Browsing by Subject "Embryogenesis"
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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 The Regulation of TCF POP-1 Asymmetry and its Function in Early Cell Fate Specification in Caenorhabditis Elegans(2010-01-12) Huang, Shu-Yi; Lin, RueylingCell-cell signaling pervades all aspects of development. Signaling pathways need to be highly robust to ensure reproducible outcomes. In the cases of several major pathways, including the Wnt signaling pathway, default repression and signal-dependent activation are both mediated by the same response elements and transcription factors. My work focuses on how the C. elegans TCF protein POP-1 is converted from a repressor into an activator upon Wnt and MAPK signaling. C. elegans embryos exhibit a nearly invariant cell lineage composed primarily of a stepwise binary diversification of anteroposterior blastomere identities. The nuclear level of TCF/POP-1 is lowered in all posterior cells in the A-P cell divisions. I showed that the ?-catenin homolog, SYS-1, exhibits reiterated asymmetry which is reciprocal to that of POP-1. SYS-1 functions as a limiting coactivator for POP-1. The SYS-1-to-POP-1 ratio is critical for both anterior and posterior cell fates. A high ratio drives the posterior cell fate whereas a low ratio drives the anterior cell fate in multiple A-P divisions throughout development. I showed genetically that while POP-1 nuclear asymmetry is mainly regulated by the MAPK pathway, SYS-1 asymmetry is regulated solely by the Wnt pathway. Besides the quantitative differences between A-P nuclei, the Wnt and MAPK pathways also modify POP-1 qualitatively, affecting the transcriptional activity of POP-1. I showed that LIT-1/WRM-1-dependent phosphorylation of POP-1 reduces its binding to the coactivator SYS-1. Deleting the PCA domain also weakens the interaction between POP-1 and SYS-1 independent of LIT-1/WRM-1 phosphorylation. The PCA domain is required for POP-1 nuclear asymmetry and all LIT-1/WRM-1-dependent phosphorylation of POP-1. Within the PCA domain, mutating POP-1 threonine 426 to aspartic acid, but not alanine, shows similar effects to deleting the PCA domain. This study shows that Wnt and MAPK signaling pathways regulate the TCF protein POP-1 both quantitatively for its nuclear asymmetry, and qualitatively for its transcriptional activity. The two pathways not only change the levels of POP-1 and SYS-1 but also modify the strength of binding, strongly favoring the formation of POP-1/SYS-1 activation complex in the posterior cell, thereby driving A-P differential cell fates.