The role of microRNAs, DNA methylation and translational control in regulation of sex specific gene expression in mouse liver
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Sex differences are widespread in both mouse and human liver, and are associated with sex differences in drug metabolism and liver pathophysiology. The secretory patterns of growth hormone (GH) is one of the major drivers of liver sex specificity, where intermittent and continuous secretion in male and female respectively lead to sex bias in the expression of more than 1000 genes in mouse liver, via a complex interplay of GH-responsive transcription factors and epigenetic mechanisms. This thesis explores three themes of molecular control in the regulation of liver sex differences: microRNAs, DNA methylation, and translational control. Studies herein identified two microRNAs, miR-1948-5p and miR-802-5p, whose expression is sex biased and regulated by GH and the transcription factor STAT5b. Small RNA sequencing confirmed the sex specificity of these two microRNAs and identified an additional 18 sex-biased microRNAs. Computational and experimental characterization of miR-1948-5p and miR-802-5p confirmed their authenticity. In vivo inhibition of these microRNAs by locked nucleic acids indicated that miR-1948-5p and miR-802-5p played a functional role in repressing female-biased genes and male-biased genes, respectively. This thesis also investigated the impact of GH and STAT5b on liver DNA methylation profiles. Reduced representation bisulfite sequencing was performed on liver tissues from four mouse models that perturbed the GH and STAT5b axis. In the wildtype liver, sex biased demethylation was positively associated with sex biased chromatin opening and gene expression. Global hypermethylation was observed in livers of mice with lit/lit mutation resulting in GH deficiency or with hepatocyte-specific deletion of the STAT5ab locus. Strikingly, these hypermethylated loci were enriched for enhancer elements and STAT5b binding sites found in wild-type mouse liver. Hypophysectomy followed by GH replacement mouse models identified differentially methylated regions that were sex-biased and rapidly methylated and demethylated in response to GH stimulation. Finally, we used ribosome profiling to validate sex-biased protein translation and identify mechanisms of translational control. In sum, this body of work provides novel insights and broadens our understanding of the diverse molecular mechanisms underlying sexual dimorphism in the liver.