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.

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For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4␣1 (HNF4␣1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4␣1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4␣-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4␣1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4␣1 in vitro and repress HNF4␣1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4␣1 and GRIP1, CBP, or p300, SMRT can titer out HNF4␣1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4␣ can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.Many key aspects of mammalian physiology, including growth, differentiation, and homeostasis, are governed by the actions of small, lipophilic compounds, including the steroid and thyroid hormones and vitamins A and D. These compounds function as ligands for many members of the nuclear receptor superfamily and modulate their transcriptional activities (for a review, see reference 69). Nuclear receptors, by definition, share a common modular architecture comprised of a highly conserved DNA binding domain (DBD) and a Cterminal ligand binding/transactivation domain (LBD), as well as more divergent N-terminal and central hinge domains (reviewed in references 42 and 53). Some receptors also possess a so-called F domain of variable length at the extreme C terminus. The various domains contain different motifs, such as activation functions (AFs), that are important in the regulation of many aspects of nuclear receptor function and, ultimately, in transcriptional activity. AF-2 (helix 12), located at the C terminus of the LBD, has been shown to play a critical role in ligand-induced activation of receptors, a role that derives from its ability to undergo ligand-dependent conformational changes necessary for recruitment of a class of accessory factors and protein complexes termed coacti...