Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.
Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms. Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well.
Sladek
F M
FM
Environmental Toxicology, University of California, Riverside, California 92521, USA. frances.sladek@ucr.edu
Ruse
M D
MD
Nepomuceno
L
L
Huang
S M
SM
Stallcup
M R
MR
D017398:Alternative Splicing; D004268:DNA-Binding Proteins; D010450:Endopeptidases / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4; D047908:Intracellular Signaling Peptides and Proteins; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism; D051958:Nuclear Receptor Coactivator 2; D056918:Nuclear Receptor Coactivators; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D047908:Intracellular Signaling Peptides and Proteins; C455500:NCOA7 protein, human; D009687:Nuclear Proteins; D051958:Nuclear Receptor Coactivator 2; D056918:Nuclear Receptor Coactivators; D010750:Phosphoproteins; D020033:Protein Isoforms; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D010450:Endopeptidases