Watson D K, Robinson L, Hodge D R, Kola I, Papas T S, Seth A
Center for Molecular and Structural Biology, Medical University of South Carolina, Charleston 29425, USA.
Oncogene. 1997 Jan 16;14(2):213-21. doi: 10.1038/sj.onc.1200839.
The ETS gene products are a family of transcriptional regulatory proteins that contain a highly conserved and structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as monomers, however it has been shown that the DNA binding activity is enhanced or modulated in the presence of other factors. By differential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that regulates the expression of many growth factor responsive genes. ELK1 and SAP1a have been shown to form ternary complexes with SRF on the SRE located in the c-fos promoter. Similarly, we examined whether the ELK1, SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and PU.1 proteins can form ternary complexes with SRF on the Egr1 SREI and II. Our results demonstrate that indeed ELK1, SAPla, FLI1 and EWS-FLI1 are able to form ternary complexes with SRF on Egr1 SREs. In addition, ELK1 and SAP1a can also form quarternary complexes on the Egr1 SREI. However, the proteins ETS1, ETS2, PEA3 and PU.1 were unable to form ternary complexes with SRF on either the Egr1 or c-fos SREs. Our data demonstrate that FLI1 and EWS-FLI1 constitute new members of a subgroup of ETS proteins that can function as ternary complex factors and further implicate a novel function for these ETS transcription factors in the regulation of the Egr1 gene. By amino acid sequence comparison we found that, in fact, 50% of the amino acids present in the B-box of SAP1a and ELK1, which are required for interaction with SRF, are identical to those present in both FLI1 (amino acids 231- 248) and EWS-FLI1 proteins. This B-box is not present in ETS1, ETS2, PEA3 or PU.1 and these proteins were unable to form ternary complexes with SRF and Egrl-SREs or c-fos SRE. Furthermore, deletion of 194 amino terminal amino acids of FLI1 did not interfere with its ability to interact with SRF, in fact, this truncation increased the stability of the ternary complex. The FLI1 protein has a unique R-domain located next to the DNA binding region. This R-domain may modulate the interaction with SRF, providing a mechanism that would be unique to FLI1 and EWS-FLI1, thus implicating a novel function for these ETS transcription factors in the regulation of the Egr1 gene.
ETS基因产物是一类转录调节蛋白家族，其包含一个高度保守且结构独特的DNA结合结构域，称为ETS结构域。几种ETS蛋白以单体形式与DNA结合，然而研究表明，在其他因子存在的情况下，DNA结合活性会增强或受到调节。通过差异显示和全基因组PCR技术，我们最近发现Erg1基因是ETS蛋白的一个靶标。Egr1启动子包含多个ETS结合位点，其中三个作为两个血清反应元件（SREI和SREII）的一部分存在。SRE是一种顺式元件，可调节许多生长因子反应性基因的表达。ELK1和SAP1a已被证明可与位于c-fos启动子中的SRE上的SRF形成三元复合物。同样，我们研究了ELK1、SAP1a、FLI1、EWS-FLI1、ETS1、ETS2、PEA3和PU.1蛋白是否能与Egr1 SREI和II上的SRF形成三元复合物。我们的结果表明，事实上ELK1、SAP1a、FLI1和EWS-FLI1能够与Egr1 SRE上的SRF形成三元复合物。此外，ELK1和SAP1a也能在Egr1 SREI上形成四元复合物。然而，ETS1、ETS2、PEA3和PU.1蛋白无法与Egr1或c-fos SRE上的SRF形成三元复合物。我们的数据表明，FLI1和EWS-FLI1构成了ETS蛋白亚组的新成员，它们可以作为三元复合因子发挥作用，并进一步暗示了这些ETS转录因子在Egr1基因调控中的新功能。通过氨基酸序列比较我们发现，实际上，SAP1a和ELK1的B-box中与SRF相互作用所需的50%氨基酸与FLI1（氨基酸231-248）和EWS-FLI1蛋白中的氨基酸相同。这个B-box在ETS1、ETS2、PEA3或PU.1中不存在，这些蛋白无法与SRF和Egrl-SREs或c-fos SRE形成三元复合物。此外，FLI1的194个氨基末端氨基酸的缺失并不影响其与SRF相互作用的能力，事实上，这种截短增加了三元复合物的稳定性。FLI1蛋白在DNA结合区域旁边有一个独特的R结构域。这个R结构域可能调节与SRF的相互作用，提供一种FLI1和EWS-FLI1特有的机制，从而暗示了这些ETS转录因子在Egr1基因调控中的新功能。
