The Roles of Forkhead Transcription Factors in Stem Cell and Myogensis




Alexander, Matthew Scott

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Vertebrate myogenesis is a highly conserved process that involves the formation, activation, proliferation, and overall regulation of myogenic progenitor cells (MPCs) that are essential for muscle formation, growth, and regeneration following injury. While the process of skeletal muscle development and regeneration has been well-described on a physiological level, the molecular mechanisms that govern the regulation of these cells are poorly understood. Through the utilization of murine transgenic models and gene disruption strategies, I have been able to elucidate important pathways involved in the regulation of MPCs during embryonic myogenesis and adult regeneration following injury. The experiments performed in satisfaction of my dissertation were aimed at defining the biological regulation of Foxk1 and characterization of a novel forkhead factor, Foxj3. Previous studies from our lab have identified the forkhead/winged helix transcription factor, Foxk1, as an essential regulator of MPC activation and quiescence. Firstly, I have undertaken a series of molecular, biochemical, and genetic studies to define the upstream regulation of the Foxk1 gene promoter. Based on evolutionary conservation of the 5' Foxk1 upstream promoter among mouse, rat, and human, I identified a conserved Sox Binding Element (SBE) that I hypothesized as being essential for the transcriptional regulation of Foxk1. I undertook a candidate-based approach, from which I identified Sox15 as being a potent transcriptional activator of Foxk1. Through cell culture, transcriptional assays, electrophorectic mobility shift assays, and transgenic founder analyses, I confirmed that this SBE is essential for the activation of Foxk1 transcription by Sox15. I demonstrated that Sox15 is essential for normal myoblast cell cycle kinetics through siRNA knockdown of endogenous Sox15 and the characterization of the Sox15 mutant mouse model. Finally, I have characterized the novel forkhead transcription factor Foxj3. I have generated a Foxj3 mutant mouse model. I have observed that Foxj3 mutant mice are growth retarded, have impaired skeletal muscle regeneration following injury, and have a significant decrease in the total percentage of Type I oxidative myofibers. When Foxj3 expression is decreased significantly, myoblasts have perturbed cell cycle kinetics and proliferate at a faster rate. Additionally, I demonstrated that Foxj3 is a direct upstream transactivator of Mef2c in skeletal muscle and an essential upstream regulator of the Mef2c-signaling pathway. In conclusion, these studies have elucidated the functional regulation of Foxk1 and its binding partner, Sin3a, in the MPC population. Additionally, I have identified a novel regulator of skeletal muscle myogenesis and myofiber identity, Foxj3, through cell culture assays and generation of the Foxj3 mutant mouse model.

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