. The liver is the primary organ responsible for producing plasma and other proteins essential for basic metabolism and detoxification of chemical compounds. Whereas research during the past decade has shown that these liver proteins are produced largely as a result of liver-specific gene expression mediated by liver-enriched transcription factors, it is not known exactly how liver-specific transcription is achieved. For example, many questions remain about the mechanism of action of hepatocyte nuclear factor 4 (HNF-4), one of the most important transcription factors for determining the hepatic phenotype and regulator of over 40 target genes, including those involved in glucose, fatty acid, and cholesterol metabolism, blood coagulation, and chemical detoxification. The goal of this proposal, therefore, is to elucidate the mechanism of liver-specific gene expression by studying three important aspects of HNF-4: (1) The applicants recently showed that due to its strong and exclusive homodimerization activity and exclusive nuclear localization, HNF-4 defines a new subgroup in the nuclear receptor superfamily. They propose to investigate the specific amino acids that determine homo- vs. heterodimerization as well as the role of protein dimerization in receptor function. This will be achieved by rationally based site-directed mutagenesis studies derived from what is already known about other receptors as well as from a naturally occurring mutation in HNF-4 that was recently found in patients with maturity-onset diabetes of the young (MODY1); (2) The applicants will examine the mechanism of transcriptional activation by HNF-4 by analyzing the interaction between HNF-4, co-activators, co-repressors, and the basal transcription machinery by in vivo and in vitro assays; and (3) The applicants will establish an in vitro system to investigate the transcriptional synergy between HNF-4 and another liver-enriched transcription factor, C/EBP alpha, on the apolipoprotein B gene promoter. Synergy between tow liver-enriched transcription factors could explain liver-specific gene expression for this and other genes. Since HNF-4 has been directly or indirectly linked to several human diseases, including atherosclerosis, hepatitis, hemophilia, hepatocarcinogenesis, and, most recently, diabetes, detailed mechanistic knowledge of HNF-4 function will not only shed light on liver-specific gene expression and the action of nuclear receptors but also provide a basis for the possible future development of therapeutic reagents for a wide variety of diseases.
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