Differential DNA methylation of the CYP11B1 and CYP11B2 genes in association with human adrenal zonation

M. Demura; F. Wang; T. Yoneda; M. Kometani; T. Sawamura; Y. Cheng; K. Ono; Y. Nakamura; H. Sasano; K. Saijoh; Y. Takeda

Background: The human adrenal cortex is composed of 3 distinct zones: the zona glomerulosa (ZG), the zona fasciculata (ZF), and the zona reticularis (ZR). Steroidogenic enzymes are expressed in a zone-specific manner. The promoters of CYP11B1 and CYP11B2 possess a number of target sites for DNA methylation that is closely associated with gene silencing.

Methods and Materials: Adrenals from 5 autopsy subjects were assessed for immunohistrochemically defined adrenal zonation, using antibodies for 17alpha-hydroxylase/17,20-lyase (CYP17), 3beta-hydroxysteroid dehydrogenase (HSD3B), and dehydroepiandrosterone (DHEA)-sulfotransferase (SULT2A1). CYP17, HSD3B, and SULT2A1 are entirely expressed in ZF/ZR, ZG/ZF, and ZR, respectively. We used laser capture microscopy to isolate DNA from each zone in adrenal tissues and performed an analysis of DNA methylation. Promoter methylation patterns of CYP11B1 and CYP11B2 in adrenal zones were examined by means of bisulfite sequencing.

Results and Discussions: CYP11B1 was completely hypomethylated in ZF while completely hypermethylated in both ZG and ZR. In contrast, CYP11B2 in ZG was hypomethylated compared with those in ZF and ZR. Inter-individual diversity was observed in DNA methylation of CYP11B2 in ZR. The important differences in DNA methylation were that CYP11B1 and CYP11B2 were hypomethylated in ZF and ZG, respectively compared with other zones and that hypomethylation of CYP11B2 in ZG was incomplete whereas that of CYP11B1 in ZF was complete. 11beta-hydroxylase (CYP11B1) has been reported to be expressed highly in ZF and weakly in ZR while aldosterone synthase (CYP11B2) has been sporadically expressed only in ZG. NR4A receptor family involved in the regulation of CYP11B2 has been shown to be differentially expressed in a zone-specific manner.

Conclusions: Taken together, DNA methylation patterns of the CYP11B1 and CYP11B2 promoters were closely associated with adrenal zonation. The DNA methylation patterns, however, do not fully explain zone-specific expression patterns of 11beta-hydroxylase and aldosterone synthase. DNA methylation is considered important for adrenal zonation in conjunction with zone-specific transcription factors. Since the expression of aldosterone synthase depends on salt intake, incomplete DNA hypomethylation of CYP11B2 in ZG is ascribed to high-sodium diets in contemporary society.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.

Endocrine Abstracts

P35