Sex differences in microRNA-mRNA networks: examination of novel epigenetic programming mechanisms in the sexually dimorphic neonatal hypothalamus

Christopher P. Morgan, Tracy L. Bale

Sexual differentiation of the male brain, and specifically the stress circuitry in the hypothalamus, is primarily driven by estrogen exposure during the perinatal period. Surprisingly, this single hormone promotes diverse programs of sex-specific development that vary widely between different cell types and across the developing male brain. The complexity of this phenomenon suggests that additional layers of gene regulation, including microRNAs (miRNAs), must act downstream of estrogen to mediate this specificity. To identify noncanonical mediators of estrogen-dependent sex-specific neural development, we assayed the miRNA complement of the mouse PN2 hypothalamus by microarray following an injection of vehicle or the aromatase inhibitor, formestane. Initially, multivariate analyses were used to test the influence of sex and experimental group on the miRNA environment as a whole. Then, we utilized traditional hypothesis testing to identify individual miRNA with significantly sex-biased expression. Finally, we performed a transcriptome-wide mapping of Argonaute footprints by high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (Ago HITS-CLIP) to empirically characterize targeting relationship between estrogen-responsive miRNAs and their messenger RNA (mRNA) targets. In this study, we demonstrated that the neonatal hypothalamic miRNA environment has robust sex differences and is dynamically responsive to estrogen. Analyses identified 162 individual miRNAs with sex-biased expression, 92 of which were estrogen-responsive. Examining the genomic distribution of these miRNAs, we found three miRNA clusters encoded within a 175-kb region of chromosome 12 that appears to be co-regulated by estrogen, likely acting broadly to alter the epigenetic programming of this locus. Ago HITS-CLIP analysis uncovered novel miRNA-target interactions within prototypical mediators of estrogen-driven sexual differentiation of the brain, including Esr1 and Cyp19a1. Finally, using Gene Ontology annotations and empirically identified miRNA-mRNA connections, we identified a gene network regulated by estrogen-responsive miRNAs that converge on biological processes relevant to sexual differentiation of the brain. Sexual differentiation of the perinatal brain, and that of stress circuitry in the hypothalamus specifically, seems to be particularly susceptible to environmental programming effects. Integrating miRNA into our conceptualization of factors, directing differentiation of this circuitry could be an informative next step in efforts to understand the complexities behind these processes.