J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M700554-JLR200 on February 13, 2008

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Journal of Lipid Research, Vol. 49, 1024-1033, May 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Cholesterol supply and SREBPs modulate transcription of the Niemann-Pick C-1 gene in steroidogenic tissues

Nicolas Gévry*, Kristina Schoonjans{dagger}, Fréderic Guay* and Bruce D. Murphy1,*

* Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Quebec, Canada J2S 7C6
{dagger} Institut de Génétique et Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale-Université Louis Pasteur, Illkirch, Communauté Urbaine de Strasbourg 67404, France

Published, JLR Papers in Press, February 13, 2008.

1 To whom correspondence should be addressed. e-mail: bruce.d.murphy{at}umontreal.ca

We tested whether sterol-regulatory element binding proteins (SREBPs) 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, whereas 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 promoter-luciferase reporter into granulosa cell lines increased the transcriptional activity of porcine, human, and mouse NPC-1 promoters. Deletion analysis of the 5' flanking region of the pig NPC-1 gene demonstrated significant promoter activity between fragments –934 and –636 bp upstream from the transcription initiation site. Sequence analysis revealed three sterol-regulatory elements (SREs) 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 attenuated the SREBP induction of promoter activity. Chromatin immunoprecipitation (ChIP) assays revealed that cholesterol depletion enriched the association of both SREBP and acetylated histone H3 with the NPC-1 promoter fragment containing the three SREs. ChIP analysis confirmed that SREBP's association with SRE and the E-box was enriched in cells cultured in cholesterol-depleted medium. We conclude that NPC-1 is sterol-regulated, achieved by SREBP acting via SRE and the E-box sequences.

Supplementary key words sterol regulatory element binding protein • gene transcription • cholesterol trafficking • granulosa cells


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