The goal of this proposal continues to be the molecular characterization of the cardiac contractile system by focusing on the mechanisms involved in the generation and maintenance of the differentiated cardiac phenotype. We have recently cloned a family muscle-and neural specific transcription factor MEF2, which belongs to the MADS super-family of regulators. The MEF2 proteins accumulate in the mesodermal cells of the early embryos of Drosophila and Xenopus. Their expression parallels, is induced by, and induces that of the bHLH myogenic regulators during myogenesis. In addition, protein complexes formed by the direct interactions between MEF2 and MyoD, or MEF2 and the T3 receptors, cooperatively induce the expression of skeletal and cardiac-specific genes, respectively. Taken together, these observations suggest that MEF2 proteins constitute the common component of a binary switch system implicated not only in the maintenance of, but also in the production of the muscle phenotype. The participation of MEF2 proteins in the cardiac morphogenetic processes will be first be evaluated by determining the temporal and spatial pattern of MEF2 gene expression during mouse development, by in situ hybridization and immunocytochemistry. Their cardiac-lineage determining properties will be assessed by testing the effects of gain or repression of MEF2 function in genetically altered cell lines and transgenic mice producing a wild-type MEF2 protein or a dominant-negative MEF2 mutant respectively. The interactive properties of MEF2 and MyoD will serve as a model system to clone the co-regulators of cardiac activity and expression in the heart. The heterodimerization domain of MEF2 will be used as a """"""""bait"""""""" to clone cardiac proteins which interact with MEF2, using the yeast two hybrid system. DNA-binding motifs for proteins which increase the DNA-binding activity of MEF2 will be selected by the CASTing procedure, as a means to identify these proteins. The cis-regulatory sequences of the MEF2 gene which is first expressed during cardiac morphogenesis will be characterized in order to clone the transcriptional regulators of gene MEF2 expression in the heart. The factors obtained by some of these procedures would have by themselves, or together with the MEF2 proteins, a cardiac-lineage determining function. These approaches should contribute significantly to the elucidation of the mechanisms involved in cardiac morphogenis.
| Andres, V; Cervera, M; Mahdavi, V (1995) Determination of the consensus binding site for MEF2 expressed in muscle and brain reveals tissue-specific sequence constraints. J Biol Chem 270:23246-9 |
