TFIIB-DIRECTED TRANSCRIPTIONAL ACTIVATION BY THE ORPHAN NUCLEAR RECEPTOR HEPATOCYTE NUCLEAR FACTOR-4

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is req uired for development and maintenance of the liver phenotype, HNF-4 ac tivates 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 n uclear 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 syst em, Functional HNF-4 was purified to homogeneity from a bacterial expr ession system, In in vitro transcription assays employing nuclear extr act from HeLa cells, which do not contain HNF-4, recombinant HNF-4 sti mulated transcription from basal promoters linked to site A. Activatio n by HNF-4 did not exhibit a ligand requirement, but phosphorylation o f HNF-4 in the in vitro transcription system was observed, The activat ion function of HNF-4 was localized to a domain displaying strong homo logy 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 consist ing of TBP, the TATA box-binding protein component of TFIID and TFIIB, 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 fi rst step involves AF-2-independent recruitment of TFIIB to the promote r complex; the second step is AF-2 dependent and entails entry of prei nitiation complex components acting downstream of TFIIB.