Author: Julie-Ann Hulin
Hulin, Julie-Ann, 2016 The role of homeobox factors Barx2 and Pax7 in Wnt signalling in muscle stem cells, Flinders University, School of Medicine
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Satellite cells are the resident stem cells of skeletal muscle and provide the mechanism by which muscle tissue is capable of repair and renewal. Satellite cells are quiescent in adults, until activated by injury to generate proliferating myoblasts. In a process known as myogenesis, the proliferating myoblasts align, fuse and undergo terminal differentiation, resulting in formation of multi-nucleated muscle fibres. The muscle regulatory factors (MRFs) are key regulators of myogenesis, and hierarchical expression of the four family members (Myf5, MyoD, myogenin and MRF4) guides progression through the stages of muscle regeneration. The homeobox factor Pax7 also plays an essential role in myogenesis and is considered the canonical marker of satellite cells. Pax7 is important for maintenance of satellite cell quiescence and prevention of precocious differentiation. Recently another homeobox protein, Barx2, has emerged as being functionally important in myoblasts. Barx2 regulates expression of muscle-specific genes, and Barx2 null mice exhibit smaller muscle fibres, muscle atrophy and defective muscle repair. In contrast to Pax7, Barx2 is upregulated during and promotes differentiation of myoblasts. The canonical Wnt signalling pathway controls myogenesis, inducing a switch from myoblast proliferation to differentiation. The central effectors of the Wnt pathway are TCF/LEF proteins and β-catenin, that together bind Wnt-responsive genes and activate transcription. Our preliminary data demonstrates that Barx2 can activate a synthetic Wnt reporter gene, TOPflash, in the C2C12 myoblast cell line. This thesis sought to understand the mechanisms underpinning this regulation and to determine if Barx2 can regulate endogenous Wnt-responsive target genes in myoblasts. Co-immunoprecipitations demonstrated that Barx2 could interact with β-catenin and TCF/LEF proteins and chromatin immunoprecipitation (ChIP) experiments showed recruitment of Barx2 to TCF/LEF binding sites in the TOPflash promoter. Barx2 expression also increased levels of nuclear β-catenin and promoted its recruitment to endogenous TCF/LEF sites. In contrast, Pax7 appeared to be antagonistic to Barx2, repressing TOPflash activity. Generation of a tetracycline-inducible Barx2 myoblast cell line (gain-of-function model), coupled with RNA-Seq and PCR array analysis of primary myoblasts isolated from Barx2 null mice (loss-of-function model), identified Axin2 and cyclinD1 as Wnt-responsive targets of Barx2. Analysis of the Axin2 promoter revealed that Barx2 bound to TCF/LEF sites, recruited β-catenin and the co-activator GRIP-1, and induced activating histone H3K-acetylation. In contrast, ectopic expression of Pax7 repressed Axin2 promoter activity, and knockdown of endogenous Pax7 allowed for greater activation of the promoter by β-catenin and Barx2. Furthermore, Pax7 interacted with the co-repressor HDAC1 and inhibited Barx2-mediated H3K-acetylation at Axin2 TCF/LEF sites. This work shows that Barx2, Pax7 and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts. These findings support a novel model in which Barx2 is a key transcriptional mediator of Wnt-driven myoblast differentiation, whilst Pax7 antagonises Wnt signalling in a role consistent with promotion of long-term satellite cell self-renewal. This provides a new regulatory pathway for regulation of muscle progenitor differentiation, and suggests that antagonism between Barx2 and Pax7 may help mediate the switch from proliferation to differentiation. Finally, canonical Wnt signalling induced Barx2 mRNA and stabilised Barx2 protein in myoblasts, suggesting a positive feed-forward loop between Barx2 and Wnt signalling. These studies prompt a biphasic model for Barx2 downstream of Wnt signals in myoblasts: Barx2 initially enhances the activation of Wnt target genes (Axin2 and possibly other negative regulators of Wnt signalling), and through this enhanced activation may help to limit the Wnt response, thus preventing the deleterious effects of excessive Wnt signalling.
Keywords: homeobox, muscle stem cells, wnt signalling, myogenesis, skeletal muscle, transcription factors, cell signalling, differentiation
Subject: Medicine thesis, Clinical Pharmacology thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Robyn Meech