A role for Sp1 in the transcriptional regulation of hepatic triacylglycerol hydrolase in the mouse.
Microsomal triacylglycerol hydrolase (TGH) hydrolyzes stored triacylglycerol in cultured hepatoma cells (Lehner, R., and Vance, D. E. (1999) Biochem. J. 343, 1-10). We studied expression of TGH in murine liver and found both protein and mRNA increased dramatically at 27 days after birth. Nuclear run-on assays demonstrated that this was due to increased transcription. We cloned 542 base pairs upstream of the transcriptional start site of the murine TGH gene. Electrophoretic mobility shift assays demonstrated enhanced binding of hepatic nuclear proteins from 27-day-old mice to the murine TGH promoter, yielding three differentially migrating complexes. DNase I footprint analysis localized these complexes to two distinct regions: site A contains a putative Sp binding site, and site B contains a degenerate E box. We transfected primary murine hepatocytes with a series of 5'-deletion constructs upstream of the reporter luciferase cDNA. Positive control elements were identified in a segment containing site A. Competitive electrophoretic mobility shift assays and supershift assays demonstrated that site A binds Sp1 and Sp3. Transcriptional activation assays in Schneider SL-2 insect cells demonstrated that Sp1 is a potent activator of the TGH promoter. These experiments directly link increased TGH expression at the time of weaning to transcriptional regulation by Sp1.
Douglas
D N
DN
Department of Biochemistry, Canadian Institute of Health Research, Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Dolinsky
V W
VW
Lehner
R
R
Vance
D E
DE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor; D018411:Spodoptera; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases; D008049:Lipase