Histone h4 acetylation involvement in sexually dimorphic aromatase expression of developing mouse brain

Abstract

During early development, sex chromosome complement (SCC) regulates a sexually dimorphic gene expression in limbic regions of the mouse brain. Furthermore, aromatase and ERβ expression are higher in amygdala neurons of XY than XX embryos at embryonic day (E) 15, before of critical period of hormone-induced brain masculinization. Epigenetic has been proposed as mediator of hormonal and genetic sexual differentiation of the brain. We aimed to study the role of SCC on the epigenetic mechanisms involved in brain sexual differentiation. Four core genotypes mouse model (FCG) was employed to evaluate by RT- qPCR the epigenetic machinery involved in DNA methylation and histone deacetylation in amygdala at E15. Moreover, the epigenetic regulation of aromatase and ERβ expression was analyzed by Chromatin Immunoprecipitation (ChIP-qPCR) from E15 amygdala primary neuronal cultures segregated by sex using anti-Acetyl-H3 and -H4 antibodies. Independent cultures were performed to evaluate the effect of pharmacological inhibition of DNA methylation (using zebularine) on the aromatase gene expression by RT-qPCR. We found that SCC regulate the sexually dimorphic expression of de novo DNA methyltransferase 3a and 3b, and histone deacetylase 2 and 8 with higher expression in XX than XY embryos. Zebularine did not change aromatase expression levels neither in male nor female cultures. However, ChIP assays showed an enrichment of Acetyl-H4 in the male aromatase promoter that was not observed in female cultures. The ERβ promoter did not show a significant enrichment of the explored marks. In summary, the acetylation of H4 could be contributing to relax chromatin structure in male amygdala neurons, thereby facilitating the access of the transcriptional machinery to the aromatase gene promoter leading to the higher expression previously observed in males. These results contribute to a better understanding of the role of epigenetics in the establishment of brain sex differences independently of hormonal masculinization.