Transcriptional regulation of the ATP citrate-lyase gene by sterol regulatory element-binding proteins.
In an attempt to identify unknown target genes for SREBP-1, total RNA from a stable Chinese hamster ovary cell line (CHO-487) expressing a mature form of human SREBP-1a (amino acids 1-487) with a LacSwitch Inducible Mammalian Expression System was subjected to a polymerase chain reaction subtraction method. One of the fragments was found to have 90 and 86% homology with rat and human ATP citrate-lyase (ACL) cDNA, respectively. When Hep G2 cells are cultured under either sterol-loaded or -depleted conditions, expression of the gene is induced approximately 2-3-fold by sterol depletion. To investigate the direct effect of SREBP-1a on transcription, luciferase assays using the promoter of the human ACL gene were performed. These deletion studies indicated that a minimum 160-base pair segment contains the information required for the transcriptional regulation brought about by enforced expression of SREBP-1a. Luciferase assays using mutant reporter genes revealed that SREBP-dependent transcriptional regulation is mediated by two nearby motifs, the SREBP-binding site (a TCAGGCTAG sequence) and the NF-Y-binding site (a CCAAT box). It was confirmed by gel mobility shift assays that recombinant SREBP-1a binds to the sequence. Data from studies with transgenic mice and reporter assays show that the ACL gene promoter is activated by SREBP-1a more strongly than SREBP-2 in contrast to the HMG CoA synthase and LDL receptor gene promoters, which exhibit the same preference for the two factors. Therefore, SREBPs transcriptionally regulates ACL enzyme activity, which generates the cytosolic acetyl CoA required for both cholesterol and fatty acid synthesis.
Sato
R
R
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. aroysato@mail.ecc.u-tokyo.ac.jp
Okamoto
A
A
Inoue
J
J
Miyamoto
W
W
Sakai
Y
Y
Emoto
N
N
Shimano
H
H
Maeda
M
M
D001275:ATP Citrate (pro-S)-Lyase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011973:Receptors, LDL / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011973:Receptors, LDL; C497387:SREBF1 protein, human; C497388:Srebf1 protein, mouse; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D007544:Isopropyl Thiogalactoside; D004247:DNA; D002784:Cholesterol; D006904:Hydroxymethylglutaryl-CoA Synthase; D001275:ATP Citrate (pro-S)-Lyase