A cAMP-responsive element binding site is essential for sterol regulation of the human lanosterol 14alpha-demethylase gene (CYP51).
Lanosterol 14alpha-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.
Halder
Sunil K
SK
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Fink
Martina
M
Waterman
Michael R
MR
Rozman
Damjana
D
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D058886:Sterol 14-Demethylase; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; C548418:CYP51A1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497387:SREBF1 protein, human; D016329:Sp1 Transcription Factor; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D017868:Cyclic AMP-Dependent Protein Kinases