Abstract 262: Complex epigenetic alterations induced by MYC during activation of the rDNA locus
Authors: Uemura Motohide , Nelson William G. , Yegnasubramanian Srinivasan , De Marzo Angelo M. 
Published: June 2012

INTRODUCTION: The nucleolus is the site of ribosome biogenesis which is tightly regulated and linked to cellular growth and proliferation. Increased rRNA synthesis and ribosome biogenesis are thought to be important molecular alterations that occur very commonly in cancer cells. The nucleolus is formed around tandem repeats (n=∼400) of the ribosomal RNA genes (18S, 5.8S and 28S) each of which contains an upstream promoter region. Only a limited number of repeat units are activated at any one time, and the inactive silent repeat units (∼50% of all repeats) are characterized by dense methylation of the CpG island of the upstream promoter. Since MYC is overexpressed in most human prostate cancers, can affect nucleolar structure, and can activate rRNA transcription, our goal is to determine precisely how MYC regulates rRNA levels. Despite MYC's ability to activate rRNA transcription, we have previously reported that MYC is unexpectedly required to maintain proper rDNA promoter methylation levels. Interestingly, maintenance of a certain number of these repeats in an inactive form is required for chromosomal stability and proper rRNA accumulation. In this study we further determine the chromatin structure of the rDNA promoter region associated with active and inactive repeats and determine the effects of forced suppression of MYC. METHODS: Chromatin immunoprecipitation (ChIP) was performed for transcription factors for rRNA transcription and several histone modifications. Isolated ChiPed DNA was followed by bisulfite sequencing (ChIP-Bis-Seq) of a region from the rDNA promoter. The functional role of MYC was explored by siRNA mediated knockdown in prostate cancer and lymphoma cell lines. RESULTS: ChIP-Bis-Seq of the rDNA promoter was used to identify two chromatin modifications associated exclusively with methylated CpG DNA of the rDNA promoter (methylated repeats), H3K9me2 and H1 linker, and, 3 transcription factors associated nearly exclusively with unmethylated repeats (MYC, Pol I, and UBTF). Forced suppression of MYC resulted in reductions in the levels of rRNA as expected, and this was accompanied by an induction in the inactive chromatin marks, H3K9me2 and H1 linker. Notably, the induction of these “heterochromatic” marks, does not result in an induction of additional rDNA promoter methylation, which is decreased overall by MYC knockdown. CONCLUSIONS: In addition to MYC's known ability to directly activate Pol I induced transcription, MYC is required to maintain active rDNA promoters by preventing accumulation of inactive chromatin marks such as H1 and H3K9me2. Despite the recruitment of H1 and H3K9me2 to the promoter after MYC suppression, this is not accompanied by an overall increase in rDNA promoter DNA methylation. This is likely the case since MYC is also required to maintain DNA promoter methylation at these sites, and promoter methylation/silencing itself is required for overall production of mature rRNA species.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 262. doi:10.1158/1538-7445.AM2011-262