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 tra nscription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic m obility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp 1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inh ibited 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 c ells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induc tion 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 syn ergistic activation suppressed by mutations that prevented HLTF binding. Mo reover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was de monstrated by co-immunoprecipitation from HeLa cell extracts and glutathion e S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 am ino acids of HLTFMet123 abolished the synergy with Sp1. This is the first d emonstration of a functional interaction between proteins of the Sp1 and SN F/SWI families.