Date of Award

5-1-2019

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Davie, Judith

Abstract

Myogenesis, the formation of muscular tissues, requires the precise expression of the Myogenic Regulatory Factors (MRFs): MYF5, MYOD, MYOG and MRF4, whose expression regulates transcription of muscle specific genes. TBX3 is a member of the T-box family of transcription factors that is expressed throughout myogenesis and is up regulated. We found that depletion of TBX3 in normal myoblast cells (C2C12) reduces migration and proliferation suggesting that TBX3 is involved in promoting cell cycle progression in. Loss of TBX3 also impairs differentiation of skeletal muscle and causes a downregulation of the MRFs resulting in the repression of the muscle-specific genes including Tnni2, Acta1, Lmod, and Mylpf. Deletion of TBX3 inhibits myofibers formation which is characterized by the loss of the expression of the differentiation marker Myosin Heavy Chain (MyHC) upon differentiation. We also showed that TBX3 regulation of myogenesis is not through the direct interaction with the MRFs MYOD and MYOG. Moreover, TBX3 acts as a transcriptional repressor in normal muscle myoblasts where it inhibits the myogenic activity of MYOD and MYOG. We found that TBX3 regulates myogenesis in part through the modulation of Wnt/β-catenin signaling pathway. Loss of TBX3 causes the recruitment of the class II histone deacetylase HDAC5 to Myod1 promoter preventing β-catenin from binding to and activating Myod1. TBX3 depleted cells also show a recruitment of the class I histone deacetylase HDAC1 to the promoter of Cyclin D1 (CCND1) inhibiting β-catenin from forming a complex with TCF/LEF and activating target genes. Activation of the Wnt pathway in TBX3 depleted cells causes a partial restoration of differentiation. We also found that loss of TBX3 causes an increase in the expression of β-catenin. Interestingly, activation of the Wnt pathway which leads to the stabilization and translocation of β-catenin also activates Axin2 in TBX3 depleted cells. Axin2 is a negative regulator of the Wnt pathway that participates in a negative feedback loop to downregulate β-catenin to normal level. Another important finding our studies revealed is that TBX3 not only permits muscle differentiation through the modulation of the Wnt/β-catenin pathway, but also through the regulation of TBX2 expression which is an inhibitor of myogenesis. TBX2 is upregulated in C2C12 cells lacking TBX3. We showed that in TBX3 depleted cells, TBX2 was recruited to muscle-specific genes promoters along with an enrichment of HDAC1. Importantly, interference with TBX2 in TBX3 depleted cells generates a partial restoration of differentiation. However, not only a complete rescue of myogenesis was achieved but also an enhancement of differentiation was obtained when we interfered with TBX2 and activated the Wnt/β-catenin pathway.

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