Interaction between the retinoid X receptor and transcription factor IIB is ligand-dependent in vivo.
The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.
Leong
G M
GM
Unit on the Molecular Mechanisms of Reproduction, NICHHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
K S
KS
Marton
M J
MJ
Blanco
J C
JC
Wang
I M
IM
Rolfes
R J
RJ
Ozato
K
K
Segars
J H
JH
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D047488:Retinoid X Receptors; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D008024:Ligands; D009687:Nuclear Proteins; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D035581:Transcription Factor TFIIB; D014157:Transcription Factors