Sex differences in mammalian gene expression result from differences in genotypic sex as well as in hormonal regulators between males and females. In rat, mouse and human liver, ~1000 genes are expressed in a sex-dependent manner, and contribute to sex differences in metabolism of drugs, steroids and lipids, and in liver and cardiovascular disease risk. In rats and mice, sex-biased liver gene expression is primarily dictated by the sexually dimorphic pattern of pituitary growth hormone (GH) release and its STAT5-dependent transcriptional activities. Studies presented in this thesis include the following. (1) A computational approach based on DNA sequence and phylogenetic conservation was developed and used to identify novel functional STAT5 binding sites - both consensus and non-consensus STAT5 sequences - near prototypic GH-responsive genes. (2) Global gene expression analysis of livers from pituitary-ablated male and female mice identified four major classes of sex-biased genes differing in their profiles of GH dependence. (3) Sex-differences in DNase-hypersensitive sites (DHS, corresponding to open chromatin regions) were identified genome-wide in mouse liver. These sex-differential DHSs were enriched for association with sex-biased genes, but a majority was distant from sex-biased genes. Furthermore, many were responsive to GH treatment, demonstrating that GH-mediated regulation involves chromatin remodeling. Analysis of sequence motifs enriched at sex-biased DHSs implicated STAT5 and novel transcription factors such as PBX1 and TAL1 in sex-biased gene regulation. (4) Genome-wide mapping of histone modifications revealed distinct mechanisms of sex-biased gene regulation in male and female liver: sex-dependent K27me3-mediated repression is an important mechanism of repression of female-biased, but not of male-biased, genes, and a sex-dependent K4me1 distribution, suggesting nucleosome repositioning by pioneer factors, is observed at male-biased, but not female-biased, regulatory sites. STAT5-mediated activation was most strongly associated with sex-biased chromatin modifications, while BCL6-mediated repression primarily occurs in association with sex-independent chromatin modifications, both at binding sites and at target genes. The relationships between sex-dependent chromatin accessibility, chromatin modifications and transcription-factor binding uncovered by these studies help elucidate the molecular mechanisms governing sex-differential gene expression, and underscore the utility of functional genomic and epigenetic studies as tools for elucidating transcriptional regulation in complex mammalian systems.

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. / 2020-10-08T00:00:00Z

Long non-coding RNAs (lncRNAs) are key regulators of gene expression, playing crucial roles in biological processes across many species, tissues and diseases. The liver is a highly responsive organ in which large changes in gene expression are perpetuated by a myriad of internal and external stimuli; as such, the liver makes an ideal system in which to study lncRNAs. Global patterns of expression, maturation and localization were established for both lncRNA and protein-coding gene (PCG) transcripts across five subcellular compartments in male and female mouse liver, both with and without exposure to TCPOBOP, a direct agonist of the nuclear receptor CAR. In contrast to PCGs, lncRNAs showed very strong enrichment for tight chromatin binding, which increased the sensitivity for lncRNA detection and facilitated discovery of many novel sex-biased and xenobiotic-responsive lncRNAs. These findings helped identify candidate regulatory lncRNAs based on their co-localization within topologically associating domains, or their transcription divergent or antisense to PCGs associated with pathways linked to liver physiology and disease. The liver cell type-specific expression of lncRNAs and PCGs was assessed by single nucleus RNA-seq (snRNA-seq). Liver sexual dimorphism was largely restricted to hepatocyte populations, where many sex-biased genes exhibited zonated expression. Changes in lncRNA and PCG expression following exposure to endogenous hormones (growth hormone) and exogenous chemicals (TCPOBOP) was assessed, identifying cell cluster-specific perturbations to native sex-bias and hepatocyte zonation-dependent gene expression, and highlighting the interconnectedness between liver sexual dimorphism and zonation of the hepatic lobule at the single nuclei level. Finally, an in vivo method for epigenetic reprogramming of lncRNAs using a dual adeno-associated virus delivery system was utilized to knockdown two TCPOBOP-inducible lncRNAs in mouse liver. The knockdown phenotype of one of these lncRNAs, established by snRNA-seq, suggests it plays a functional role in regulating cholesterol metabolism and transport, triglyceride catabolism, and pyruvate metabolism in mouse liver. Together, these studies characterize hepatic lncRNA expression patterns, on both the sub-cellular and single cell levels, and present a strategy for interrogating the roles of specific lncRNAs in liver tissue in vivo. / 2023-01-18T00:00:00Z