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 anticancer action by remodeling DNA methylation and gene expression. 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 functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

转录因子（TF）介导的基因调节在癌发生过程中经常被破坏。 TF 结合位点的 DNA 甲基化状态可能决定相应基因的转录活性。二苯乙烯类多酚，例如紫檀芪 (PTS)，已被证明可以通过重塑 DNA 甲基化和基因表达来发挥抗癌作用。然而，这些影响背后的机制仍不清楚。在此，我们探讨了 PTS 处理的 MCF10CA1a 侵袭性乳腺癌细胞中致癌 TF OCT1 结合与从头DNA 甲基转移酶 DNMT3B 结合之间的动态。使用染色质免疫沉淀 (ChIP) 和下一代测序，我们确定了 47 个基因调控区域，这些区域在 PTS 的作用下 OCT1 结合减少，而 DNMT3B 结合增加。大多数这些基因被发现具有致癌功能。我们选择了三个候选物PRKCA、TNNT2和DANT2 ，进行进一步的机制研究，考虑到 PRKCA 与众多癌症驱动过程和途径的功能和监管联系，以及TNNT2和DANT2增强剂中 DNMT3B 占用率增加最高的一些因素。 PTS 导致PRKCA、TNNT2和DANT2位点内的 DNMT3B 募集，这些位点也表现出 OCT1 结合减少。随着 DNMT3B 结合的增加，OCT1 显着减少，并伴有PRKCA启动子、 TNNT2和DANT2增强子高甲基化以及基因沉默。有趣的是，在 DNMT3B-CRISPR 敲除的 MCF10CA1a 乳腺癌细胞中，没有检测到对 PTS 反应的基因 DNA 高甲基化。 它表明PRKCA、TNNT2和DANT2在 PTS 上发生依赖于 DNMT3B 的甲基化。我们的研究结果让我们更好地了解可能有助于二苯乙烯类多酚抗癌作用的机制参与者及其基因靶标。