Epigenetic regulation of the estrogen receptor alpha promoter in the cerebral cortex following ischemia in male and female rats

Abstract

Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERα) dependent manner. ERα is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERα mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERα expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERα expression after ischemia. Twenty-four hours after surgery, mRNA or genomic DNA was collected from 1 mm micropunches taken from 300 μm brain sections for quantitative reverse transcription-polymerase chain reaction (RT-PCR) or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20 μm sections were processed for ERα immunohistochemistry. In OVX females, ERα mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERα in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5′ untranslated region (UTR) of the rat ERα gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERα 5′UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERα expression in response to MCAO between males and females and that methylation of the ERα gene corresponds with mRNA levels in the brain.