| Hepatocyte nuclear factor 4alpha (HNF4alpha), an orphan member of the nuclear receptor superfamily, regulates the expression of a wide array of genes important for glucose, amino acid and lipid metabolism, as well as genes important for blood composition and nutrient transport in organisms ranging from cniderians to man. HNF4alpha expression is highly tissue-restricted, with high levels of expression found in liver and kidney, and moderate levels in the pancreas and intestine. HNF4alpha plays critical roles in the development of these tissues, and has been linked to a number of human diseases including a type II diabetes and hemophilia B Leyden.; Unlike many orphan receptors, HNF4alpha displays considerable apparent constitutive activity, suggesting that either no endogenous ligand is required for HNF4alpha activity or that a putative ligand for HNF4alpha would likely serve to decrease its activity (i.e. a negative ligand). In the dissertation, data are presented which suggest that the apparent constitutive activity is at least partially attributable to an ability to interact with and respond to coactivators in vivo. Surprisingly, however, HNF4alpha was also shown to functionally interact with a corepressor under the same conditions, arguing against the presence of a traditional ligand under the conditions used. Alternatively, the unusually large F domain of HNF4alpha1 was found to play an inhibitory role, promoting corepressor recruitment while also abrogating optimal coactivator interaction. Finally, a naturally occurring HNF4alpha splicing variant, HNF4alpha2---which differs from HNF4alpha1 solely by the presence of a ten amino acid insert within the F domain---was shown to possess enhanced transcriptional activity which correlates with an increased ability to recruit coactivators, suggesting that modulation by the F domain may represent an alternative mode of regulation to ligand dependence for HNF4alpha.; In a preliminary search for additional alternative regulatory mechanisms, the p21-activated kinase, gamma-PAK, was identified as a kinase that phosphorylates HNF4alpha1 in vitro. Given that this phosphorylation occurs in several strategic sites, including a region of HNF4alpha1 hypothesized to be important for cofactor interaction as well as F domain function, phosphorylation by gamma-PAK may represent a physiologically and mechanistically relevant means by which to modulate HNF4alpha function. |
