Transcriptional Regulation of the Murine Acetyl-CoA Synthetase 1 Gene through Multiple Clustered Binding Sites for Sterol Regulatory Element-binding Proteins and a Single Neighboring Site for Sp1

Yukio Ikeda, Joji Yamamoto, Masashi Okamura, Takahiro Fujino, Sadao Takahashi, Kazuhisa Takeuchi, Timothy Osborne, Tokuo T. Yamamoto, Sadayoshi Ito, Juro Sakai

Research output: Contribution to journalArticle

Abstract

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5′-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (∼2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility " supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.

Original languageEnglish (US)
Pages (from-to)34259-34269
Number of pages11
JournalJournal of Biological Chemistry
Volume276
Issue number36
DOIs
StatePublished - Sep 7 2001
Externally publishedYes

Fingerprint

Acetate-CoA Ligase
Sterol Regulatory Element Binding Proteins
Sterols
Genes
Binding Sites
Sterol Regulatory Element Binding Protein 1
Reporter Genes
Transfection
Cultured Cells
Assays
Cells
Electrophoretic mobility
Acetyl Coenzyme A
5' Flanking Region
Cloning
Hep G2 Cells
Luciferases
Drosophila
Sequence Analysis
Organism Cloning

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Transcriptional Regulation of the Murine Acetyl-CoA Synthetase 1 Gene through Multiple Clustered Binding Sites for Sterol Regulatory Element-binding Proteins and a Single Neighboring Site for Sp1. / Ikeda, Yukio; Yamamoto, Joji; Okamura, Masashi; Fujino, Takahiro; Takahashi, Sadao; Takeuchi, Kazuhisa; Osborne, Timothy; Yamamoto, Tokuo T.; Ito, Sadayoshi; Sakai, Juro.

In: Journal of Biological Chemistry, Vol. 276, No. 36, 07.09.2001, p. 34259-34269.

Research output: Contribution to journalArticle

Ikeda, Yukio ; Yamamoto, Joji ; Okamura, Masashi ; Fujino, Takahiro ; Takahashi, Sadao ; Takeuchi, Kazuhisa ; Osborne, Timothy ; Yamamoto, Tokuo T. ; Ito, Sadayoshi ; Sakai, Juro. / Transcriptional Regulation of the Murine Acetyl-CoA Synthetase 1 Gene through Multiple Clustered Binding Sites for Sterol Regulatory Element-binding Proteins and a Single Neighboring Site for Sp1. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 36. pp. 34259-34269.
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abstract = "Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5′-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (∼2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility {"} supershift{"} analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.",
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AU - Takeuchi, Kazuhisa

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