Skeletal muscle wasting occurs as a corollary of numerous physiological conditions, including denervation, immobilization, and aging. The E3 ubiquitin ligases MuRF1 and MAFbx are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation. Data in this thesis provides interesting new evidence that MuRF1 may also act as a transcriptional modulator of atrophy-induced genes or atrogenes. The transcriptional regulation of MuRF1 and MAFbx were characterized using a reporter gene system and exhibited repressed activity in C2C12 cells overexpressing MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRFs), MyoD and myogenin, caused significant activation of the MuRF1 and MAFbx reporter gene constructs, while co-overexpression of MuRF1 with MRFs resulted in reversal of MRF induction of reporter gene activity. Interestingly, ectopic expression of a catalytically dead MuRF1 RING mutant failed to reverse MRF activation of the reporters, suggesting that ubiquitin ligase activity may be necessary for MuRF1 transcriptional regulation. To further investigate a potential mechanism of MuRF1 regulation of MRF activity, Western blot analysis was performed to analyze MRF protein levels in C2C12 cells overexpressing MuRF1 and MuRF1 RING mutant. Cells with ectopic MuRF1 or MuRF1 RING mutant showed repressed levels of myogenin. Additionally, cells overexpressing MuRF1 and MuRF1 RING mutant treated with MG132 showed only a partial rescue of myogenin protein levels. Finally, chromatin immunoprecipitation was performed to analyze occupancy of MRFs at the MuRF1 promoter. Overexpression of MRFs resulted in increased MuRF1 promoter immunoprecipitation (IP) and amplification, while co-overexpression of MuRF1 with MRFs resulted in a reversal of promoter IP and amplification. These findings suggest that MuRF1 may regulate MRF transcriptional activity in a non-canonical fashion giving insight into a potentially new mechanism by which MuRF1 may act to transcriptionally regulate atrophy-induced gene expression.
| Identifer | oai:union.ndltd.org:unf.edu/oai:digitalcommons.unf.edu:etd-1785 |
| Date | 01 January 2017 |
| Creators | Kakareka, Karina Marie |
| Publisher | UNF Digital Commons |
| Source Sets | University of North Florida |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | UNF Theses and Dissertations |
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