Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4 alpha 1

Transcription factors, such as nuclear receptors, often exist in various fo rms that are generated by highly conserved splicing events. Whereas the fun ctional significance of these splicing variants is often not known, it is k nown that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate thos e interactions, however, are not well characterized, nor is the role of spl icing variants. In this study, transient transfection, yeast two-hybrid, an d GST pulldown assays are used to show not only that nuclear receptor hepat ocyte nuclear factor 4 alpha 1 (HNF4 alpha 1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommon ly large F domain in the C terminus of the receptor inhibits that interacti on. 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 r esults also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4 alpha 2) abrogates that inhibit ion in vivo and in vitro. A series of protease digestion assays indicates t hat there may be structural differences between HNF4 alpha 1 and HNF4 alpha 2 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 a nd the LED of HNF4 alpha 1 and -alpha 2 and that that contact is different in the HNF4 alpha 1 and HNF4 alpha 2 isoforms. Finally, we propose a model in which the F domain of HNF4 alpha 1 acts as a negative regulatory region for transactivation and in which the alpha 2 insert ameliorates the negativ e effect of the F domain. A conserved repressor sequence in the F domains o f HNF4 alpha 1 and -alpha 2 suggests that this model may be relevant to oth er nuclear receptors as well.