Estrogen receptor regulation of brain sexual differentiation

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PROJECT SUMMARY/ABSTRACT In sexually reproducing species, males and females display different social and reproductive behaviors, such as mating and aggression. These behaviors typically require no training, indicating they are developmentally programmed in the brain. In many mammalian species, including primates and rodents, sexual differentiation of the brain is regulated by nuclear receptor transcription factors (TFs), which bind gonadal steroid hormones, such as testosterone and estrogens. In mice, a perinatal surge of testosterone permanently masculinizes a key brain region controlling sex-typical behaviors, called the bed nucleus of the stria terminalis (BNST). Within the perinatal BNST, testosterone is converted to estradiol, which activates estrogen receptor α (ERα). Previous genetic knockout and pharmacological studies demonstrate perinatal ERα signaling is necessary and sufficient for masculinization of BNST circuitry and behavior. Specifically, ERα activation leads to a male-bias in BNST cell survival and axon guidance, particularly to the anteroventral periventricular nucleus (AVPV), between postnatal day 4 (P4) and P10. This project seeks to identify and characterize ERα genomic binding sites and target genes involved in BNST sexual differentiation. Recently our group discovered that estradiol regulates the expression of the netrin receptor, Unc5b, in adult BNST ERα+ cells. Unc5b is robustly expressed in the neonatal BNST and has previously been shown to regulate neuron survival and axon guidance. Aim 1 of this project investigates whether estradiol regulation of Unc5b contributes to BNST sexual differentiation, using mice lacking Unc5b expression in ERα+ cells (Esr1cre/+;Unc5blx/lx). The experiments in Aim 1 test the hypothesis that estradiol regulation of Unc5b contributes to male-biased BNST cell survival and/or AVPV innervation. Because ERα likely masculinizes the BNST through multiple biological pathways, Aim 2 of this project seeks to identify the complete repertoire of ERα genomic binding sites and target genes in the developing BNST. Previously, I discovered the first ERα genomic binding sites in the adult brain, using a recently published low-input TF profiling method, called CUT&RUN (Cleavage Under Targets & Release Under Nuclease). This approach revealed brain-specific ERα binding sites are enriched near genes involved in neurodevelopmental processes. Aim 2 of this application uses CUT&RUN to measure estradiol-regulated ERα binding sites in the perinatal BNST/hypothalamus. Aim 2 also measures perinatal estradiol-regulated gene expression in P4 BNST ERα+ cells. Using a CRISPR-mediated activation (CRISPRa) system in primary neurons, distal ERα binding sites will be causally linked to the expression of perinatal estradiol-regulated genes. Overall, the project will reveal how nuclear receptors regulate genes involved in brain sexual differentiation and, in doing so, will provide novel insight into the molecular basis of sex-biased mental health conditions and neurodevelopmental diseases.