Egr1 is a sex-specific regulator of neuronal chromatin, synaptic plasticity, and behaviour [ATAC-Seq]

Sex differences are found in brain structure and function across species, and across brain disorders in humans. The major source of brain sex differences is differential secretion of steroid hormones from the gonads across the lifespan. Specifically, ovarian hormones oestrogens and progesterone are known to dynamically change structure and function of the adult female brain, having a major impact on psychiatric risk. However, due to limited molecular studies in female rodents, very little is still known about molecular drivers of female-specific brain and behavioural plasticity. Here we show that overexpressing Egr1, a candidate oestrous cycle-dependent transcription factor, induces sex-specific changes in ventral hippocampal neuronal chromatin, gene expression, and synaptic plasticity, along with hippocampus-dependent behaviours. Importantly, Egr1 overexpression mimics the high-oestrogenic phase of the oestrous cycle, and affects behaviours in ovarian hormone-depleted females but not in males. We demonstrate that Egr1 opens neuronal chromatin directly across the sexes, although with limited genomic overlap. Our study not only reveals the first sex-specific chromatin regulator in the brain, but also provides functional evidence that this sex-specific gene regulation drives neuronal gene expression, synaptic plasticity, and anxiety- and depression-related behaviour. Our study exemplifies an innovative sex-based approach to studying neuronal gene regulation in order to understand sex-specific synaptic and behavioural plasticity and inform novel brain disease treatments. Male and ovariectomized, or intact proestrus female C57BL/6J mice underwent stereotaxic surgery at 7 weeks of age and received either Egr1 overexpression (AAV9-hSYN-Egr1-IRES-eGFP-WPRE3), Egr1 knockdown (AAV9-hSYN-Egr1-shRNA-eGFP-WPRE), eGFP control (AAV9-hSYN-eGFP-WPRE), or scrambled shRNA control (AAV9-hSYN-scramble-eGFP-WPRE) AAV targeted to the ventral hippocampus to determine the sex-dependent effects of Egr1 overexpresssion or knockdown on the neurons in this brain region. Following 3 weeks of incubation, mice were sacrificed at 10-11 weeks of age and bilateral ventral hippocampi were flash frozen in liquid nitrogen. Nuclei were then extracted from frozen tissue and neuronal nuclei were obtained by FACS using an antibody targeting the neuronal nuclei-specific NeuN protein. The ATAC-seq libraries were prepared from 50,000 NeuN+ nuclei for each sample. 150 bp, paired-end sequencing was performed in one lane of an S1 flow cell on the NovaSeq 6000 instrument, yielding 40-75 million reads per sample.