TRANSCRIPTIONAL REGULATION OF THE TATA-LESS NADPH CYTOCHROME-P-450 OXIDOREDUCTASE GENE

Multiple cis-acting DNA sequences regulating expression of the rat liv er NADPH-cytochrome P-450 oxidoreductase gene have been identified in transient transfection assays using promoter deletion constructs linke d to the chloramphenicol acetyl transferase gene. The TATA-less promot er possesses nine CC-boxes which contain the consensus sequence for th e transcription factor Spl. While loss of the seven distal CC-boxes ha d minimal effect on transcriptional activity, deletion of the next 35 bp, from -206 to -172, resulted in similar to 90% loss of promoter act ivity. Contained within this region is an Spl binding site indicating that either (1) this particular consensus sequence was essential for t ranscription, (2) the two proximal GC boxes act in concert, or (3) a y et unidentified regulatory element resides within this 35-bp stretch. In addition, transfection experiments demonstrated that two separate d istal regions (-622 to -1167 and -1500 to -2300) contain negative regu latory elements which down-regulate gene transcription in a position-i ndependent manner. Mobility-shift analyses and DNase footprinting iden tified sequences in the proximal region of the promoter that bound pro teins present in nuclear extracts. Four protected segments were observ ed within the first 100 bp upstream of the transcription start site; t hese include (1) the region encompassing the transcription start site (-7 to +4), (2) the region normally occupied by a TATA-box (-38 to -18 ), (3) the bases from -78 to -60 which contain the regulatory element CACC, and (4) bases -105 to -92 which include an Spl binding site. Hen ce, regulation of the NADPH-cytochrome P-450 oxidoreductase gene is co ntrolled by both positive and negative regulatory elements, and, of th e nine Spl consensus sites, the two proximal sites are sufficient to s upport basal transcription. (C) 1996 Academic Press, Inc.