methylation. To test this hypothesis while controlling for insertion site effects, we established a system using Flp/FRT to construct single integration sites in a host cell line (HCT116 colon cancer cell line), so that exogenous fragments from CpG-island promoters can be introduced into the same genomic locus or chromatin micro-environment. The entire RIL promoter region (−713 to +664, 96 CpG sites), or isolated fragments containing the hypermethylated upstream (−713 to −378, 11 CpG sites) and downstream regions (+214 to +664, 20 CpG sites), and the central part (−406 to +237, 66 CpG sites) were integrated (separately) into a single genomic locus at 3q13.31, corresponding to the intron of an expressed gene. Cells collected at 1, 2 and 3 months after transfection were examined by bisulfite-sequencing. No significant de-novo methylation of the transfected constructs was observed. We next tested a genomic region highly methylated in all tissues examined except germ cells, the insulin-6 promoter, as well as 3 motifs in this promoter computationally associated with DNA methylation, but both constructs remained completely methylation free after transfection. Thus, multiple sequences associated with very high levels of DNA methylation in normal and neoplastic tissues can be completely methylation free when inserted into a different chromatin context. These results have important implications for the pathogenesis of aberrant methylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-84.