A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 73-96). Also available in electronic version.

Thesis (Ph.D.) - University of St Andrews, March 2008.

Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 69-81). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11868

The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators Yap, Taz, Vgll family which bind Tead1-4 transcription factors. Recently the Hippo pathway has been shown to regulate muscle cell fate. Yap enhances the proliferation of myoblasts and inhibits their differentiation, Taz comparatively enhances both aspects. The mechanisms in which they regulate muscle cell fate and muscle development are poorly defined. In this thesis I determined the endogenous gene expression of Hippo transcription factors during myogenesis. Secondly their proteomic binding partners in myoblasts and myotubes. Thirdly, the gene sets targeted by YAP1 S127A, TAZ S89A and Vgll3 in myoblasts. Finally, I cloned chicken Yap1 to identify its role during embryonic muscle development, followed by retroviral YAP1 S127A overexpression during chicken embryonic limb development. The results demonstrated that the Hippo transcriptional regulators are mainly up-regulated during muscle regeneration in vivo. YAP1 and TAZ were mainly found to regulate the same gene sets and have the same binding partners. TAZ additionally had unique binding partners and gene sets that may promote muscle differentiation. Furthermore, Vgll3 also had many overlapping genes with YAP and TAZ, suggesting it is part of the Hippo pathway, but negatively regulates Hippo pathway gene expression. Yap expression during chicken embryonic development was observed in muscle related regions including the somites and limb buds. However, retroviral overexpression of YAP S127A did not lead to an overt phenotype but still up-regulated transcription musculoskeletal related genes. Collectively my data provides insight into how the Hippo transcriptional regulators control myogenesis, their binding partners and their transcription targets. Additionally, have characterised the expression of chicken Yap during embryonic muscle development.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

Cachexia or muscle atrophy is a condition that is associated with a variety of diseases such as chronic heart failure and cancer. In North America, Europe and Japan, more than 8 million patients suffer from cachexia, and it is estimated that cachexia is the cause of death in 30% of cancer patients. Unfortunately, there is no available treatment for cachexia. Bexarotene, a retinoid X receptor (RXR) agonist, is a FDA approved drug used to treat cancer and is able to induce myogenic differentiation in embryonic stem cells. In this study, we investigated the mechanism by which bexarotene enhances myogenic differentiation. The Akt signaling pathway is required for myogenesis and thus we examined its involvement in bexarotene-enhanced myogenic differentiation. We showed that bexarotene, through the activation of RXR signaling, regulates Akt2 expression to enhance myoblast differentiation and fusion. Additionally, we showed that Akt2, but neither Akt1 nor Akt3, is required for bexarotene-enhanced differentiation. Furthermore, we showed that the activation of RXR signaling by bexarotene correlates with a specific histone acetylation mark at the Akt2 locus. More importantly, we demonstrated that bexarotene is able to rescue myoblast differentiation in an in vitro cachexia system. Taken together, our data revealed the significance of Akt2 in bexarotene-enhanced myogenic differentiation and the potential of using bexarotene as a treatment for cachexia.

The MEF2 family of transcription factors (MEF2A, MEF2C, and MEF2D) are crucial during skeletal muscle differentiation. Although the roles of MEF2D isoforms are well established, the roles of MEF2A and MEF2C are not as well understood. This thesis, we investigated the expression, localization, and function of MEF2A and MEF2C, using specific antibodies. While MEF2A is expressed in both proliferating and differentiated myoblasts, protein levels of MEF2C were only detected during differentiation. During early stages of differentiation MEF2A is expressed in both the cytoplasm and the nucleus. However during later stages of differentiation, it is localized predominately in the nucleus. MEF2C appears to be localized differently depending on which isoform is being investigated. Using an affinity purification and mass spectrometry based approach we identified PRMT1 as a unique interacting protein with MEF2A during skeletal muscle differentiation. PRMT1 is a protein arginine methyltransferase which mediates the addition of methyl groups onto various proteins including histone H4 arginine 3 (H3R4) which is associated with gene activation. Both MEF2A and PRMT1 occupy genomic targets of MEF2A. Inhibition of PRMT1 with a specific inhibitor delays C2C12 myoblast differentiation in the early stages of differentiation but no effect was observed during late stage differentiation. The MEF2 family of transcription factors show distinct but overlapping function during skeletal muscle differentiation.

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