Competitive Cofactor Recruitment by Orphan Receptor Hepatocyte Nuclear Factor 4α1: Modulation by the F Domain

Abstract

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...

Regulation of Vascular Endothelial Growth Factor D by Orphan Receptors Hepatocyte Nuclear Factor-4α and Chicken Ovalbumin Upstream Promoter Transcription Factors 1 and 2

“…p160 proteins typically link other coactivators to the transcription initiation complex, whereas CBP/p300 can directly acetylate histones and/or other components of the transcription initiation complex (47). Similarly, orphan receptor HNF-4a was shown to interact with CBP/ p300 and GRIP-1 (48,49). In line with these studies, we found that CBP and GRIP-1 were associated with HNF-4a at the proximal vegf-D promoter (Fig.…”

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

“…Furthermore, whereas studies using truncated mutants assume that N-terminal deletion of the DNA binding domain or Cterminal deletion of the F-domain have no effect on ligand binding to HNF-4␣, the validity of these assumptions has never been demonstrated. The latter is especially surprising, since the C-terminal F-domain is a well recognized negative regula- tor of HNF-4␣ transactivation and is thought to directly interact with the ligand binding domain E (8,16,22,38). Since high affinity ligand binding and ligand-induced conformational changes are hallmarks of ligand-dependent nuclear receptors, fluorescence binding assays and circular dichroism were used in the present study to directly address these issues by examining ligand specificity and conformational responsiveness to ligand.…”

“…Since the discovery of HNF-4␣ over a decade ago (1), there has been considerable controversy regarding whether this orphan nuclear transcription factor is (8,11,14,19) or is not (12,16,18) ligand-activated. Due to problems with crystallizing the full-length HNF-4␣ (aa 1-455) or HNF-4␣ mutants containing an intact C-terminal F-domain (17,18), almost nothing is known regarding the ligand specificity, structure, or conformational dependence of the full-length HNF-4␣ (aa 1-455).…”

“…When isolated from recombinant bacteria, all such N-and C-terminal truncation mutants contained entrapped LCFA (17,18), especially palmitoleic acid (C16:1). However, the C-terminal F-domain of HNF-4␣ is known to be a negative regulator of HNF-4␣ transactivation in intact cells (8,16,22,38), and the assumption that its deletion had no effect on HNF-4␣ structure or LCFA/LCFA-CoA binding was never tested before. In order to resolve whether ligand affinity was affected by deleting the negative regulatory F-domain from the C terminus of HNF-4␣, an HNF-4␣ truncated mutant lacking N-terminal domains A-D and F-domain (i.e.…”

“…HNF-4␣ exhibits constitutive but not full activity in the absence of added exogenous ligands (reviewed in Ref. 1) (8,(11)(12)(13)(14)(15)(16). The molecular basis for this constitutive activity remains to be determined but may include ligand-based mechanisms involving dietary or endogenous long chain fatty acids (LCFA) (17,18), endogenously synthesized coenzyme A (CoA) thioesters of long chain fatty acids (LCFA-CoAs) (8,11,14,19), and/or the F-domain itself functioning as a modulator (16).…”