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

Jui Cheng Hsieh, G. Kerr Whitfield, Anish K. Oza, Hope T L Dang, Jack N. Price, Michael A. Galligan, Peter Jurutka, Paul D. Thompson, Carol A. Haussler, Mark R. Haussler

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Abstract

The vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D3 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 (Δ134, Δ113, Δ102, Δ90, Δ84, Δ80, and Δ60) was generated and expressed in bacteria. Only the Δ134 and Δ113 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-RXRVDRE 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 α- helix, where they could contact transcriptional coactivators or basal transcription factors.

Original languageEnglish (US)
Pages (from-to)16347-16358
Number of pages12
JournalBiochemistry
Volume38
Issue number49
DOIs
StatePublished - Dec 7 1999

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Hsieh, J. C., Whitfield, G. K., Oza, A. K., Dang, H. T. L., Price, J. N., Galligan, M. A., Jurutka, P., Thompson, P. D., Haussler, C. A., & Haussler, M. R. (1999). 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. Biochemistry, 38(49), 16347-16358. https://doi.org/10.1021/bi9916574