Role of epigenetic modifications in the aberrant CYP19A1 gene expression in polycystic ovary syndrome

Abstract

Introduction: In this study, the global DNA methylation, histone acetylation and methylation levels of cumulus cells (CCs) in infertile polycystic ovary syndrome (PCOS) patients and the correlation of these epigenetic modifications with the expression of the ovarian aromatase gene (as an important marker in the etiology of PCOS) were investigated.

Material and methods: A cross-sectional study was conducted on 24 patients (12 PCOS patients and 12 healthy women), who underwent ovarian stimulation. Nucleosome ELISA was performed, in order to identify the global occupancy level of Mecp2 (as a marker of DNA methylation) and H3K9me2/H3K9ac as histone modification markers in chromatin fractions obtained from CCs. The CYP19A1 gene expression was measured by qRT-PCR. The level of DNA incorporation of MeCP2, histone modification markers and binding of estrogen receptor β (ERβ) to CYP19A1 regulatory sequences were examined by ChIP-QPCR assay.

Results: The data demonstrate a significant increase in global occupancy levels of MeCP2 and H3K9ac markers and a decrease of H3K9me2 to chromatin in CCs of PCOS patients vs. control group. Furthermore, CYP19A1 gene expression, and the incorporation of H3K9ac in PII, PI.3, and PI.4 promoters of CYP19A1 in PCOS, were higher than those of controls. Also, significant hypomethylation of H3K9 at PII and DNA hypomethylated at PII and PI.3 promoters and differential binding of ERβ to three promoters were observed in PCOS patients (p < 0.05).

Conclusions: Aromatase expression can be affected by epigenetic modifications and differential ERβ binding to the proximal CYP19A1 promoters. These mechanisms may be involved in the enhanced aromatase transcription during ovarian stimulation in PCOS patients.

Keywords: CYP19A1; cumulus cell; epigenetic; estrogen receptor β; polycystic ovary syndrome.

Figure 1

Differential incorporation of epigenetic marks (MeCP2, H3K9me2, and H3K9ac) into chromatin of cumulus cells of PCOS patients vs. control normalized to histone H1 content

Figure 2

Relative mRNA expression of CYP19A1 gene in human cumulus cells of patients with PCOS and control groups

Figure 3

Analysis of the epigenetic profile of CYP19A1 gene. A – Schematic diagram of CYP19A1 gene promoters (PII, PI.3, and PI.4) examined by chromatin immunoprecipitation (ChIP) assay. Regions amplified by qPCR are shown by arrows, and nucleotide numbers are relative to the transcription start site (TSS). B – Incorporation of MeCP2, H3K9me2, and H3K9ac into PII, PI.3, and PI.4. C – Incorporation of ERβ into three evaluated regulatory regions of the CYP19A1 gene. The results are expressed relative to a 1/100 dilution of input chromatin (mean ± SEM)

Figure 4

Correlations between CYP19A1 mRNA expression levels and DNA methylation or histone modifications in CCs analyzed using Pearson’s correlation *Correlation is significant at the 0.05 level, **correlation is significant at the 0.01 level.