Grayson J, Bassel-Duby R, Williams R S
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8573, USA.
J Cell Biochem. 1998 Sep 1;70(3):366-75.
Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation.
先前的研究表明，作为肌肉特异性转录增强子的功能成分，CCAC与富含A/T的核苷酸序列基序在体内存在协同相互作用。利用来自肌红蛋白和肌肉肌酸激酶（MCK）基因增强子的CCAC和富含A/T的元件，Sp1和肌细胞特异性增强子因子2（MEF-2）被鉴定为识别这些位点的同源结合蛋白。通过仅在存在A/T序列基序的情况下，用核提取物在体外形成的DNA结合复合物中对两种蛋白质进行免疫检测、在存在与谷胱甘肽珠结合的谷胱甘肽-S-转移酶-Sp1融合蛋白的情况下对重组MEF-2进行共沉淀以及在酿酒酵母中进行双杂交试验，证明了Sp1和MEF-2之间的物理相互作用。MEF-2在体外和体内与Sp1的相互作用具有特异性，而与血清反应因子（一种相关的MADS结构域蛋白）未观察到这种相互作用。在原本缺乏这些因子的昆虫细胞中强制表达Sp1和MEF-2可促进含有这两种蛋白质结合位点的启动子的协同转录激活。这些数据扩展了谱系限制（MEF-2）和普遍存在（Sp1）的转录因子之间功能和物理相互作用的范围，这可能对肌源性分化很重要。
