| Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. In many cell types, including cardiac and skeletal muscle, the basic helix-loop-helix (bHLH) family of transcription factors have been shown to play critical roles in cellular differentiation. These proteins generally regulate transcription by binding as homo- or heterodimers to E-box (‘CANNTG’) elements. The focus of this thesis project was to determine the role of the E-box/bHLH dependent transcriptional regulatory pathway in SMC differentiation.; Mutagenesis studies revealed that two E-boxes in the smooth muscle α-actin (SM α-actin) promoter were required for proper regulation of the promoter in transgenic mice, providing the first demonstration of E-box dependent regulation of an SMC differentiation marker gene in vivo. Further studies utilizing co-transfection and chromatin immunoprecipitation approaches demonstrated that Class I bHLH proteins are likely involved in SM α-actin regulation and that at least a subset of Class I bHLH proteins, including HEB and E2-2, activated the SM α-actin promoter synergistically with serum response factor, SRF. Interestingly, the dominant negative/inhibitory HLH proteins, Id2, Id3, and Twist, negatively regulated the SM α-actin promoter.; To further understand regulation of SMC differentiation by Class I bHLH proteins, Class I bHLH-interacting proteins were sought using a yeast two-hybrid approach. This screen resulted in the identification of several factors including the homeoprotein, Mox1, and members of the PIAS (protein inhibitor of activated STAT) family, PIAS1/xβ. Initial characterization of Mox1 and PIAS1/x proteins using a variety of approaches including RT-PCR analysis and co-transfection assays suggested that these factors are likely to play an important role in SMC differentiation.; Taken together, the results presented in this thesis support a model in which regulation of the SM α-actin promoter occurs, at least in part, through complex combinatorial interactions between several important classes of transcription factors including HLH proteins, homeoproteins, PIAS family members, and SRF and suggests that these factors may be important in control of SMC differentiation and phenotypic modulation. |
