RUI: MEF2 and Regulation of Transcription of the Skeletal Muscle Actin Gene

前体细胞发育为骨骼肌的肌形成过程是由分子机制的复杂相互作用调控的。了解这些机制的一种方法是研究调节肌肉特异性基因转录的dna -蛋白质和蛋白质-蛋白质相互作用。本项目将研究转录反激活因子MEF2及其与DNA的结合在促进肌生成调节因子MyoD激活转录中的作用。哺乳动物骨骼肌形成需要肌生成调节因子（MRF）和肌生成增强因子2 （MEF2）家族的成员。MEF2s由四种不同的基因A-D表达，形成同源二聚体和异源二聚体。不同亚型的细胞特异性和启动子特异性作用尚不清楚，已知MRF和MEF2因子协同作用；也就是说，当MRF和MEF2同时存在时，由最小启动子驱动的转录比单独添加它们各自的作用所能解释的转录增强得更大。在人工测试启动子上，即使只有一个家族的结合位点存在，或者MEF2或MRF缺乏转激活结构域，这种协同作用也会发生。骨骼肌肌动蛋白（SKMA）基因的启动子与MEF2/MRF协同作用的启动子不同，MEF2和MRF的结合位点不是彼此非常靠近，而是彼此相隔很远。SKMA基因的最高表达效率需要MEF2结合位点的存在，然而，使用瞬时转染系统（MEF2和MyoD在非肌肉细胞中通过转基因表达），还不可能证明MEF2对MyoD激活SKMA启动子的贡献。相反，在相同条件下，由两个紧密间隔的MEF2和MyoD结合位点组成的控制修饰的肌酸激酶（MCK）启动子很容易证明协同作用。因此，MEF2对转录效率的贡献方面仍有待解释。SKLAA、MCK和MEF2位点阴性启动子的瞬时转染和荧光素酶报告基因试验将用于检验以下假设。1)小鼠SKMA启动子的高效转录依赖于MEF2位点和转录活性MEF2。2) MEF2的不同同工异构体或异源二聚体对特定启动子的激活能力不同，并且对所有启动子的激活作用也不尽相同。3) MEF2可以通过其转录激活活性和转录失活来促进启动子激活。这两种贡献的相对意义都是同工异构体和启动子特异性的。为了验证这些假设，转染将在非肌肉细胞中进行，其中包含每种不同启动子的报告结构和Myo的表达结构[)，而MEF2同种异构体的表达结构以及MEF2活性的抑制剂和刺激物将根据需要进行变化。从这些实验中得出的结论将有助于我们理解基因表达是如何被调控的。他们将允许构建更广泛的监管模型，然后可以通过进一步的实验进行测试。参与研究过程将有助于本科生和硕士学位学生作为科学家的发展，并将激励学生继续攻读高级学位或进入劳动力市场，成为分子生物学的研究助理或社区大学教育工作者。

SharpThe process of myogenesis, by which precursor cells develop into skeletal muscle, is regulated by an intricate interplay of molecular mechanisms. One approach to understanding these mechanisms is to study the DNA-protein and protein-protein interactions that regulate transcription of muscle-specific genes. This project will investigate the role of the transcriptional transactivator MEF2 and its binding to DNA in facilitating the activation of transcription by the myogenesis regulatory factor MyoD. Members of both the myogenesis regulatory factor (MRF) and the myogenesis enhancing factor 2 (MEF2) families are required for skeletal myogenesis in mammals. MEF2s are expressed from four different genes, A-D, and form homo- and heterodimers. The cell- and promoter-specific roles of the different isoforms are not known, It is known that MRF and MEF2 factors act synergistically; that is, transcription driven by a lest promoter is enhanced to a greater extent when both a MRF and a MEF2 are present than can be explained by adding the effects of each of them alone. On artificial test promoters, this synergism can occur even when the binding site for only one family is present, or when either MEF2 or MRF lacks a transactivation domain. The promoter of the skeletal muscle actin (SKMA) gene differs from the promoters for which MEF2/MRF synergism has been tested thus far in that the binding sites for MEF2 and MRF, rather than being situated very near each other, are widely separated from each other. Maximum efficiency of expression of the SKMA gene requires the presence of a MEF2 binding site, However, it has not been possible to demonstrate a contribution of MEF2 to the activation of the SKMA promoter by MyoD using a transient transfection system in which MEF2 and MyoD are expressed from transgenes in non-muscle cells. In contrast, under the same conditions, synergism is easily demonstrable on a control modified muscle creatine kinase (MCK) promoter comprising two closely spaced MEF2 and MyoD binding sites. Thus aspects of the contribution of MEF2 to transcriptional efficiency remain to be explained. Transient transfection and luciferase reporter assays with SKLAA, MCK, and MEF2 site-negative promoters will be used to test the following hypotheses. 1) Efficient transcription from the mouse SKMA promoter is dependent upon the MEF2 site and upon transc6ptionally active MEF2. 2) Different isoforms or heterodimers of MEF2 have different potencies with respect to activation of a given promoter, and the contribution of a given isoform is not the same for all promoters. 3) MEF2 can contribute to promoter activation both through its transactivational activity and when it is inactive transcriptionally. The relative significance of the two contributions is both isoform and promoter specific. To test these hypotheses, transfections will be carried out in non-muscle cells with reporter constructs for each of the different promoters and an expression construct for Myo[), while inclusion of expression constructs for the MEF2 isoforms, as well as inhibitors and stimulators of MEF2 activity, will be varied as appropriate.The conclusions drawn from these experiments will contribute to our understanding of how gene expression is regulated. They will allow the construction of broader regulatory models that can then be tested by further experimentation, Involvement in the research process will contribute to the development of undergraduate and Masters Degree students as scientists and will motivate students to continue for advanced degrees or to enter the workforce as research associates or community college educators in molecular biology.