Fishes comprise approximately 24,000 existing species and are among the most diverse and successful groups of vertebrates. These organisms represent an extensive array of biological characteristics and maintain considerable genetic diversity. It appears that much of the complexity of these organisms is a result of successive rounds of genetic duplications resulting in multiple copies of many important genes. The presence of multiple gene copies is believed to have had a large impact on the evolution of fish and their diversity. The retention of multiple genes in fish provides the opportunity to gain insights into how genes evolve through specific processes. This research will examine whether multiple vitamin D receptor genes (VDRs) present in most fish species have evolved new functions and/or partitioned ancestral functions subsequent to duplication. Through three specific aims, critical vitamin D receptor structure-function dynamics, spatial-temporal patterns of VDR gene expression, and role of vitamin D receptor during embryo development will be investigated. The proposed approach provides a systematic and comprehensive investigation that enables a detailed evaluation of functional change of the vitamin D receptor across a number of evolutionary diverse fish species. Results of this work will support a scientific understanding of the evolution of fish endocrinology by enhancing the conceptual framework of how retention, loss or diversification of gene duplicates contributes to endocrine innovation in these species. Training of undergraduate and graduate students as part of this research plan will provide a unique opportunity to educate and engage young scientists. In addition to yearly training, students will participate in the summer research program at the Mount Desert Island Biological laboratory (MDBL) in Maine. The MDIBL facility has a long-standing dedication to undergraduate education with an emphasis on the exploration of timely questions involving marine and freshwater biology in a diverse array of aquatic fauna.
Teleost (Boney) fish comprise ~27,000 extant species and are the most diverse and successful groups of vertebrates. Much of the complexity of the teleost genome is a result of successive rounds of whole genome duplications (WGD). The retention of multiple gene pairs in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. This study examines whether duplicated vitamin D receptor genes (VDRs) have acquired novel function(s) and/or partitioned ancestral sub-functions subsequent to successive duplication events. VDRα and VDRβ paralogs were cloned from the Japanese medaka (Oryzias latipes) and the zebrafish (Danio rerio), two distantly related teleosts. Vitamin D binding analysis demonstrates that the VDR paralogs maintain a high binding affinity for 1α, 25-dihydroxyvitamin D3 (1, 25D3) that is similar to mammalian VDR. Concentration-response curves of VDR transactivation with 1, 25D3 suggest that while the ligand potency is highly similar, maximal receptor activation is significantly different between the two VDR forms. In addition, both VDRα paralogs demonstrate preferential interaction with DNA binding sites compared to the VDRβ paralogs in response to 1, 25D3. Protein-protein interactions were investigated using co-transfection and mammalian two-hybrid, and mutation of co-regulator activation domains. Our results suggest that functional differences between VDR paralogs are driven through differential interactions between receptors and their co-regulators, including RXR and the SRC family of nuclear receptor coactivators. We speculate that the observed functional differences are due to subtle conformational differences between the two paralogs The retention of multiple gene pairs in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In the next component of this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution to infer derived and ancestral functions of teleost VDR’s. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish that diverged before the teleost 3R event. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across evolution. Concentration response curves in transient transfection assays reveal similar EC50s, however maximum transactivational efficacy varies. Protein-Protein interactions were investigated using co-transfection and mammalian 2-hybrid assays, and mutations of coregulator activation domains. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. We combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that functional differences between VDR are influenced by differential interactions with essential co-regulators. We speculate that we may be observing a change in the pharmacodynamic relationships between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators. Lastly, It has been speculated that the adoption of lithocholic acid (LCA) as a functional ligand for the vitamin D receptor (VDR) was an adaptation to facilitate the detoxification of the bile acid. However, the evolutionary history of this partnership is not well understood due to the lack of data from non-mammalian vertebrates. Here, using VDRs cloned from species representing key nodes in vertebrate evolution, and our previous work with 1, 25D3 as a comparison, we assess how the critical molecular functions of VDR have evolved over time in response to LCA. Competitive binding assays demonstrate that the VDR ligand affinities across species were comparable to that of human VDR. However, VDR transactivation in response to LCA was limited to select species. Subsequent assays were conducted to examine downstream events following ligand binding in order to characterize the functional evolution of the LCA-VDR partnership. We demonstrate that LCA and 3-keto LCA both function as full agonists only human VDR and the teleost VDRα paralogs. Interaction between the bile acids and VDRβ paralogs did not facilitate transactivation or RXR heterodimerization, and display attenuated coactivator recruitment. Similarly, VDR from basal vertebrates were not able to mediate any response to LCA or 3-keto LCA beyond ligand binding. Bioinformatics analysis suggests that functional differences between VDRs are driven though differential interactions between VDRs and essential coactivators. Taken together, our results suggest that the ability of LCA to function as a VDR ligand evolved before the bile acid itself, most likely though a process of exaptation followed by co-option once the need to detoxify LCA arose.
