Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme.
The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.
Rice
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DA
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Kirkman
M S
MS
Aitken
L D
LD
Mouw
A R
AR
Schimmer
B P
BP
Parker
K L
KL
D000306:Adrenal Cortex Neoplasms; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I; D015972:Gene Expression Regulation, Neoplastic; D005796:Genes; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D011401:Promoter Regions, Genetic; D014162:Transfection
D013006:Growth Hormone; D002786:Cholesterol Side-Chain Cleavage Enzyme; D003850:Deoxyribonuclease I