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Updated: Feb 16, 2026
Methylated DNA Immunoprecipitation
Published on: January 2, 2009
Purkinje Cell Protein 4 Expression Is Associated With DNA Methylation Status in Aldosterone-Producing Adenoma
Kazuhiro Kobuke1, Kenji Oki1, Celso E Gomez-Sanchez2
1Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Context:
Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation.
Objective:
Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels.
Methods:
Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells.
Results:
The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells.
Conclusions:
We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.
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