The N-terminal domain of transcription factor IIB is required for direct interaction with the vitamin D receptor and participates in vitamin D-mediated transcription.
The interaction of the vitamin D receptor (VDR) with transcription factor IIB (TFIIB) represents a potential physical link between the VDR-DNA complex and the transcription preinitiation complex. However, the functional relevance of the VDR-TFIIB interaction in vitamin D-mediated transcription is not well understood. In the present study, we used site-directed mutagenesis to demonstrate that the structural integrity of the amino-terminal zinc finger of TFIIB is essential for VDR-TFIIB complex formation. Altering the putative zinc-coordinating residues (C15, C34, C37, or H18) to serines abolished TFIIB interaction with the VDR as assessed in a yeast two-hybrid system and by in vitro protein interaction assays. This N-terminal, VDR-interactive domain functioned as a selective, dominant-negative inhibitor of vitamin D-mediated transcription. Expressing amino acids 1-124 of human TFIIB (N-TFIIB) in COS-7 cells or in osteoblastic ROS17/2.8 cells effectively suppressed 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induced transcription, but had no effect on basal or glucocorticoid-activated transcription. A mutant N-terminal domain [N-TFIIB(C34S:C37S)] that does not interact with VDR had no impact on 1,25-(OH)2D3-induced transcription. Interestingly, both in vitro and in vivo protein interaction assays showed that the VDR-TFIIB protein complex was disrupted by the 1,25-(OH)2D3 ligand. Mechanistically, these data establish a functional role for the N terminus of TFIIB in VDR-mediated transcription, and they allude to a role for unliganded VDR in targeting TFIIB to the promoter regions of vitamin D-responsive target genes.
Masuyama
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St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, Missouri 63104, USA.
Jefcoat
S C
SC
MacDonald
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014807:Vitamin D / Q000378:metabolism*; D016335:Zinc Fingers
D018167:Receptors, Calcitriol; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D014807:Vitamin D; D002117:Calcitriol