Members of the nuclear factor 1 transcription factor family regulate rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD AKR1C9) gene expression: a member of the aldo-keto reductase superfamily.
Rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD; AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, inactivates nearly all steroid hormones by converting 5alpha- and 5beta-dihydrosteroids to their respective 3alpha,5alpha- and 3alpha,5beta-tetrahydrosteroids and protects against circulating steroid hormone excess. It is highly expressed in rat liver comprising 0.5-1.0% of the soluble protein. Previously, we identified a powerful distal enhancer resident at about -4.0 kb to -2.0 kb in the 5'-flanking region of the 3alpha-HSD/DD gene. We now report the functional dissection of this enhancer. Transfection of nested deletions of the 5'-end of the gene promoter linked to chloramphenicol acetyltransferase (CAT) into HepG2 cells located the enhancer activity between (-4673 to -4179 bp). Further internal and 5'-end deletion mutants revealed that a 73-bp fragment (from -4351 to -4279 bp) contained a major enhancer element. This fragment spanned two imperfect direct repeats GTGGAAAAACCCAGGAA and GTGGAAAAAACCCAGGAA and contained three direct repeats of GGAAAAA. This fragment also contained three potential half-nuclear factor 1 (NF1) sites (TGGA-NNNNNGCCA) and a putative CCAAT-enhancer binding protein (C/EBP) binding site. The 73-bp fragment enhanced CAT activity from the basal 3alpha-HSD/DD gene promoter. Recombinant C/EBPalpha and C/EBPbeta did not bind to this fragment. Electrophoretic mobility shift assays showed that HepG2 and rat liver nuclear extracts bound to this 73-bp fragment. The 73-bp protein complex was competed out by a NF1 oligonucleotide and was supershifted by an NF1 antibody. When the 73-bp fragment was fused to an alpha1-globin promoter-CAT construct and cotransfected with CCAAT transcription factor 1 (CTF1)/NF1 into Drosophila Schneider SL2 insect cells (which lack NF1-like proteins) trans-activation of CAT activity was observed. These results indicate that members of the NF1 transcription factor family regulate high constitutive expression of the rat 3alpha-HSD/DD gene that is responsible for steroid hormone inactivation. The potential role of NF1 in regulating other AKR genes that have protective roles is discussed.
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Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
Penning
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics; D006801:Humans; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D005914:Globins; D010088:Oxidoreductases; D007658:Ketone Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase