Our grant application tests the hypothesis that sex differences in the brain are regulated by epigenetic events during a perinatal critical period. In many vertebrates, including mice, sex-specific neural circuitry develops under the control of estrogen signaling during the first few days of life. Treating neonatal females with estrogen irreversibly masculinizes adult social behavior and gene expression. However, the molecular strategies used by estrogen to exert lasting effects on the brain are poorly understood. The goal of this proposal is to identify sex differences in gene expression and chromatin in two sexually dimorphic brain regions. The posterior division of the bed nucleus of the stria terminalis (BNST), and the medial amygdala (MeA) are highly interconnected brain regions that develop under the control of neonatal estrogen and regulate innate sex- specific social behaviors such as mating and aggression. We hypothesize that neonatal estrogen generates male-specific chromatin states that fundamentally alter the cellular identity of neurons and thus their function in behavioral circuitry. We will test this hypothesis through genome-wide analysis of gene expression and chromatin specifically in ERα-expressing neurons in both pups and adults. In Specific Aim 1 we will determine the sex-specific gene programs in the BNST/MeA and explore how these programs are acutely modulated by distinct adult hormonal profiles in males and females. In Specific Aim 2 we will identify cis-regulatory elements, such as enhancers, in ERα neurons from BNST/MeA, and investigate sex differences in transcription factor occupancy of these elements. In Specific Aim 3 we will test the requirement for a novel sexually dimorphic transcription factor in generating sex differences in gene expression and behavior. Taken together, our findings will reveal how estrogen signaling during early life permanently influences adult gene expression and ultimately, sex-specific behaviors. This work will provide insight into how a transient event during a critical developmental period can have significant impact on the brain and behavior in adulthood. This critical period permanently affects brain structures and function, suggesting that sex differences in psychiatric disorders, such as autism and depression, may originate during sexual differentiation of the brain.