Transcriptional regulation of the bovine CYP17 (P-450(17)alpha) gene. Identification of two cAMP regulatory regions lacking the consensus cAMP-responsive element (CRE).
Regions within the 5'-flanking sequence of the bovine CYP17 (P-450(17)alpha) gene which are required for cAMP-dependent regulation of transcription have been localized by transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells. Two sequences have been found which individually confer cAMP responsiveness to reporter genes; they are located at -243/-225 and -80/-40 base pairs (bp). Obvious sequence homology between these two regions is not apparent. Gel shift competition analysis indicates that nuclear protein(s) binding to the -243/-225-bp region can be competed for by the addition of a double-stranded oligonucleotide containing a consensus cAMP-responsive element (CRE) from the human chorionic gonadotropin alpha gene, whereas addition of this CRE does not abolish protein-DNA complexes formed with fragments containing the -80/-40-bp sequence. Gel shift and Southwestern analysis indicate that the -243/-225-bp region of the P-450(17)alpha gene and the CRE both bind a 47-kDa protein and that the CRE binds additional proteins (43 and 68 kDa) not apparently recognized by the -243/-225-bp sequence. Thus cAMP-dependent regulation of the bovine P-450(17)alpha gene appears to involve two independent cis-regulatory regions, neither of which contains a consensus CRE. Based on protein binding analysis, one of these regions (that including -80/-40 bp) is distinct from the consensus CRE while the other (that containing -243/-225 bp) may be related to the consensus CRE.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
J
J
Lund
R
R
Ahlgren
D H
DH
Wu
M
M
Kagimoto
E R
ER
Simpson
M R
MR
Waterman
D000310:Adrenal Gland Neoplasms; D000818:Animals; D002417:Cattle; D002678:Chimera; D006063:Chorionic Gonadotropin / Q000235:genetics; D002872:Chromosome Deletion; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D005809:Genes, Regulator; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D013250:Steroid Hydroxylases / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D006063:Chorionic Gonadotropin; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D012313:RNA; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase