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

RUI：MEF2 和骨骼肌肌动蛋白基因转录的调控

• 批准号: 9983140
• 负责人: Sandra Sharp
• 金额: $ 24万
• 依托单位: California State University-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9983140&HistoricalAwards=false
• 关键词: RUI; MEF2; Regulation; Transcription; Skeletal

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.

肌肉发生的过程，即前体细胞发育成骨骼肌的过程，受到复杂的分子机制相互作用的调节。理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式激活因子MEF 2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。肌生成调节因子（MRF）和肌生成增强因子2（MEF 2）家族的成员都是哺乳动物骨骼肌生成所必需的。MEF 2由四种不同的基因A-D表达，并形成同源和异源二聚体。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MEF 2因子协同作用;即，当MRF和MEF 2都存在时，由启动子驱动的转录被增强到比通过单独添加它们中的每一个的作用所能解释的更大程度。在人工测试启动子上，即使当仅存在一个家族的结合位点时，或者当MEF 2或MRF缺乏反式激活结构域时，也可以发生这种协同作用。骨骼肌肌动蛋白（SKMA）基因的启动子与迄今为止已经测试的MEF 2/MRF协同作用的启动子的不同之处在于MEF 2和MRF的结合位点彼此相距很远，而不是彼此非常接近。SKMA基因表达的最大效率需要MEF 2结合位点的存在。然而，使用瞬时转染系统（其中MEF 2和MyoD由非肌肉细胞中的转基因表达），还不可能证明MEF 2对MyoD激活SKMA启动子的贡献。相比之下，在相同条件下，在包含两个紧密间隔的MEF 2和MyoD结合位点的对照修饰的肌肉肌酸激酶（MCK）启动子上容易证明协同作用。因此，MEF 2对转录效率的贡献方面仍有待解释。使用SKLAA、MCK和MEF 2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)从小鼠SKMA启动子的有效转录依赖于MEF 2位点和转录活性MEF 2。2)MEF 2的不同同种型或异源二聚体对于给定启动子的激活具有不同的效力，并且给定同种型的贡献对于所有启动子是不相同的。3)MEF 2可以通过其反式激活活性和当其在转录上无活性时促进启动子激活。这两个贡献的相对意义是同种型和启动子特异性的。为了检验这些假设，将在非肌肉细胞中用每个不同启动子的报告构建体和Myo的表达构建体进行转染[），同时包括MEF 2同种型的表达构建体以及MEF 2活性的抑制剂和刺激剂将视情况而变化。他们将允许更广泛的监管模式，然后可以通过进一步的实验进行测试的建设，在研究过程中的参与将有助于本科和硕士学位的学生作为科学家的发展，并将激励学生继续深造或进入劳动力作为研究助理或社区大学教育工作者在分子生物学。