Abstract 1185: EZH2-mediated H3K27 trimethylation silencing of microRNAs in hepatocellular carcinoma

Abstract

Abstract Although the biological pathways by which misexpressed microRNAs (miRNAs) contribute to the development of hepatocellular carcinoma (HCC) have been extensively investigated, little is known about the upstream regulatory mechanisms. Epigenetic gene silencing in cancer cells is mediated by promoter DNA methylation and at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 9 dimethylation (H3K9me2). We have previously demonstrated that EZH2-mediated H3K27me3 is a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation (Nat Genet 2008;40:741-50). Here we used an integrative genome-wide location and expression analysis to investigate the epigenetic control of miRNA expression in HCC cells and explored the involvement of transcription factors in this process. Chromatin immunoprecipitation coupled with human promoter microarrays revealed that 8, 20, and 15% of the interrogated miRNA promoters were enriched with H3K27me3, H3K9me2 and DNA methylation in Hep3B HCC cells, respectively. Some of these enriched miRNAs e.g. miR-101 and -194 have been reported to be down-regulated in primary HCCs, providing evidence on epigenetic silencing of miRNAs in HCC. Consistent with our previous observation on protein-coding genes, most of the miRNAs enriched with H3K27me3 had no detectable DNA hypermethylation; and most miRNAs showing DNA hypermethylation had no enrichment for H3K27me3. There was also minimal overlap between H3K27me3 and H3K9me2 binding on miRNA promoters. Down-regulation of EZH2 decreased global H3K27me3 level and restored expression of the H3K27me3-targeted miRNAs while 5-aza-2’-deoxycytidine and trichostatin A treatment alone or in combination did not reactivate their expression, further suggesting the independence of EZH2-mediated H3K27me3 in miRNA silencing. To identify the transcription factors involved in the formation of H3K27me3 modification, the -0.5 to +1kb regulatory regions of ∼2000 H3K27me3-bound protein-coding and miRNA genes were submitted to transcription factor binding predictions by TRANSFAC Pro. Interestingly, the binding sites for YY1, known to recruit polycomb for H3K27me3 modifications, were recurrently over-represented in these loci. Quantitative RT-PCR showed that YY1 and EZH2 were over-expressed (defined as greater than 2-fold increase) in 73% (38/52) and 87% (45/52) of HCCs compared with the paired non-tumor tissues. Overall, their expressions were significantly higher in tumor tissues (p < 0.0001) and positively correlated in these 52 pairs of HCCs (p < 0.05). In conclusion, our findings suggest that YY1 may play an important role in EZH2-mediated H3K27me3 for miRNA silencing in HCC. Acknowledgements: This study was partially supported by the General Research Fund 462309, RFCID 09080042 and Collaborative Research Fund CUHK04/CRF/08. 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 1185. doi:10.1158/1538-7445.AM2011-1185