Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain.
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 4alpha1 (HNF4alpha1, 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 HNF4alpha1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4alpha-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4alpha1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4alpha1 in vitro and repress HNF4alpha1 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 HNF4alpha1 and GRIP1, CBP, or p300, SMRT can titer out HNF4alpha1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4alpha 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.
Ruse
Michael D
MD
Biochemistry and Molecular Biology Graduate Program, University of California-Riverside, Riverside, CA 92521, USA.
Privalsky
Martin L
ML
Sladek
Frances M
FM
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001667:Binding, Competitive / Q000502:physiology; D050882:CREB-Binding Protein; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050881:E1A-Associated p300 Protein; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D056985:Nuclear Receptor Co-Repressor 2; D051958:Nuclear Receptor Coactivator 2; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; D008024:Ligands; C403128:MLX protein, human; C498001:NCOA2 protein, human; C539130:NCOR2 protein, human; C498002:Ncoa2 protein, mouse; C539131:Ncor2 protein, mouse; D009687:Nuclear Proteins; D056985:Nuclear Receptor Co-Repressor 2; D051958:Nuclear Receptor Coactivator 2; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C501970:Tcfl4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse