JBC Transcription and Nuclear Factor Monoclonals

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J Biol Chem, Vol. 274, Issue 28, 19573-19580, July 9, 1999

Functional Interactions between Sp1 or Sp3 and the Helicase-like Transcription Factor Mediate Basal Expression from the Human Plasminogen Activator Inhibitor-1 Gene

Hao DingDagger , Abderrafi M. BenotmaneDagger , Guntram Suske, Désiré CollenDagger , and Alexandra BelayewDagger

From the Dagger  Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium and the  Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, 35037 Marburg, Germany

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.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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