Muscle-Specific Regulation of Serum Response Factor by Differential DNA Binding Affinity and Cofactor Interactions
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Serum response factor (SRF) is a MADS-box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the CArG box consensus sequence CC(A/T)6GG. Because SRF expression is not muscle-restricted, its expression alone cannot account for the muscle-specificity of some of its target genes. To further understand the role of SRF in muscle-specific transcription, two distinct approaches were taken. First, tandem multimers of different CArG boxes with flanking sequences were analyzed in transgenic mice. CArG elements from the SM22 and skeletal a-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the cfos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type-specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread expression bound SRF more strongly than those that directed muscle-restricted expression. Therefore, sequence variations among CArG boxes influence cell type-specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo. Second, a novel transcriptional cofactor for SRF called Myocardin was characterized. Myocardin belongs to the SAP domain family of nuclear proteins, is expressed specifically in cardiac and smooth muscle cells, and is a potent activator of cardiac and smooth muscle genes, including SM22. Myocardin activates through CArG boxes, and its activation is dependent on its interaction with the MADS box domain of SRF. Myocardin is the founding member of a new class of muscle-specific transcription factors and provides another mechanism whereby SRF can convey myogenic activity to muscle-specific genes. These results describe two mechanisms for muscle-specific activation of target genes by SRF. Muscle-specific genes contain CArG boxes with relatively low affinities for SRF, and thus are only able to respond to the high levels of SRF found in muscle. Also, Myocardin, a muscle-specific transcription factor, is able to associate with SRF and cooperatively activate transcription of muscle genes.