Gene structure and promoter for Crad2 encoding mouse cis-retinol/3 alpha-hydroxysterol short-chain dehydrogenase isozyme

Cis-retinol/androgen dehydrogenase type 2 (CRAD2) has been shown to catalyz e the dehydrogenation of retinols, including 9-cis retinol, and also to exh ibit 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities. To exami ne the function of this enzyme and regulation of its gene, the Crad2 gene w as cloned from a mouse genomic DNA library and characterized. The complete mouse CRAD2-coding region was found in four exons spanning an approximately 5 kb region. The nucleotide sequences of the exons encoding 316 amino acid s were identical to those of the previously reported mouse Crad2 cDNA. Prim er extension analysis and RNase protection assay were used to map the major transcription initiation sites to the positions lying 87 and 89 base pairs upstream of the ATG translation start codon. The region proximal to the in itiation sites exhibited the absence of both TATAA and CAAT boxes. This reg ion had hepatocyte nuclear factor binding sites, consistent with its predom inant expression in the liver. Computer analysis of an approximately 7.5 kb 5'-flanking region also suggested the presence of binding sites for AP-1, SREBP1, HSF2, c-Rel, c-Myc, CREBP, GATA, Ets, E2F, and Oct-1, suggesting th at various factors including retinoic acid, cholesterol, various kinds of s tress, the cell cycle, and cyclic AMP may regulate the expression of this g ene. Fluorescence in-situ hybridization analysis showed that Crad2 is locat ed at the terminus of mouse chromosome 10, an area that corresponds to band 10D3, suggesting that RDH-related SDRs may be located together in the clus ter locus. Northern blot hybridization and RT-PCR analysis demonstrated tha t CRAD2 was expressed not in early embryonic stages, and not in embryonic s tem cells, but instead in the gastrointestinal tract during later embryonic development and adult stage. In conclusion, we have presented the first co mplete structural analysis, including that of the promoter and chromosomal location, of a member of the retinol/androgen dehydrogenase subfamily of th e group of the short-chain dehydrogenase/reductase (SDR) isozymes. Our find ings will provide the basis for in-vitro or in-vivo studies concerning the regulation of retinol and androgen metabolism and enable determination of t he mechanism of diseases related to retinol, retinal, retinoic acid, and an drogen. (C) 2000 Elsevier Science B.V. All rights reserved.