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Abstract

Genome-wide expression and methylation studies in patients with pancreatic adenocarcinoma (PDAC) indicate that numerous genes involved in the development of cancer are highly methylated in their promoter regions but are nevertheless strongly transcribed. The mechanisms underlying the relationship between altered DNA methylation and increased transcription, as well as the effects on cancer development remain elusive.

Recent systematic investigations have shown that many transcription factors (TFs) which lack methyl-CpG binding domains (MBDs) can also bind to methylated DNA in vitro and in vivo. As a consequence, the binding preference of such TFs to mCpG leads to the activation of gene expression, the splicing regulation, and the chromatin remodeling. Based on these observations, it’s hypothesized that TFs that specifically bind to highly methylated promoters are involved in the regulation of transcription activity.

With the help of protein microarrays covering 667 DNA binding domains of TFs, the binding patterns of the methylated/unmethylated promoter together with TFs were analysed after incubating the promoter on the TF-microarray. The analysis results showed that the transcription factors NFATc1/2/3 (Nuclear Factor of Activated T cells 1/2/3) of the NFAT family preferentially bound to the methylated promoters. Afterward, NFATc1 was selected for further investigation since it was upregulated in PDAC tissues compared with healthy tissues. The viability, colony, and migration assays indicated that NFATc1 played an oncogenic role in pancreatic cancer cell lines (Panc1 and Miapaca2). To better understand how NFATc1 regulates transcription, mRNA profiling of NFATc1-knockdown cells was used to determine the down-regulated genes. The decreased expression of ALDH1A3 was confirmed further by q-PCR. Next, in silico analysis revealed that multiple methylated/unmethylated binding sites of NFATc1 were in the promoter region of ALDH1A3. In addition, luciferase assay and chromatin immunoprecipitation (ChIP) verified that NFATc1 directly regulated the transcription of ALDH1A3. Moreover, the in vitro methylation and the ex vivo demethylation assay also showed that NFATc1 regulated the transcription of ALDH1A3, though the promoter region was methylated.

In summary, this work reveals that NFATc1 plays an oncogenic role in pancreatic cancer cell lines (Panc1 and Miapaca2) and regulates the transcription of ALDH1A3, though DNA methylation is in its promoter region. The elucidation of the methylation-dependent binding of NFATc1 provides insights for a better understanding of methylation-mediated biological processes.