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

California State University-Los Angeles, Los Angeles CA

Investigators

Abstract

Sharp The 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.