Identification of promoter elements in the mouse 21-hydroxylase (Cyp21) gene that require a functional cyclic adenosine 3',5'-monophosphate-dependent protein kinase.
The constitutive and cAMP-induced expression of the mouse steroid 21-hydroxylase gene (Cyp21) are impaired in adrenal cell mutants harboring mutations in cAMP-dependent protein kinase (cAMPdPK). These requirements for a functional cAMPdPK have been mapped to the proximal 330 basepairs of the Cyp21 promoter. This study attempts to identify specific promoter elements of Cyp21 that require cAMPdPK for constitutive activity by comparing their abilities to enhance the expression of a reporter gene in Y1 adrenocortical tumor cells and Y1 Kin mutants defective in cAMPdPK activity. As determined in transient transfection assays, Cyp21 promoter elements at -65, -140, -170, -210, and -280 each enhanced the expression of a human GH reporter gene in parent Y1 cells. The relative order of effectiveness of each of these elements was: -170 >> -280 > -140 > -65 > or = -210. The -170 element was 25-fold more effective in enhancing gene expression from the reporter construct in Y1 cells than in Kin mutant cells; the elements at -65, -140, and -210 were 3-fold more effective in Y1 cells than in Kin mutant cells; the -280 element was equally effective in the parent and Kin mutant clones. These studies suggest that the promoter elements at -170, -65, -140, and -210 mediate the requirement for a functional cAMPdPK in the expression of Cyp21. As determined by gel mobility shift assays with these elements, the dependence of the Cyp21 promoter elements on a functional cAMP-dependent protein kinase did not result from decreased expression or binding affinities of their respective DNA-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
A M
AM
Parissenti
K L
KL
Parker
B P
BP
Schimmer
D000302:Adrenal Cortex / Q000166:cytology / Q000201:enzymology; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology; D004790:Enzyme Induction; D005796:Genes; D006801:Humans; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D013255:Steroid 21-Hydroxylase; D011494:Protein Kinases