The long-term goal of this project is to elucidate the molecular mechanism of liver-specific gene expression. The immediate goal is to determine the mechanism of action of one critical liver-enriched transcription factor, hepatocyte nuclear factor 4a (HNF4a). Found primarily in the liver, kidney, intestine and pancreas, HNF4a is a highly conserved member of the superfamily of ligand-dependent transcription factors (nuclear receptors) that regulates many target genes essential to basic metabolism as well as xenobiotic and drug metabolism. Whereas a ligand has not yet been definitively identified for HNF4a, it has been linked to several human diseases including hemophilia, diabetes, atherosclerosis and cancer via regulation of hepatitis B viral genes and P450 (cyp) genes. HNF4a is also known to be an essential gene in organisms ranging from insect to man and to be essential for the adult liver phenotype. Despite the obvious importance of HNF4a, much remains to be elucidated about its mechanism of action. To address this, the following three Specific Aims will be pursued: 1) Investigation of the role of the unusually large F domain in HNF4a function. While our current results indicate that the F domain plays a role in recruiting co-regulatory molecules, little else is known about the structure or function of this unique domain. We will use a variety of in vivo and in vitro methods to investigate the interaction between the F domain and other regions of HNF4a as well as other proteins; 2) Identification and analysis of HNF4a interacting proteins. Studies will be continued on the interaction between HNF4a and known co-regulators using biochemical and molecular biological techniques. Newly identified interacting proteins will also be investigated; 3) Identification and analysis of HNF4a phosphorylation sites. We will finish mapping the more than 13 phosphorylation sites in HNF4a and elucidate their function in vitro and in vivo in cell-based assays.
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