DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in response to pterostilbene
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA733126 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14667019.fastq.gz | Fastqsanger.gz | |||
| SRR14667020.fastq.gz | Fastqsanger.gz | |||
| SRR14667021.fastq.gz | Fastqsanger.gz | |||
| SRR14667022.fastq.gz | Fastqsanger.gz | |||
| SRR14667023.fastq.gz | Fastqsanger.gz |
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Project description: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.
2021-08-31 | GSE175639 | GEO
Project description: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.
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Project description:Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation. Oct1 is an AR interacting partner and regulates the transcriptional activity of AR. In order to investigate the Oct1 function in prostate cancer cells, we performed gene expression in AR-positive prostate cancer cell lines after siOct1 or pyrrole-imidazole (PI) polyamide targeting Oct1-binding treatment. We also treated cells with vehicle or androgen to analyze the effects of Oct1 on AR function. Observation of androgen dependent gene expression changes after treatment with siOct1 or polyamide targeting Oct1 with microarray.
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Project description:3D spheroids of primary human hepatocytes (3D PHH) keep a differentiated phenotype with retained metabolic function and proteome fingerprint for weeks in culture. As a result, 3D PHH are gaining ground as a model for mechanistic studies on liver homeostasis and in vitro to in vivo (IVIV)predictions in drug discovery. However, the function of drug-transporting proteins has not yet been studied in the 3D PHH. Here we used the organic cation transporter 1 (OCT1/SLC22A1) as a model to explore both transporter kinetics and long-term regulation of transporter activity via different pathways. The 3D PHH were cultured for one week and then used for short- and long-term studies. The OCT1 transporter kinetics was assessed using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, the 3D PHH were exposed to xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomics analysis was used to track prototypical changes of other regulated proteins. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14.14±3.97 using three donors. Established OCT1 inhibitors reduced the uptake of ASP+ in the 3D PHH spheroids, resulting in 40-60% of the maximal uptake rate of ASP+. The long-term exposure studies showed that OCT1 is relatively stable to the activation of a broad range of pathways known to regulate ADME proteins. Importantly, while expression levels of other ADME proteins, such as CYP3A4 and MDR1 were regulated as expected, OCT1 levels remained stable. In conclusion, our results show for the first time that 3D PHH spheroids express fully active OCT1 and that transporter kinetics can be studied in 3D PHH. We also confirm that OCT1 remains stable and functional during activation of various pathways that alter the expression and function of other ADME proteins.
2024-08-10 | PXD045733 | Pride
Project description:Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation. Oct1 is an AR interacting partner and regulates the transcriptional activity of AR. In order to investigate the Oct1 function in prostate cancer cells, we performed gene expression in AR-positive prostate cancer cell lines after siOct1 or pyrrole-imidazole (PI) polyamide targeting Oct1-binding treatment. We also treated cells with vehicle or androgen to analyze the effects of Oct1 on AR function.
2016-04-01 | GSE77650 | GEO
Project description:Embryonic stem cells (ESCs) co-express Oct4/POU5F1 and an Oct4 paralog known Oct1/POU2F1. To study the role of Oct1 in embryonic stem cell transcriptional control and pluripotency, we constructed germline and inducible-conditional Oct1 deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal, growth rates and metabolic signatures. However loss of Oct1 results in defective lineage specification. ESCs lacking Oct1 fail to form beating cardiomyocytes in culture, generate neurons poorly, form smaller, less differentiated teratomas, and are incapable of generating chimeric mice. Upon RA-mediated neuronal differentiation, Oct1 deficient cells fail to properly induce developmentally poised genes. Simultaneously, genes for alternative developmental pathways, most notably for the development of extra-embryonic tissues, are inappropriately expressed. ChIP experiments show that Oct1 does not occupy pluripotency genes or differentially expressed developmental targets in ESCs. Additionally, Oct1 occupies developmental targets as cells differentiate and Oct4 is lost. These results are consistent with a role for Oct1 in promoting the expression of lineage-specific genes in differentiating cells while simultaneously repressing genes specific for alternative lineages.
2017-05-24 | GSE85062 | GEO
Project description:Embryonic stem cells (ESCs) co-express Oct4/POU5F1 and an Oct4 paralog known Oct1/POU2F1. To study the role of Oct1 in embryonic stem cell transcriptional control and pluripotency, we constructed germline and inducible-conditional Oct1 deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal, growth rates and metabolic signatures. However loss of Oct1 results in defective lineage specification. ESCs lacking Oct1 fail to form beating cardiomyocytes in culture, generate neurons poorly, form smaller, less differentiated teratomas, and are incapable of generating chimeric mice. Upon RA-mediated neuronal differentiation, Oct1 deficient cells fail to properly induce developmentally poised genes. Simultaneously, genes for alternative developmental pathways, most notably for the development of extra-embryonic tissues, are inappropriately expressed. ChIP experiments show that Oct1 does not occupy pluripotency genes or differentially expressed developmental targets in ESCs. Additionally, Oct1 occupies developmental targets as cells differentiate and Oct4 is lost. These results are consistent with a role for Oct1 in promoting the expression of lineage-specific genes in differentiating cells while simultaneously repressing genes specific for alternative lineages.
2017-05-24 | GSE85061 | GEO
Project description:The tumor suppressor BRCA1 regulates DNA damage responses and multiple other processes. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal RING domain. Here we show that BRCA1 promotes oxidative metabolism via degradation of Oct1, a transcription factor with pro-glycolytic/tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells towards a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNAseq confirms the deregulation of metabolic genes. BRCA1 mediates direct Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenografts. Oct1 protein levels correlate positively with tumor aggressiveness, and inversely with BRCA1, in primary breast cancer samples. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism.
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