Submitted on November 28, 2007
Revised on January 18, 2008
Accepted on February 12, 2008
Cholesterol supply and sterol regulatory element binding proteins modulate transcription of the Niemann-Pick C1 gene in steroidogenic tissues
Nicolas Gévry, Kristina Schoonjans, Fréderic Guay, and Bruce D. Murphy
CRRA, Université de Montréal, St-Hyacinthe, QC J2S7C6
Corresponding Author: bruce.d.murphy{at}umontreal.ca
WWe tested whether Sterol Regulatory Element Binding Proteins (SREBP’s) mediate sterol-regulated transactivation of the Niemann Pick C-1 (NPC-1) gene. Loading granulosa cells with 22- or 25-hydroxycholesterol decreased NPC-1 mRNA 5 while culturing in cholesterol-depleted medium or inhibition of cholesterol biosynthesis increased NPC-1 promoter activity and NPC-1 mRNA abundance. Cotransfection of SREBP1a, SREBP1c and SREBP2 and the NPC-1 promotorluciferase reporter into granulosa cell lines increased transcriptional activity of porcine, human and mouse NPC-1 promoters. Deletion analysis of the 5' flanking 10 region of the pig NPC-1 gene demonstrated significant promoter activity between fragments -934 and –636 bp upstream from the transcription initiation site, while sequence analysis revealed three sterol regulatory elements (SRE) clustered between-558 and -650 bp. Each site, along with E-box sequences, bound recombinant SREBP in electromobility shift assays. Mutation of all three sites 15 attenuated SREBP induction of promoter activity. Chromatin immunoprecipitation assays (ChIP) revealed that cholesterol depletion enriched association of both SREBP and acetylated histone H3 with the NPC-1 promoter fragment containing the three SRE. ChIP analysis confirmed that SREBP association with SRE and the E-box was enriched in cells cultured in cholesterol-depleted medium. We conclude 20 that NPC-1 is sterol-regulated, achieved by SREBP acting via SRE and the E-box sequences.
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