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Epigenetic regulation of enhancer regions in breast cancer cells in response to pterostilbene

University of British Columbia

Epigenetic alterations are estimated to be linked to 30% of sporadic breast cancer cases. Interestingly, certain dietary polyphenols, such as pterostilbene found abundantly in blueberries, have been shown to regulate gene expression and reverse tumour development by altering epigenetic patterns. Our group has proposed the involvement of DNA methyltransferase 3B (DNMT3B) and oncogenic transcription factor OCT1 as vital players in polyphenol-mediated targeting of oncogenes. We have also identified enhancers as important regulatory regions with altered DNA methylation in response to polyphenols. However, the genome wide effects of pterostilbene-mediated alterations in the occupancy of DNMT3B and OCT1 in enhancer regions of oncogenes remains to be elucidated. In this study, following chromatin immunoprecipitation (ChIP) sequencing analyses of highly invasive MCF10CA1a breast cancer cells treated with 7μM pterostilbene for 9 days, we discovered that pterostilbene treatment leads to altered occupancy of DNMT3B and OCT1 at enhancer regions of genes with oncogenic functions. In addition, trimethylation at lysine 36 of histone 3 (H3K36me3) enrichment was measured to indicate decrease in gene transcriptional activity. QPCR and pyrosequencing were performed to assess gene expression and DNA methylation of the selected oncogenes, respectively. We identified 20 candidate genes whose enhancers showed increased binding of DNMT3B, decreased occupancy of OCT1 and reduced enrichment of H3K36me3 in MCF10CA1a upon pterostilbene exposure compared with control untreated cells (p<0.05). Of those 20 candidates, we selected 4 genes for further analyses (i.e., PITPNC1, TNNT2, DANT2 and LINC00910). Using pyrosequencing, we found that PITPNC1 and TNNT2 enhancer regions, encompassing 5 and 3 CpG sites respectively, showed 8-16% and 6-19% increase in DNA methylation throughout the region upon pterostilbene treatment. DANT2, a long noncoding RNA, was hypermethylated by 3-7% across 7 CpG sties and LINC00910 was hypermethylated by 2-28% across 8 CpG sites. These changes coincided with 84%, 87%, 41% and 92% down-regulation of PITPNC1, TNNT2, DANT2 and LINC00910, respectively, upon pterostilbene treatment. This work provides novel insights into the mechanisms of dietary polyphenols in driving epigenetic silencing of enhancer regions within genes with oncogenic functions in breast cancer cells.

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2020-05-04

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74280

Human Nutrition

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/