Role of Hepatocyte Nuclear Factor 4 alpha in Hepatocyte Proliferation

Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the master regulator of hepatocyte differentiation. It is involved in the up-regulation of genes involved in many classic hepatic functions including: bile acid metabolism, xenobiotic metabolism, glucose homeostasis, lipid metabolism, coagulation factor synthesis, etc. However, the role of HNF4α in regulation of hepatocyte proliferation was not known. The primary goal of this dissertation was to investigate the role of HNF4α in the regulation of hepatocyte proliferation. In these studies we utilized two novel inducible, hepatocyte specific, HNF4α knockdown mouse models. Past models of HNF4α dificiency result in deletion within the first few weeks of birth and lead to lethality within 6-8 weeks. This makes it difficult to address a role for HNF4α in hepatocyte proliferation because the liver is still growing and differentiating within this time frame. Hepatocyte-specific deletion of HNF4α in adult mice resulted in fat accumulation (steatosis), glycogen depletion, and increased hepatocyte proliferation with a significant increase in liver/body weight ratio without complimentary liver regeneration due to hepatocyte cell death. Global gene expression analysis (microarray and RNA-Seq) revealed that a significant number of the 300+ down-regulated genes are involved in hepatic differentiation, many of which are known HNF4α targets. Interestingly, a significant number of the 500+ up-regulated genes are associated with cell proliferation and cancer. Further, a combined bioinformatics analysis of ChIP-sequencing and RNA-sequencing data indicated that a substantial number of up-regulated genes are putative HNF4α targets. We have used chromatin immunoprecipitation (ChIP) to confirm three of these targets: Ect2, Osgin1, and Hjurp. Ingenuity Pathway Analysis (IPA)-mediated functional analysis revealed the most significantly activated gene network after HNF4α deletion is regulated by c-Myc. To determine the role of HNF4α in pathogenesis of hepatocellular carcinoma (HCC), we performed the classic initiation-promotion experiment using diethylnitrosamine (DEN). Deletion of HNF4α resulted in extensive promotion of DEN-induced hepatic tumors, which were highly proliferative and less differentiated. Further, the HCC observed in HNF4α-deleted mice exhibited significant up-regulation of c-Myc and its target genes. We hypothesized that HNF4α inhibits hepatocyte proliferation by repression of target genes. One possible mechanism is that HNF4α influences the epigenetic state of a given gene to promote or inhibit gene expression. We studied various epigenetic modifications associated with gene activation and repression on a 9 kb segment of the promoter of Ect2, a validated HNF4α negative target gene also known to activate hepatocyte proliferation. ChIP analysis performed on chromatin isolated from control and HNF4α KO livers indicated that a deletion of HNF4α resulted in an epigenetic switch in histone modifications at the Ect2 promoter from an inhibited to an active state; primarily affecting acetylation of histone H3K9. These data indicate that HNF4α inhibits hepatocyte proliferation, is a potential tumor suppressor in the liver, and plays a critical role in chemical carcinogenesis. We also provide data that support the hypothesis that HNF4α may be functioning through an influence on the epigenetic state of select genes