The hepatocyte nuclear factor-4 (HNF-4) contains two transcription activation domains. One domain, activation function-1 (AF-1), consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic α-helical structure. Transcriptional analysis of sequential point mutations of the negatively charged residues (Asp and Glu) revealed a stepwise decrease in activity, while mutation of all acidic residues resulted in complete loss of transcriptional activity. Mutations of aromatic and hydrophobic amino acids surrounding the negatively charged residues had a much more profound effect than mutations of acidic amino acids, since even a single mutation of these residues resulted in a dramatic decrease in transactivation, thus demonstrating the importance of hydrophobic residues in AF-1 activity. Like other acidic activators, the AF-1 of HNF-4 binds the transcription factor IIB and the TATA-binding protein directly in vitro. In addition, the cAMP-response-element-binding-protein, a transcriptional adapter involved in the transactivation of a plethora of transcription factors, interacts with the AF-1 of HNF-4 and co-operates in the process of transactivation by HNF-4. The different protein targets of AF-1 suggest that the AF-1 of HNF-4 may be involved in recruiting both general transcription factors and chromatin remodelling proteins during activation of gene expression.
Abbreviations used: AF-1/AF-2, activation function 1/2; β-gal, β-galactosidase; CAT, chloramphenicol acetyltransferase; CREB, cAMP-responsive-element-binding protein; CBP, CREB-binding protein; DTT, dithiothreitol; GST, glutathione S-transferase; HepG2 cells, human hepatoma cells; HNF-4, hepatocyte nuclear factor-4; NP40, Nonidet P40; PBST, PBS containing 0.1% Tween 20, 5% non-fat dry milk and 0.5% BSA; TBP, TATA-binding protein; TFIIB, transcription factor IIB.
This paper is dedicated to the memory of Christos Cladaras, an inspired scientist, who died suddenly on 10 August 1994.
Present address: Aristotle University of Thessaloniki, Faculty of Sciences, School of Biology, Department of Genetics, Development and Molecular Biology, GR-540 06, Thessaloniki, Greece.