Journal of Biological Chemistry
Volume 273, Issue 45, 6 November 1998, Pages 29950-29957
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NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS
Critical Structural Elements and Multitarget Protein Interactions of the Transcriptional Activator AF-1 of Hepatocyte Nuclear Factor 4*

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The nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is an important regulator of several genes involved in diverse metabolic and developmental pathways. Mutations in the HNF-4Agene are responsible for the maturity-onset diabetes of the young type 1. Recently, we showed that the 24 N-terminal residues of HNF-4 function as an acidic transcriptional activator, termed AF-1 (Hadzopoulou-Cladaras, M., Kistanova, E., Evagelopoulou, C., Zeng, S., Cladaras C., and Ladias, J. A. A. (1997) J. Biol. Chem. 272, 539–550). To identify the critical residues for this activator, we performed an extensive genetic analysis using site-directed mutagenesis. We showed that the aromatic and bulky hydrophobic residues Tyr6, Tyr14, Phe19, Lys10, and Lys17 are essential for AF-1 function. To a lesser degree, five acidic residues are also important for optimal activity. Positional changes of Tyr6 and Tyr14 reduced AF-1 activity, underscoring the importance of primary structure for this activator. Our analysis also indicated that AF-1 is bipartite, consisting of two modules that synergize to activate transcription. More important, AF-1 shares common structural motifs and molecular targets with the activators of the tumor suppressor protein p53 and NF-κB-p65, suggesting similar mechanisms of action. Remarkably, AF-1 interacted specifically with multiple transcriptional targets, including the TATA-binding protein; the TATA-binding protein-associated factors TAFII31 and TAFII80; transcription factor IIB; transcription factor IIH-p62; and the coactivators cAMP-responsive element-binding protein-binding protein, ADA2, and PC4. The interaction of AF-1 with proteins that regulate distinct steps of transcription may provide a mechanism for synergistic activation of gene expression by AF-1.

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*

This work was supported by American Heart Association Grant 9750343N and National Institutes of Health Grants HL52666 and AI34270 (to J. A. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.