Characterization of unique DNA-binding and transcriptional-activation functions in the carboxyl-terminal extension of the zinc finger region in the human vitamin D receptor.
The vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D(3) and mediates its actions on gene transcription by heterodimerizing with retinoid X receptors (RXRs) on direct repeat (DR+3) vitamin D responsive elements (VDREs) located in target genes. The VDRE binding function of VDR has been primarily ascribed to the zinc finger region (residues 24-87). To define the minimal VDRE binding domain for human VDR (hVDR), a series of C-terminally truncated hVDR mutants (Delta134, Delta113, Delta102, Delta90, Delta84, Delta80, and Delta60) was generated and expressed in bacteria. Only the Delta134 and Delta113 mutants bound the VDRE (predominantly as monomers), suggesting that, in addition to the conserved zinc finger region of hVDR, as many as 25 amino acids in a C-terminal extension (CTE) participate in DNA binding. Site-directed mutagenesis of conserved charged residues in full-length hVDR was then performed to dissect the functional significance of the CTE (residues 88-112) in the context of the complete hVDR-RXR-VDRE interaction. Functional assays revealed that E98K/E99K, R102A/K103A/R104A, and K109A/R110A/K111A mutant hVDRs possessed dramatically reduced DNA binding and transcriptional activities, whereas distinct point mutants, such as K103A, bound to DNA normally but lacked transcriptional activity. Therefore, the boundary for the minimal DNA-binding domain in hVDR extends C-terminal of the zinc fingers to Lys-111, with clusters of highly conserved charged amino acids playing a crucial role in binding to the DR+3 element. Further, individual residues in this region (e.g., Lys-103) may lie on the opposing face of a DNA-binding alpha-helix, where they could contact transcriptional coactivators or basal transcription factors.
Hsieh
J C
JC
Department of Biochemistry, College of Medicine, The University of Arizona, Tucson 85724, USA.
Whitfield
G K
GK
Oza
A K
AK
Dang
H T
HT
Price
J N
JN
Galligan
M A
MA
Jurutka
P W
PW
Thompson
P D
PD
Haussler
C A
CA
Haussler
M R
MR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016335:Zinc Fingers / Q000235:genetics
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D018167:Receptors, Calcitriol; D015534:Trans-Activators