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Hundreds of genes have been described as having aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. Here, we report the identification of RIL, a gene that maps to a region frequently deleted in leukemia, as a frequent methylation target in cancer. Restoring RIL expression resulted in reduced cancer cell growth and clonogenicity, and sensitized them to apoptosis. In search for a cause of RIL hypermethylation, we identified a 12-bp polymorphic sequence around the transcription start site that creates a long allele. Bisulfite sequencing of cancers heterozygous for RIL showed that the short allele is ∼10-fold more methylated than the long (P<0.001). EMSA showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfections of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing levels of transcription from the short allele with eventual spreading of de novo methylation. By contrast, the long allele showed ∼5-30 fold higher levels of expression over time as measured by luciferase and ∼2.4 fold lower levels of methylation as measured by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that gene hypermethylation in cancer is a non-random event, which is dictated by protein-DNA interactions at specific promoters.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]