A cAMP-Responsive Element Binding Site Is Essential for Sterol Regulation of the Human Lanosterol 14α-Demethylase Gene (CYP51)

Lanosterol 14α-demethylase (CYP51) is involved in the cholesterol biosynthesis pathway, producing follicular fluid meiosis-activating sterol. The promoter region of the human CYP51 gene contains a cluster of regulatory elements including GC box, cAMP response element (CRE), and sterol regulatory element (SRE). To understand the mechanism of sterol-dependent regulation of this gene, several constructs of the promoter with the reporter gene have been tested in JEG-3 cells containing overexpressed human sterol regulatory element binding protein (SREBP)-1a. The wild-type construct showed maximal SREBP-dependent activation, most of which is retained when the GC box is mutated/deleted. Activation is abolished when either CRE or SRE are removed/mutated. Furthermore, mutation of CRE abolishes SREBP-dependent activation after overexpression of SREBP-1a and CRE binding protein (CREB). This shows that CRE is essential, and that under ex vivo conditions CREB and SREBP cooperate in transactivating CYP51. Interestingly, protein kinase A shows a marked stimulation of the CYP51 promoter activity when overexpressed together with SREBP-1a but not when overexpressed with CREB, suggesting phosphorylation of SREBP-1a. Using a DNA probe containing all three regulatory elements, it is found that SREBP-1a, a CREB-like factor, and specificity protein (Sp1) all probably bind the CYP51 promoter. While SREBP-1a and the CRE-bound proteins are essential for the SREBP-dependent response, Sp1 apparently functions only to maximize sterol regulation of CYP51. To date this is the first gene in which cooperation between SREBP and a CREB/CRE modulator/activating transcription factor family transcription factor is shown to be essential and sufficient for SREBP-dependent activation.