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

• Published in: Biochimica et biophysica acta Vol. 1801; no. 5; pp. 587 - 592
• Main Authors: Schiavoni, Gianluca, Bennati, Anna Maria, Castelli, Marilena, Fazia, Maria Agnese Della, Beccari, Tommaso, Servillo, Giuseppe, Roberti, Rita
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2010
• Subjects: 3beta-hydoxysterol delta14-reductase; Animals; Base Sequence; Cholesterol; 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; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; SREBP-2; Sterol Regulatory Element Binding Protein 2 - genetics; Sterol Regulatory Element Binding Protein 2 - metabolism; TM7SF2; NF-Y; 3beta-hydoxysterol delta14-reductase; SRE; SREBP-2; ChIP; 25-OH chol; Cholesterol; TM7SF2; LPDS; Promoter; Lov; FBS; EMSA; LDLR
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2010.01.013

TM7SF2 gene encodes 3β-hydroxysterol Δ 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.