Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box

Biochim Biophys Acta. 2010 May;1801(5):587-92. doi: 10.1016/j.bbalip.2010.01.013. Epub 2010 Feb 4.

Abstract

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA Mutational Analysis
  • Gene Expression Regulation
  • Humans
  • Membrane Proteins* / genetics
  • Membrane Proteins* / metabolism
  • Molecular Sequence Data
  • Oxidoreductases Acting on CH-CH Group Donors* / genetics
  • Oxidoreductases Acting on CH-CH Group Donors* / metabolism
  • Promoter Regions, Genetic*
  • Regulatory Sequences, Nucleic Acid*
  • Sterol Regulatory Element Binding Protein 2 / genetics
  • Sterol Regulatory Element Binding Protein 2 / metabolism*

Substances

  • Membrane Proteins
  • SREBF2 protein, human
  • Sterol Regulatory Element Binding Protein 2
  • Oxidoreductases Acting on CH-CH Group Donors
  • TM7SF2 protein, human