# Sex chromosome–dependent epigenetic regulation underlies sex-specific H4 acetylation at the aromatase promoter in the developing mouse amygdala

## TL;DR

This study shows that sex chromosomes influence epigenetic regulation in the mouse brain, leading to sex-specific differences in aromatase expression during early development.

## Contribution

The study identifies sex chromosome-dependent epigenetic regulation of aromatase in the developing mouse amygdala.

## Key findings

- XX embryos show higher expression of DNA methyltransferases and histone deacetylases in a region-specific manner.
- Acetyl-H4 enrichment at the Cyp19a1 promoter was observed only in male amygdala neuronal cultures.
- Zebularine did not significantly affect aromatase expression in amygdala neurons.

## Abstract

Sexual differentiation of the brain is a complex ontogenetic process orchestrated by genetic and hormonal influences, leading to sex‑specific physiological and behavioral traits in adulthood. In mammals, the sex chromosome complement (SCC) contributes to this process by encoding unequal genetic information in XX and XY cells. Furthermore, SCC upregulates aromatase and estrogen receptor β (ERβ) expression in amygdala neurons of XY compared to XX embryos at embryonic day (E) 14. These molecules are critically implicated in the steroid-dependent programming of neural circuits during the subsequent critical window of sexual differentiation (E17-PN10). Since epigenetic mechanisms play a key role in specific target gene expression forming a layer of gene regulation, we aimed to contribute to a better understanding of their impact on the sexual differentiation of the brain.

Four Core Genotypes mouse model was employed to study the epigenetic machinery involved in DNA methylation and histone deacetylation in different brain regions (amygdala, hypothalamus, and cortex) to elucidate the underlying epigenetic landscape at E14 by RT-qPCR. Amygdala primary neuronal cultures were then established to evaluate the epigenetic regulation of Cyp19a1 (aromatase) and Esr2 (ERβ) expression. To assess this, pharmacological inhibition of DNA methylation, using zebularine, as well as Chromatin Immunoprecipitation (ChIP-qPCR) assays were performed.

Sex-specific expression of DNA methyltransferases 3a and 3b, along with histone deacetylases 2 and 8, was higher in XX than XY embryos in a region- and developmental stage- dependent manner. Pharmacological inhibition of DNA methylation did not significantly alter aromatase expression in male or female amygdala neuronal cultures under the conditions tested. However, ChIP-qPCR assays revealed a selective enrichment of Acetyl-H4 at the Cyp19a1 promoter in male cultures that was not observed in females. No significant enrichment of the examined epigenetic marks was detected at the Esr2 promoter.

Acetylation of histone H4 contributes to promoting the higher Cyp19a1 expression previously observed in male neurons. Our findings support a model in which SCC plays a role in the epigenetic regulation of aromatase, a key enzyme involved in hormone-driven sexual differentiation of the male brain. Furthermore, the presence of two X chromosomes shapes a distinct epigenetic landscape in the brain during early development, highlighting the influence of chromosomal sex on the neurodevelopmental programming.

The online version contains supplementary material available at 10.1186/s13293-026-00854-4.

Sex chromosomes regulate epigenetic machinery during early brain development in mice.

XX embryos show higher expression of DNA methyltransferases (Dnmt3a, Dnmt3b) and histone deacetylases (Hdac2, Hdac8) in a region-specific manner.

Zebularine-mediated DNA inhibition did not significantly affect aromatase (Cyp19a1) expression in amygdala neurons.

Acetyl-H4 enrichment at Cyp19a1 promoter was observed only in male amygdala neuronal cultures, indicating histone acetylation drives sex differences in aromatase expression.

Sex chromosome-dependent epigenetic mechanisms may shape aromatase regulation, contributing to sexual differentiation of the brain.

The online version contains supplementary material available at 10.1186/s13293-026-00854-4.