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.
Mesh Terms:
Acetyltransferases, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Carrier Proteins, Cell Cycle Proteins, Cell Extracts, Cell Line, Cell Nucleus, Chromatin, DNA, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Hela Cells, Hepatocyte Nuclear Factor 4, Histone Acetyltransferases, Humans, Immediate-Early Proteins, Macromolecular Substances, Mediator Complex, Mediator Complex Subunit 1, Membrane Proteins, Peptide Chain Initiation, Translational, Phosphoproteins, Protein Binding, Repressor Proteins, Trans-Activators, Transcription Factors, Transcriptional Activation, p300-CBP Transcription Factors
Acetyltransferases, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors, Carrier Proteins, Cell Cycle Proteins, Cell Extracts, Cell Line, Cell Nucleus, Chromatin, DNA, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Hela Cells, Hepatocyte Nuclear Factor 4, Histone Acetyltransferases, Humans, Immediate-Early Proteins, Macromolecular Substances, Mediator Complex, Mediator Complex Subunit 1, Membrane Proteins, Peptide Chain Initiation, Translational, Phosphoproteins, Protein Binding, Repressor Proteins, Trans-Activators, Transcription Factors, Transcriptional Activation, p300-CBP Transcription Factors
Mol. Cell. Biol.
Date: Aug. 01, 2002
PubMed ID: 12101254
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