DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in response to pterostilbene. 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).

转录因子（Transcription Factor, TF）所介导的基因调控过程，在癌变进程中常发生紊乱。转录因子结合位点的DNA甲基化状态，可决定对应基因的转录活性。芪类多酚类化合物，如紫檀芪（Pterostilbene, PTS），已被证实可通过重塑抑癌基因与癌基因的DNA甲基化及基因表达谱，发挥抗癌作用。然而，这类化合物发挥抗癌作用的具体分子机制仍未明确。本研究以经紫檀芪处理的侵袭性乳腺癌MCF10CA1a细胞为模型，探究了致癌性转录因子OCT1与从头DNA甲基转移酶DNMT3B（de novo DNA methyltransferase, DNMT3B）的结合动态变化。通过染色质免疫共沉淀（Chromatin Immunoprecipitation, ChIP）结合下一代测序技术，本研究鉴定出47个基因调控区域：经PTS处理后，这些区域的OCT1结合水平显著下降，而DNMT3B结合水平显著富集。经分析，其中多数基因具有致癌及促转移功能。编码蛋白激酶Cα（protein kinase C alpha, PKC-α）的PRKCA基因，因其与多条致癌通路及癌症驱动过程存在功能与调控关联，被选为后续机制研究的靶基因。另外两个基因——肌钙蛋白T2（Troponin T2, TNNT2）与DXZ4关联非编码转录本2（DXZ4 Associated Non-coding Transcript 2, DANT2）——也被纳入研究，因其DNMT3B结合富集程度位居前列，且被发现具备新的致癌功能。PTS可将DNMT3B招募至溶剂对照组（载体处理）细胞中原本由OCT1结合的PRKCA启动子区域，以及TNNT2与DANT2的增强子位点。OCT1结合水平的显著下降伴随DNMT3B结合水平的升高，同时伴随着PRKCA启动子区域、TNNT2与DANT2增强子区域的高甲基化，以及对应基因的沉默。值得注意的是，在DNMT3B CRISPR敲除的MCF10CA1a乳腺癌细胞中，并未检测到PTS处理后上述基因调控区域的高甲基化现象，这表明PTS处理后PRKCA、TNNT2与DANT2的甲基化依赖于DNMT3B。本研究的发现有助于进一步阐明芪类多酚类化合物发挥抗癌作用的核心分子机制及其靶基因。实验整体设计：检测紫檀芪（PTS）处理后，MCF10CA1a乳腺癌细胞中DNMT3B与OCT1的结合占据情况。