Tretiakova A, Steplewski A, Johnson E M, Khalili K, Amini S
Center for NeuroVirology and NeuroOncology, MCP Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102, USA.
J Cell Physiol. 1999 Oct;181(1):160-8. doi: 10.1002/(SICI)1097-4652(199910)181:1<160::AID-JCP17>3.0.CO;2-H.
Direct interaction between transcription factors may provide a mechanism for the regulatory function of these proteins on transcription of the responsive genes. These interactions may be facilitated if the target DNA sequences for the participant regulatory proteins are overlapped or positioned in close proximity to each other within the promoter of the responsive genes. In earlier studies, we identified a cellular protein, named Puralpha, which upon binding to the MB1 regulatory DNA sequence of the myelin basic protein (MBP) gene, stimulates its transcription in central nervous system (CNS) cells. Here, we provide evidence for binding of the ubiquitous DNA binding transcription factor, Sp1, to the MB1 DNA motif at the region that partially overlaps with the Puralpha binding site. We demonstrate that binding of Puralpha to its target sequence is enhanced by inclusion of Sp1 in the binding reaction. Under this condition, binding of Sp1 to the MB1 regulatory sequence remained fairly unchanged, and no evidence for the formation of Puralpha:MB1:Sp1 was observed. This observation suggests that transient interaction of Puralpha and Sp1 may result in stable association of Puralpha and the MB1 element. In support of this notion, results from immunoprecipitation/Western blot studies have established association of Puralpha and Sp1 in nuclear extracts from mouse brain. Of interest, Puralpha appears to bind to the phosphorylated form of Sp1 which is developmentally regulated and that coincides with the periods when MBP gene expression is at its maximum level. Results from cotransfection studies revealed that ectopic expression of Puralpha and Sp1 synergistically stimulates MBP promoter activity in CNS cells. The importance of these findings in stage-specific expression of MBP during brain development is discussed.
转录因子之间的直接相互作用可能为这些蛋白质对响应基因转录的调控功能提供一种机制。如果参与调控的蛋白质的靶DNA序列在响应基因启动子内相互重叠或位置紧邻，这些相互作用可能会更容易发生。在早期研究中，我们鉴定出一种细胞蛋白，命名为Puralpha，它与髓鞘碱性蛋白（MBP）基因的MB1调控DNA序列结合后，可刺激其在中枢神经系统（CNS）细胞中的转录。在此，我们提供证据表明，普遍存在的DNA结合转录因子Sp1可与MB1 DNA基序在部分与Puralpha结合位点重叠的区域结合。我们证明，在结合反应中加入Sp1可增强Puralpha与其靶序列的结合。在此条件下，Sp1与MB1调控序列的结合保持相当稳定，且未观察到Puralpha:MB1:Sp1形成的证据。这一观察结果表明，Puralpha和Sp1的瞬时相互作用可能导致Puralpha与MB1元件的稳定结合。支持这一观点的是，免疫沉淀/蛋白质印迹研究结果证实了小鼠脑核提取物中Puralpha和Sp1的结合。有趣的是，Puralpha似乎与Sp1的磷酸化形式结合，而Sp1的磷酸化形式受发育调控，且与MBP基因表达达到最高水平的时期一致。共转染研究结果显示，Puralpha和Sp1的异位表达协同刺激CNS细胞中MBP启动子的活性。本文讨论了这些发现在脑发育过程中MBP阶段特异性表达中的重要性。
