Abstract
Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5'-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, School of Medicine, 607 Light Hall, Nashville, TN 37232-0146
We demonstrate that 1) one of the proteins binding to the sequence between Ϫ111 and Ϫ100 bp within the bovine CYP11A promoter is Sp1 or a protein antigenically related to it, 2) mutations in this region either eliminate or markedly reduce both binding of Sp1 and cAMP-dependent transcription mediated by this element in Y1 adrenocortical cells, 3) there is at least one additional sequence between Ϫ70 and Ϫ50 bp of the bovine CYP11A gene which binds Sp1 and supports cAMPinduced transcription, and 4) the cAMP-induced transcription mediated by the Sp1-binding sequences of the bovine CYP11A gene is dependent on the cAMP-dependent protein kinase (PKA) catalytic subunit
Mutations in the Ϫ118/Ϫ100 Sequence Affect Formation of DNA-Sp1 Complexes—The proximal promoter region of the bovine CYP11A gene (Fig. 1A) that supports cAMP-induced transcription in Y1 adrenocortical cells (Ahlgren et al, 1990) contains potential binding sites for the general transcription factor Sp1 (Kadonaga et al, 1986), steroidogenic factor, SF-1 (Morohashi et al, 1992; Lala et al, 1992), and adrenal specific factor, adrenal-specific protein (ASP) (Kagawa and Waterman, 1992). Deletion analyses of this region have previously shown that the sequence between Ϫ118 and Ϫ100 is sufficient for supporting cAMP-dependent transcription in both Y1 (Momoi et al, 1992) and bovine ovarian luteal cells (Begeot et al, 1993)
Summary
§ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, School of Medicine, 607 Light Hall, Nashville, TN 37232-0146. Serial deletions of the proximal 896 base pairs (bp) of the 5Ј-flanking region of the bovine CYP11A gene identified a cAMP-response sequence (CRS) between Ϫ183 and Ϫ83 bp (Ahlgren et al, 1990) This element was found to reside within Ϫ118 and Ϫ100 bp and was shown to be sufficient for cAMP-dependent transcription of a reporter gene in mouse adrenocortical Y1 tumor cells (Momoi et al, 1992) as well as in bovine ovarian luteal cells (Begeot et al, 1993). We demonstrate that 1) one of the proteins binding to the sequence between Ϫ111 and Ϫ100 bp within the bovine CYP11A promoter is Sp1 or a protein antigenically related to it, 2) mutations in this region either eliminate or markedly reduce both binding of Sp1 and cAMP-dependent transcription mediated by this element in Y1 adrenocortical cells, 3) there is at least one additional sequence between Ϫ70 and Ϫ50 bp of the bovine CYP11A gene which binds Sp1 and supports cAMPinduced transcription, and 4) the cAMP-induced transcription mediated by the Sp1-binding sequences of the bovine CYP11A gene is dependent on the cAMP-dependent protein kinase (PKA) catalytic subunit
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