Abstract 4804: Identification of a functional network of human epigenetic silencing factors

Abstract

Abstract Epigenetic silencing directs transcriptional shutoff of specific genes during development and cellular differentiation. This process is mediated by epigenetic “marks” including DNA methylation and a variety of posttranslational histone modifications (e.g. methylation). Errors in placement or removal of epigenetic marks can drive epigenetic silencing and tumorigenesis. Inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme families (DNMTi and HDACi, respectively) can reverse epigenetic silencing and produce anti-tumor effects, possibly through reactivation of silent tumor suppressor genes (so-called epigenetic therapy). As these inhibitors show little specificity within enzyme families, their precise mechanisms of action are not well understood. To identify cellular factors involved in maintenance of epigenetic silencing, HeLa cells harboring epigenetically silent GFP reporter genes were interrogated with an siRNA library targeting predicted epigenetic regulators, including potential activators, silencers, chromatin remodelers, and ancillary factors. Using this approach, individual, or combinatorial requirements for specific epigenetic silencing factors can be detected by measuring GFP reactivation after siRNA-based factor knockdown. In our analyses, we identified a specific subset of epigenetic factors that are candidates for participation in a functional epigenetic silencing network in human cells. These factors include the histone deacetylase HDAC1, the de novo DNA methyltransferase DNMT3A, components of the Polycomb PRC1 complex (RING1, HPH2), and the histone lysine methyltransferases KMT1E and KMT5C. Roles were also detected for two TRIM protein family members, the cohesin component Rad21, and the histone chaperone CHAF1A (CAF-1 p150). Remarkably, combinatorial knockdown of factors was not required for reactivation, indicating little functional redundancy. Consistent with this interpretation, knockdown of either KMT1E or CHAF1A resulted in a loss of multiple histone repressive marks and concomitant gain of activation marks on the promoter during reactivation. These results reveal how functionally diverse factors may cooperate to maintain gene silencing during normal development or in disease. Furthermore, the findings suggest an avenue for discovery of new targets for epigenetic therapies. 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 4804.