Abstract
Regulation of the human CYP11A gene encoding cytochrome P450scc, which catalyzes the first step of steroid synthesis, is regulated by many trans-acting transcription factors including steroidogenic factor 1 (SF-1). Transfection experiments in human adrenal NCI-H295 cells demonstrate regulation of the P450scc gene promoter region that contains several putative SF-1 binding sites. Cotransfection of SF-1 with a luciferase reporter construct containing the P450scc gene 5'-flanking region from nucleotides -1676 to +49 increased promoter activity, and deletion of the nucleotide sequence from position -1676 to -1620, which removes a putative cAMP response element (CRE), did not affect the stimulatory response to SF-1. As well, further deletion of the promoter region to nucleotide -110, which contains only one SF-1 binding site, still retained the ability to respond to exogenous SF-1. However, mutation of the remaining site which abolished SF-1 protein/DNA interaction also abrogated any functional response to the factor. All the P450scc reporter constructs which responded to SF-1 were further stimulated by exogenous p300 and CREB-binding protein (CBP), suggesting interaction between SF-1 and p300/CBP. As well, mutation of the binding site that abrogated the response to SF-1 also abolished the response to p300 and CBP. Cotransfection of the adenovirus E1A oncoprotein, which has been shown to interact with p300/CBP and interfere with its function, decreased the stimulatory effect of SF-1 and p300/CBP. Cotransfection of a mutated E1A protein, RG2, which does not interact with p300/CBP, did not alter the stimulatory effect of SF-1 and p300/CBP on the P450scc promoter. Deletion of the region from amino acid residues 2-67 in E1A, which has been postulated to interact with p300/CBP, also abolished the inhibitory effect of E1A, whereas deletion of the region from residues 120 to 140 had no effect. Two regions of CBP from amino acids 1 to 451 and from 1460 to 1891 were demonstrated to interact with SF-1 in vitro. Coexpression of fragments of the p300 protein fused to the VP16 protein in the presence of SF-1 and the -110 P450scc reporter construct indicated in vivo the interaction of two regions of p300 with SF-1, thus confirming the in vitro results. Taken together these results indicate that regulation of the human P450scc gene by SF-1 is mediated by p300/CBP. Due to the many putative roles of SF-1 to regulate many genes, its interaction with p300/CBP is potentially a key component effecting important physiological processes.
Highlights
Effect of steroidogenic factor 1 (SF-1) and CREB-binding protein (CBP)/p300 on the Human P450scc Promoter—Previous gene transfer experiments have demonstrated the ability of the 5Ј-flanking region of the human P450scc gene to confer significant promoter activity in NCIH295 cells [14]
Since it is possible that the response of promoter activity to SF-1 is concentration-dependent, Western blot analysis was performed to measure the level of exogenous SF-1 (Fig. 2)
These results were later confirmed in mouse leydig MA-10 [11] and I-10 cells [12]; experiments in human JEG-3 cells localized a cAMP response element to a different region further downstream and implicate an Sp1 site [13, 58]
Summary
The orphan nuclear receptor steroidogenic factor 1 (SF-1)1 known as adrenal 4-binding protein (Ad4BP) has been demonstrated to promote cell-specific expression of the human P450scc gene promoter in Y1 cells [15,16,17]. Reporter constructs under the control of the 5Ј-flanking DNA of the human P450scc gene, cotransfected with an SF-1 expression vector, show the ability of the nuclear receptor to regulate P450scc promoter activity in NCI-H295 cells.
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