title: TFIIB-directed transcriptional activation by the orphan nuclear receptor hepatocyte nuclear factor 4.
abstract: The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is required for development and maintenance of the liver phenotype. HNF-4 activates several hepatocyte-specific genes, including the gene encoding apolipoprotein AI (apoAI), the major protein component of plasma high-density lipoprotein. The apoAI gene is activated by HNF-4 through a nuclear receptor binding element (site A) located in its liver-specific enhancer. To decipher the mechanism whereby HNF-4 enhances apoAI gene transcription, we have reconstituted its activity in a cell-free system. Functional HNF-4 was purified to homogeneity from a bacterial expression system. In in vitro transcription assays employing nuclear extract from HeLa cells, which do not contain HNF-4, recombinant HNF-4 stimulated transcription from basal promoters linked to site A. Activation by HNF-4 did not exhibit a ligand requirement, but phosphorylation of HNF-4 in the in vitro transcription system was observed. The activation function of HNF-4 was localized to a domain displaying strong homology to the conserved AF-2 region of nuclear receptors. Dissection of the transcription cycle revealed that HNF-4 activated transcription by facilitating assembly of a preinitiation complex intermediate consisting of TBP, the TATA box-binding protein component of TFIID and TFIID, via direct physical interactions with TFIIB. However, recruitment of TFIIB by HNF-4 was not sufficient for activation, since HNF-4 deletion derivatives lacking AF-2 bound TFIIB. On the basis of these results, HNF-4 appears to activate transcription at two distinct levels. The first step involves AF-2-independent recruitment of TFIIB to the promoter complex; the second step is AF-2 dependent and entails entry of preinitiation complex components acting downstream of TFIIB.
authors.affiliation: Department of Cardiovascular Molecular Biology, Lederle Laboratories, Pearl River, New York 10965, USA.
authors.forename: S
authors.initials: S
authors.lastname: Malik
authors.forename: S K
authors.initials: SK
authors.lastname: Karathanasis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008024:Ligands; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016632:Apolipoprotein A-I; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors