Changes in Epigenetic Status in the Promoter Region of Steroidogenic Acute Regulatory (StAR) Gene in Rat Granulosa Cells During Luteinization.

Abstract

Ovulatory LH surge rapidly induces gene expression of StAR, which is a rate-limiting step for progesterone synthesis in luteal cells. Recent evidence has shown that epigenetic mechanisms such as histone modification and DNA methylation are essentially involved in the regulation of gene expression. The present study investigated the involvement of epigenetic mechanisms in the rapid increase in StAR mRNA expression induced by ovulatory LH surge in luteinized granulosa cells during luteinization. To induce ovulation, 21-days-old immature rats were injected with PMSG (15 IU) followed by hCG (15 IU) injection 48 h later. The ovaries were removed and luteinized granulosa cells were collected before hCG (0 h), and 1, 2, 4, 8, and 12 h after hCG injection. StAR mRNA levels gradually increased after hCG injection, reached the peak at 4h, and decreased thereafter. Histone acetylation status and histone methylation status in the StAR promoter region (-492 bp ~ -42 bp) were analyzed by chromatin immunoprecipitation assay among cells collected at 0, 4 and 12 h after hCG injection. Histone H4 acetylation levels were significantly increased at 4 h compared with 0 h, and then decreased to the basal level at 12 h, whereas histone H3 acetylation status was not changed at any times studied. The levels of tri-methyl histone H3K4, which promotes transcription, were gradually increased after hCG injection and significantly higher at 12 h than at 0 h. The levels of tri-methyl histone H3K9, which suppresses transcription, were gradually decreased and significantly lower at 12 h than at 0 h. DNA methylation status was analyzed in the StAR promoter region including the distal region (17 CpG sites, -1675 bp ~ -42bp) by sodium bisulfite genomic sequencing method. Six CpG sites in the promoter region (-503 bp ~ -42 bp) were demethylated and the other 11 CpG sites in the distal region (-1675 bp ~ -754 bp) were methylated. This DNA methylation profile did not change at any times studied during luteinization induced by hCG injection. These results led us to study whether histone acetylation of the promoter region is involved in StAR mRNA expression. We used RLC-16 cells, which show low histone acetylation status of the StAR promoter and do not express StAR gene. To experimentally induce histone acetylation, cells were treated with histone deacetylase (HDAC) inhibitors. Treatment with HDAC inhibitors induced StAR mRNA expression with induction of histone acetylation of the StAR promoter region. In conclusion, our results show that increases in histone acetylation levels and changes in histone methylation status in the promoter region by ovulatory LH surge in addition to DNA hypomethylation status of the promoter are closely associated with the rapid increase in StAR mRNA expression in luteinized granulosa cells during luteinization. The changes in epigenetic status in the StAR promoter region may be involved in StAR gene expression through the change in chromatin structure in rat granulosa cells during luteinization. (poster)