CLONING, SEQUENCING, AND FUNCTIONAL-ANALYSIS OF THE 5'-FLANKING REGION OF THE RAT 3-ALPHA-HYDROXYSTEROID DIHYDRODIOL DEHYDROGENASE GENE/

Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HS D/DD) is a member of the aldo-keto reductase gene superfamily. It disp lays high constitutive expression and inactivates circulating steroid hormones and suppresses the formation of polycyclic aromatic hydrocarb on anti- and syn-dio[-epoxides (ultimate carcinogens). To elucidate me chanisms responsible for constitutive expression of the 3 alpha-HSD/DD gene a rat genomic library obtained from adult Sprague-Dawley female liver (HaeIII partial digest) was screened, using a probe correspondin g to the 5'-end of the cDNA (-15 to +250), and a 15.8-kb genomic clone was isolated, Sequencing revealed that 6.3 kb contained exon 1 (+16 t o +138 bp) plus additional introns and exons. The transcription start site (+1) was located by primer extension analysis, and the initiation codon, ATG, was located at +55 bp, The remaining 9.5 kb represented t he 5'-flanking region of the rat 3 alpha-HSD/DD gene. A 1.6-kb fragmen t of this region was sequenced. A TATTTAA sequence (TATA box) was foun d at 33 bp upstream from the major transcription start site, cis-actin g elements responsible for the constitutive expression of the rat 3 al pha-HSD/DD gene were located on the 5'-flanking region by transient tr ansfection of reporter-gene (chloramphenicol acetyl transferase, CAT) constructs into human hepatoma cells (HepG2). CAT assays identified th e basal promoter between (-199 and +55 bp), the presence of a proximal enhancer (-498 to -199 bp) which stimulated CAT activity 6-fold, the existence of a powerful silencer (-755 to -498 bp), and a strong dista l enhancer (-4.0 to -2.0 kb) which increased CAT activity by 20-40-fol d. A computer search of available consensus sequences for trans-acting factors revealed that a cluster of Oct-sites were uniquely located in the silencer region, Using the negative response element (-797 to -49 8 bp) as a probe and nuclear extracts from HepG2 cells, three bands we re identified by gel mobility shift assay, indicating the presence of protein binding sites in this proposed negative response element. All three bands were supershifted with anti-Oct-1 mAb, suggesting that Oct -1 may be the repressor. The 5'-flanking region also contained an AP-1 site, an estrogen response element, and a glucocorticoid response ele ment, which together may comprise a steroid response unit. Although no sequence homology was found to exist between the 5'-flanking region o f the rat 3 alpha-HSD/DD gene and its human orthologue the DD2 gene, t rans-acting factor consensus sequences comprising Oct sites and steroi d response units were conserved. This implies that the expression of t he two genes may be regulated by POU-domain transcription factors and steroid hormones, respectively.