Browsing by Subject "Serum Response Factor"
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Item Biochemical and Functional Analysis of Members of the Myocardin Family During Cardiovascular Development(2006-12-20) Oh, Jiyeon; Olson, Eric N.The various stages of muscle development are characterized by distinct patterns of gene expression precisely controlled by combinatorial interaction between a large number of muscle-specific and ubiquitous transcription factors. Myocardin is a cardiac and smooth muscle-specific transcriptional coactivator of serum response factor (SRF) that forms a ternary complex with SRF on DNA and provides its strong transcriptional activation domain (TAD) to SRF. SRF has been shown to stimulate expression of smooth and cardiac muscle genes in association with GATA transcription factors, which play important roles in cardiac and smooth muscle development. I show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. To determine the functions of MRTF-B in vivo, I generated MRTF-B mutant mice by targeted inactivation of the MRTF-B gene. I show that mice homozygous for an MRTF-B loss-of-function mutation die during mid-gestation from a spectrum of cardiovascular defects. These abnormalities are accompanied by a failure in differentiation of smooth muscle cells within the branchial arch arteries, which are derived from the neural crest. The phenotype of MRTF-B mutant mice is distinct from that of mice lacking myocardin and MRTF-A, revealing unique roles for these SRF coactivators in the development of different subsets of smooth muscle cells in vivo.Item Mechanistic Analysis of SRF and the Myocardin Family of Coactivators During Muscle Development(2005-12-20) Li, Shijie; Olson, Eric N.The precise mechanism of how specification and differentiation of different muscle types are controlled by a large number of transcription factors has been a long-standing question in developmental biology. Using animal models with tissue-specific deletions of various transcription factors, coupled with biochemical studies, the molecular mechanisms regulating muscle development and growth are being elucidated. Serum response factor (SRF), a muscle-enriched transcription factor, activates the expression of numerous muscle genes by recruiting a variety of partner proteins. The function of SRF in each muscle type in vivo is clouded by the fact that SRF mutant mice die before gastrulation without the formation of mesoderm. Generating a tissue-specific deletion of the SRF gene, I found that SRF is required for skeletal muscle growth and maturation. Myocardin was identified as a cardiac and smooth muscle-specific transcriptional coactivator of SRF. Mice lacking myocardin die during early embryogenesis due to cardiovascular defects, which are caused by the failure of vascular smooth muscle to differentiate. Together with the data that overexpression of myocardin in non-muscle cells can activate the smooth muscle gene program, we demonstrate that myocardin is both required and sufficient for smooth muscle differentiation. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, which also interact with SRF and stimulate its transcriptional activity, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. Consistent with the role of SRF during skeletal muscle development, a dominant-negative form of MRTF-A interferes with skeletal muscle development in transgenic mice. To further elucidate MRTF-A's function, I generated MTTF-A mutant mice by gene homologous recombination. Female MRTF-A mutant mice fail to nurture their offspring due to mammary defects. While milk is produced at a normal level, mammary myoepithelial cells, which are similar to smooth muscle cells and required for milk ejection, fail to differentiate and undergo programmed cell death during lactation. Taken together, these data indicated that SRF regulates specification or maturation of different muscle types by interacting with various members of the myocardin family of coactivators.Item Muscle-Specific Regulation of Serum Response Factor by Differential DNA Binding Affinity and Cofactor Interactions(2003-04-01) Chang, Priscilla Shin-Ming; MacDonald, Raymond J.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.Item Roles of Myocardin-Related Transcription Factors in Muscle and Brain(2010-01-12T18:55:05Z) Mokalled, Mayssa H.; Olson, Eric N.Partnerships between DNA binding transcription factors and transcriptional cofactors govern gene transcription in various developmental and tissue contexts, particularly during cardiovascular and neuronal development. This dissertation aims at studying the in vivo relevance of the partnership between Serum response Factor (SRF) and its Myocardin Related Transcription Factor (MRTF) coactivators during development. I present here my studies on the functions of MRTFs during brain and muscle development. First, I show that MRTF-A and -B redundantly control neuronal migration and neurite outgrowth during brain development. Conditional deletion of these genes in the mouse brain disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. I also describe a previously unrecognized role for the MRTF/SRF pathway in the regulation of the Pctaire-1/Cdk5 kinase cascade to govern actin dynamics. I conclude that MRTFs function as essential coregulators of SRF to control actin dynamics during neuronal development via the Cdk5/Pctaire-1 kinase cascade. I also explore the role of MRTF-A and -B in cardiac development and function. Ablation of these MRTF genes in the embryonic heart causes` a range of cardiac defects, reflecting the sensitivity of cardiac function to MRTF gene dosage. Moreover, I show that the gene encoding Pctaire-1 kinase, whose functions in the heart are unknown, is also a target of MRTF/SRF signaling and a regulator of sarcomere assembly in the heart. Furthermore, by creating mice lacking the Myocardin related factor MASTR, I explore the in vivo developmental functions of MASTR. Germline deletion of MASTR alone does not cause any obvious defects in mice. However, deletion of MASTR in an MRTF-A null background causes perinatal lethality, which appears to be due to defective skeletal muscle growth and development. Thus, the results of my thesis research demonstrate that MRTFs are essential regulators of multiple developmental processes in brain, heart, and skeletal muscle. At the cellular level, MRTFs are essential regulators of the actin cytoskeleton. Disruption of MRTF functions, whether in neurons or in muscle cells, causes major cytoskeletal defects that impair brain and muscle development and function. [Keywords: MRTF; skeletal muscle; cardiac development; actin dynamics; neurite outgrowth; neuronal migration; MASTR; Pedtaire-1; myocardin; sarcomere assembly]