Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene
Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene
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DOI：
10.1074/jbc.274.28.19573
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发表时间：
1999-07-09
影响因子：
4.8
通讯作者：
Belayew, A
中科院分区：
文献类型：
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作者：
Ding, H;Benotmane, AM;Belayew, A
人纤溶酶原激活物抑制物-1(PAI-1)的基础表达是由一个名为B box的启动子元件介导的，它与解旋酶样转录因子(HLTF)结合，与SNF/SWI蛋白同源。对一组B盒突变体进行的电泳迁移率分析表明，两个HLTF位点位于GT盒结合Sp1和Sp3的侧翼并部分重叠。影响Sp1/SP3或两个HLTF位点的突变分别被6倍和2.5倍抑制，报告基因在HeLa细胞中的瞬时表达与PAI-1启动子融合。在Sp1/Sp3缺失的昆虫细胞中，PAI-1-LacZ与Sp1或Sp3共表达导致14-26倍的诱导，而HLTF没有作用。同时存在Sp1或Sp3和短的HLTF形式(起始于Met-123)提供了额外的2-3倍的协同激活，该激活被阻止HLTF结合的突变所抑制。此外，通过HeLa细胞提取物的免疫共沉淀和谷胱甘肽S转移酶下拉实验证明了HLTFMet123与Sp1/Sp3之间的相互作用是不依赖于DNA的。相互作用区域被映射到每个蛋白质的羧基末端区域；HLTFMet123最后85个氨基酸的缺失取消了与Sp1的协同作用。这是首次证明Sp1和SnF/SWI家族蛋白之间的功能相互作用。
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.