TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4.
The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.
Malik
Sohail
S
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021, USA. maliks@rockvax.rockefeller.edu
Wallberg
Annika E
AE
Kang
Yun Kyoung
YK
Roeder
Robert G
RG
D000123:Acetyltransferases / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D051548:Histone Acetyltransferases; D006801:Humans; D017874:Immediate-Early Proteins; D046911:Macromolecular Substances; D056892:Mediator Complex; D056913:Mediator Complex Subunit 1; D008565:Membrane Proteins; D010442:Peptide Chain Initiation, Translational / Q000502:physiology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D004247:DNA; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D051548:Histone Acetyltransferases; D017874:Immediate-Early Proteins; D046911:Macromolecular Substances; D056892:Mediator Complex; D056913:Mediator Complex Subunit 1; D008565:Membrane Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C490043:IFRD2 protein, human; C490042:Ifrd2 protein, mouse; C538855:MED1 protein, human; C503845:MED24 protein, human; C403128:MLX protein, human; C538856:Med1 protein, mouse; C501970:Tcfl4 protein, mouse