Rat liver 3α-hydroxysteroid/dihydrodiol dehydrogenase (3α-HSD/DD) is a member of the aldo-keto reductase gene superfamily. It displays high constitutive expression and inactivates circulating steroid hormones and suppresses the formation of polycyclic aromatic hydrocarbon anti- and syn-diol-epoxides (ultimate carcinogens). To elucidate mechanisms responsible for constitutive expression of the 3α-HSD/DD gene a rat genomic library obtained from adult Sprague-Dawley female liver (HaeIII partial digest) was screened, using a probe corresponding 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 to +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 the 5′-flanking region of the rat 3α-HSD/DD gene. A 1.6-kb fragment of this region was sequenced. A TATTTAA sequence (TATA box) was found at 33 bp upstream from the major transcription start site. cis-acting elements responsible for the constitutive expression of the rat 3α-HSD/DD gene were located on the 5′-flanking region by transient transfection of reporter-gene (chloramphenicol acetyl transferase, CAT) constructs into human hepatoma cells (HepG2). CAT assays identified the 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 distal enhancer (-4.0 to -2.0 kb) which increased CAT activity by 20–40-fold. 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 -498 bp) as a probe and nuclear extracts from HepG2 cells, three bands were 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 element, which together may comprise a steroid response unit. Although no sequence homology was found to exist between the 5′-flanking region of the rat 3α-HSD/DD gene and its human orthologue the DD2 gene, trans-acting factor consensus sequences comprising Oct sites and steroid response units were conserved. This implies that the expression of the two genes may be regulated by POU-domain transcription factors and steroid hormones, respectively.
This work was supported in part by Grant CA55711 from the National Cancer Institute. A preliminary report describing this work was presented at the 85th Annual Meeting of the American Association for Cancer Research, April 10–13, 1994, San Francisco, California. The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number U32864.