Ras signaling and transcriptional synergy at a flexible Ets-1/Pit-1 composite DNA element is defined by the assembly of selective activation domains
Ras signaling and transcriptional synergy at a flexible Ets-1/Pit-1 composite DNA element is defined by the assembly of selective activation domains
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DOI：
10.1074/jbc.m302433200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Gutierrez-Hartmann, A
中科院分区：
文献类型：
--
作者：
Duval, DL;Jean, A;Gutierrez-Hartmann, A
PIT-1和Ets-1与一个复合元件结合，协同激活和靶向RAS-丝裂原激活的蛋白激酶信号转导大鼠催乳素启动子。这些转录反应似乎依赖于三个分子特征：Ets-1/Pit-1复合元件的组织，这两个因子通过Pit-1同源结构域(氨基酸199291)和Ets-1调节III结构域(氨基酸190-257)的物理相互作用，以及它们的转录激活结构域(TADS)的组装。在这里，我们表明ETS-1/Pit-1复合元件的组织在RAS刺激和协同作用方面具有显著的灵活性。具体地说，假定的单体Pit-1结合位点可以被Pit-1单体或二聚体的真正结合位点取代，并且这些结合位点允许28bp的分离。此外，我们表明Ets-1和Pit-1的物理相互作用不是RAS反应或协同所必需的，因为Ets-1中Pit-1相互作用表面的阻断突变在体外减少了Ets-1/Pit-1的结合，并没有显著影响Ets-1对RAS反应或协同作用的刺激。我们还展示了不同的TAD亚型和Pit-1 TAD亚区的不同用途，以调节协同作用或RAS反应。具体地说，来自Gal4、VP16或ETS-2调控III结构域的TAD与ETS-1 DNA结合域构建物连接，恢复了对这些TAD/ETS-1 DNA结合域融合的协同作用。相反，删除已定义的Pit-1TAD(氨基酸2-80)保留了协同作用，但不是RAS反应。因此，我们进一步将Pit-1氨基端TAD定义为区域1(R1，氨基酸2-45)和区域2(R2，氨基酸46-80)。R1似乎调节基础反应和协同反应，而RAS反应被映射到R2。综上所述，RAS的反应性和Pit-1/Ets-1的协同作用是通过在一个灵活的复合元件上组装不同的TADs来调节的，这表明这两种转录反应背后的机制不同，Pit-1R2亚区代表了一个新的组织特异性RAS反应TAD。
Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199 291) and the Ets-1 regulatory III domain (amino acids 190-257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2-80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2-45) and region 2 (R2, amino acids 46-80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD.