DNA methylation and demethylation underlie the sex difference in estrogen receptor alpha in the arcuate nucleus

Abstract

IntroductionNeurons expressing estrogen receptor (ER) in the arcuate nucleus (ARC) of the hypothalamus sex-specifically control energy homeostasis and bone density. Females have more of these neurons than do males, but how this sex difference develops is unknown. ObjectiveWe tested the hypothesis that DNA methylation and/or demethylation control the development of a sex difference in ER in the ARC. MethodsER immunoreactive neurons were quantified at birth and at weaning in male, female and testosterone-treated female mice that received neonatal, intracerebroventricular injections of vehicle or zebularine, a DNA methyltransferase inhibitor. Methylation status of Esr1 was determined in the ARC and ventromedial hypothalamus (VMH) using bisulfite conversion of DNA followed by pyrosequencing. Small interfering RNAs against ten-eleven translocases were used to examine effects of demethylation on ER cell number. ResultsA sex difference in ER cell number in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Zebularine treatment eliminated the sex difference in ER in the ARC at weaning by decreasing ER in females to male-like levels. Previously, the same treatment increased ER in males in the VMH. A promoter region of Esr1 exhibited sex differences in opposite directions in percent of total methylation in the ARC (females > males) and VMH (males > females). Moreover, neonatal inhibition of demethylation increased ER in the ARC of males. ConclusionDNA methylation and demethylation regulate ER cell number in the ARC, and methyl marks may paradoxically activate Esr1 in this region.