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 i ts actions on gene transcription by heterodimerizing with retinoid X recept ors (RXRs) on direct repeat (DR+3) vitamin D responsive elements (VDREs) lo cated 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 truncate d hVDR mutants (Delta 134, Delta 113, Delta 102, Delta 90, Delta 84, Delta 80, and Delta 60) was generated and expressed in bacteria. Only the Delta 1 34 and Delta 113 mutants bound the VDRE (predominantly as monomers), sugges ting 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 bindin g. Site-directed mutagenesis of conserved charged residues in full-length h VDR was then performed to dissect the functional significance of the CTE (r esidues 88-112) in the context of the complete hVDR-RXR-VDRE interaction. F unctional assays revealed that E98K/E99K, R102A/K103A/R104A, and K109A/ R11 0A/K111A mutant hVDRs possessed dramatically reduced DNA binding and transc riptional activities, whereas distinct point mutants, such as K103A, bound to DNA normally but lacked transcriptional activity. Therefore, the boundar y 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 D NA-binding or-helix, where they could contact transcriptional coactivators or basal transcription factors.