The liver is the largest internal organ and performs a wide variety of functions to maintain organismal homeostasis. While some liver functions are carried out by all hepatocytes, other functions are restricted to certain populations of hepatocytes within the liver. This phenomenon, called zonal gene regulation or liver zonation, controls may metabolic processes within the liver including ammonia detoxification; glucose homeostasis; bile acid and glutamine synthesis; and metabolism of xenobiotics, lipids, and amino acids. The liver also expresses many genes in a developmental or sex-biased manner. Some genes are expressed at higher levels early or late in development, or alternatively, in male or female liver. Several years ago, our lab identified a transcription factor called Zinc finger and homeoboxes 2 (Zhx2) based on its ability to control the silencing of genes that are normally expressed in the fetal liver. Zhx2 belongs to a small gene family that also includes Zhx1 and Zhx3. These four exon genes have a rather unique structure in that their entire protein coding region is located on an unusually large third exon. Preliminary studies indicate that these proteins are found only in vertebrates. I have performed a comprehensive analysis of Zhx proteins across a number of chordate species to determine their relationship throughout chordate evolution. Using multiple sequence alignment and phylogenetic tree-building, my studies have found that the primordial Zhx gene is most related to Zhx3 and that this gene exists in lower chordates including lancelet, sea squirt, and sea lamprey. Additional studies from our lab showed that Zhx2 regulates numerous hepatic genes in the adult liver, including cytochrome p450 (Cyp) genes as well as other genes that exhibit sex-biased expression. Previous studies have demonstrated that female-biased expression of Cyp2a4, is controlled, in part, by Zhx2. I have extended these studies to perform a comprehensive analysis of Cyp2a4 and the highly related Cyp2a5 gene. Despite the high similarity of these two Cyp genes, my data indicate that these genes exhibit different zonal expression patterns and are differentially regulated in the regenerating liver. In the course of these studies, I discovered and characterized antisense transcripts for both Cyp2a4 and Cyp2a5. Both Cyp2a4as and Cyp2a5as have positively correlated expression patterns compared to their sense counterparts. In contrast to Cyp2a4 and Cyp2a5, Cyp2a4as and Cyp2a5as show sex-biased expression patterns earlier in development, suggesting that they might contribute to later sex-biased patterns established for Cyp2a4 and Cyp2a5.

The growing obesity epidemic in America carries with it numerous health risks, including diabetes, increased serum lipid levels, and excess fat accumulation in the liver. If these conditions persist or become exacerbated, they may lead to the development of cardiovascular disease, the current leading cause of death among Americans, or to nonalcoholic fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma (HCC), one of the deadliest forms of cancer. Better understanding of the genes involved in these diseases can lead to improved identification of at-risk individuals and treatment strategies. Our lab previously identified zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression. The BALB/cJ mouse strain has a hypomorphic mutation in the Zhx2 gene, causing a 95% reduction in Zhx2 protein expression. The near ablation of Zhx2 in BALB/cJ mice confers protection from cardiovascular disease when fed a high fat diet, yet these mice show increased hepatic lipid accumulation and liver damage. Microarray data indicates Zhx2 may be involved in the regulation of numerous genes involved in lipid metabolism. Recent GWAS studies indicate ZHX2 may contribute to the risk of cardiovascular disease and liver damage in humans as well. In this dissertation, I characterize the role of Zhx2 expression in the liver and how it affects the risk of both cardiovascular disease and liver damage. I generated liver-specific Zhx2 knockout mice and confirmed Zhx2 regulates several novel targets that could contribute to the fatty liver phenotype seen in BALB/cJ mice. Further studies revealed that hepatic Zhx2 expression is necessary for proper sex-specific expression of several Cyptochrome P450 (CYP) genes and could contribute to gender differences in disease susceptibility. Lastly, I performed studies into the functional role of the Zhx2 target gene Elovl3. A mouse model of HCC revealed that Elovl3 is completely repressed in HCC tumors. Cell viability and cell cycle assays indicate that Elovl3 expression slows cell proliferation and may be important for proper cell cycle checkpoints. Together, these data indicate that Zhx2 and/or its targets could be clinically relevant in the detection, prevention, or treatment of cardiovascular disease, fatty liver, and HCC.