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Paralogous Vitamin D Receptors in Teleosts: Transition of Nuclear Receptor Function

Integrated Toxicology and Environmental Health Program, Nicholas School of the Environment and Earth Sciences (D.L.H., B.B., D.E.H.) and Department of Pharmacology and Cancer Biology (J.M.H.), Duke University, Durham, North Carolina 27708; GlaxoSmithKline Discovery Research (L.M., J.M.M., J.T.M.), Research Triangle Park, North Carolina 27709; and Department of Environmental and Molecular Toxicology (S.H.W.L., S.W.K.), North Carolina State University, Raleigh, North Carolina 27695

Address all correspondence and requests for reprints to: Seth W. Kullman, Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, North Carolina 27695. E-mail: swkullma{at}ncsu.edu.

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we describe the cloning and functional characterization of two novel vitamin D receptor (VDR) paralogs from the freshwater teleost medaka (Oryzias latipes). VDR sequences were identified through mining of the medaka genome database in which gene organization and structure was determined. Two distinct VDR genes were identified in the medaka genome and mapped to defined loci. Each VDR sequence exhibits unique intronic organization and dissimilar 5' untranslated regions, suggesting they are not isoforms of the same gene locus. Phylogenetic comparison with additional teleosts and mammalian VDR sequences illustrate that two distinct clusters are formed separating aquatic and terrestrial species. Nested within the teleost cluster are two separate clades for VDR and VDRβ. The topology of teleost VDR sequences is consistent with the notion of paralogous genes arising from a whole genome duplication event prior to teleost radiation. Functional characterization was conducted through the development of VDR expression vectors including Gal4 chimeras containing the yeast Gal4 DNA binding domain fused to the medaka VDR ligand binding domain and full-length protein. The common VDR ligand 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] resulted in significant transactivation activity with both the Gal4 and full-length constructs of medaka (m) VDRβ. Comparatively, transactivation of mVDR with 1,25(OH)₂D₃ was highly attenuated, suggesting a functional divergence between these two nuclear receptor paralogs. We additionally demonstrate through coactivator studies that mVDR is still functional; however, it exhibits a different sensitivity to 1,25(OH)₂D₃, compared with VDRβ. These results suggest that in mVDR and VDRβ have undergone a functional divergence through a process of sub- and/or neofunctionalization of VDR nuclear receptor gene pairs.