Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene.
Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.
Ding
H
H
Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
Benotmane
A M
AM
Suske
G
G
Collen
D
D
Belayew
A
A
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C100023:HLTF protein, human; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor