GSE175639
DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in response to pterostilbene
Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anti-cancer action by remodeling DNA methylation and gene expression patterns of tumor suppressor genes and oncogenes. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF, OCT1, binding and de novo DNA methyltransferase, DNMT3B, binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic and pro-metastatic functions. PRKCA, a gene encoding for protein kinase C alpha (PKC-alpha), was selected for further mechanistic investigation due to its functional and regulatory connection with numerous oncogenic pathways and cancer-driving processes. Two additional genes, troponin T2 (TNNT2) and DXZ4 associated non-coding transcript 2 (DANT2), were also investigated as they demonstrated some of the highest increase in DNMT3B occupancy and novel oncogenic functions. PTS led to the recruitment of DNMT3B to the PRKCA promoter region and TNNT2 and DANT2 enhancers at loci that are occupied by OCT1 in vehicle-treated cells. Substantial decrease in OCT1 occupancy with increased DNMT3B binding were accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, hypermethylation of the regulatory regions of the genes in response to PTS was not detected in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells, which indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS treatment. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anti-cancer action of stilbenoid polyphenols. Overall design: Examination of occupancy of DNMT3B and OCT1 in MCF10CA1a breast cancer cells after treatment with pterostilbene (PTS).