LAUSR

Abstract Purpose: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We reported that angiotensin II could change the DNA methylation status around transcription factor binding sites and a transcription start site (TSS) and activate expression of the aldosterone synthase gene (CYP11B2). In order to clarify the effect of potassium on the methylation status of CYP11B2 H295R cells were treated with high concentration of potassium and the methylation status of CYP11B2 was examined. Methods: H295R cells were treated with 16mM potassium for 7 days and observed the dynamic changes in DNA methylation patterns of the CYP11B2 promoter for 28 days. The gene expression of CYP11B2 was measured by real time quantitative PCR. Isolated DNAs from H295R cells were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions. Non-treated cells served as a control. Human CYP11B2 ELISA, chromatin accessibility by real-time PCR (CHART-PCR), DNA methylation and demethylation activity assay were done as previously reported. Results: DNA demethylation at CpG1 (Ad1) was detected within 2 days after stimulation. DNA at CpG2 (Ad5) and CpG3 and DNA around a transcription start site (CpG-1/-2) were demethylated at day 4 in cells treated with potassium. CYP11B2 mRNA levels in cells treated with potassium were significantly higher than those in non-treated cells at days 7 and 9. Similar changes were observed in the levels of CYP11B2 protein. CHART-PCR revealed that potassium treatment increased chromatin accessibility around Ad1 at days 2, 4, 7, 9 and 11, with accessibility increasing from 67% to 70%. Potassium treatment significantly reduced DNA methylation activity at days 7 and 9. Demethylation activity was not affected by potassium. Conclusion: Our data indicated that binding of transcription factors initiates chromatin relaxation and transcription, which are followed by DNA demethylation around transcription factor binding sites and a TSS in the CYP11B2 promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of CYP11B2 expression from an inactive to an active state.