Abstract Aberrant DNA methylation of promoter CpG islands (CGIs) is deeply involved in cancers and other disorders. Contrary to our initial expectations, aberrant methylation is now recognized to possess several unique characteristics different from mutations [Ushijima and Asada, Cancer Sci, 2010]. One of the remarkable differences is target gene specificity. Specific cancers have aberrant methylation of specific genes, and specific inducers of methylation, such as Helicobacter pylori infection, induce methylation of specific genes [Nakajima et al, Int J Cancer, 2009]. As the mechanism of target gene specificity (namely, instructive mechanism), low transcription levels and trimethylation of histone H3 lysine27 (H3K27me3) in precursor cells are known to promote methylation induction. However, factors that protect genes from aberrant methylation are unknown. To identify protective factors, we first isolated 5,510 and 521 genes resistant and susceptible, respectively, to aberrant methylation induction by methylated DNA immunoprecipitation (MeDIP)-on-chip analysis of nucleosome-free regions of promoter CGIs in normal prostatic epithelial cells and four prostate cancer cell lines. Then, by gene expression analysis, the susceptible genes were shown to have low transcription levels in normal prostatic epithelial cells. Finally, by ChIP-on-chip analysis in normal prostatic epithelial cells, we revealed that, even among the genes with low transcription, genes with high active histone modifications and binding of RNA polymerase II (Pol II) had marked resistance to aberrant methylation induction, and confirmed that genes with high H3K27me3 had marked susceptibility to aberrant methylation induction. On the other hand, another repressive histone modification, trimethylation of histone H3 lysine9 (H3K9me3), was not associated with resistance or susceptibility. By multivariate analysis, Pol II binding showed stronger influence on the resistance than active histone modifications, and H3K27me3 showed independent influence on the susceptibility. The same was true in normal mammary epithelial cells for 5,430 and 733 genes resistant and susceptible, respectively, to aberrant methylation in breast cancers [Takeshima et al, Genome Res, 2009]. These results indicated that the presence of Pol II, active or stalled, protects promoter CGIs from aberrant DNA methylation, independently of transcription levels. 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 176.