TGF-β-dependent active demethylation and expression of the p15ink4b tumor suppressor are impaired by the ZNF217/CoREST complex

Abstract

In this study we examine the mechanisms of dynamic DNA methylation of the p15(ink4b) tumor suppressor gene. Using conventional ChIP and ChiPseq, we identify the p15(ink4b) promoter as a target for the ZNF217 oncogene, the CoREST complex, and DNMT3A. Treatment of cells with TGF-β triggers active demethylation involving loss of ZNF217/CoREST/DNMT3A and the corecruitment of SMAD2/3, CBP, and the DNA glycosylase TDG. Knockdown of TDG, or its functional homolog MBD4, prevents TGF-β-dependent demethylation of p15(ink4b). DNA immunoprecipitation of 5mC and 5hmC indicates that 5mC undergoes conversion to 5hmC prior to activation of p15(ink4b). Remarkably, overexpression of ZNF217 inhibits active demethylation and expression of the p15(ink4b) gene by preventing recruitment of SMAD2/3 and TDG. These findings suggest that active demethylation is essential for regulating a subset of TGF-β-dependent genes. Importantly, disruption of active demethylation by the ZNF217 oncogene may be a paradigm for other oncogenic signals on DNA methylation dynamics.