Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box.
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.
Schiavoni
Gianluca
G
Department of Internal Medicine, Laboratory of Biochemistry, University of Perugia, via del Giochetto, 06122 Perugia, Italy.
Bennati
Anna Maria
AM
Castelli
Marilena
M
Della Fazia
Maria Agnese
MA
Beccari
Tommaso
T
Servillo
Giuseppe
G
Roberti
Rita
R
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; C497390:SREBF2 protein, human; D051782:Sterol Regulatory Element Binding Protein 2; D044925:Oxidoreductases Acting on CH-CH Group Donors; C113387:TM7SF2 protein, human