Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
AN ABSTRACT OF THE DISSERTATION OF Priya Londhe, for the Doctor of Philosophy degree in Biochemistry and Molecular Biology, presented on 30th July, 2013 at Southern Illinois University Carbondale. TITLE: UNRAVELING THE MOLECULAR MECHANISMS OF THE CLASS II TRANSACTIVATOR IN SKELETAL MUSCLE MAJOR PROFESSOR: Dr. Judy Davie The inflammatory cytokine, interferon gamma, IFN-gamma orchestrates a diverse array of fundamental physiological processes and exhibits complex effects on myogenesis. IFN-gamma also induces the class II transactivator, CIITA, which is a critical mediator of IFN-gamma mediated repression and activation. The aims in my dissertation are directed towards understanding the role of IFN-gamma and CIITA in muscle. Stimulation by IFN-gamma in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. We show that the IFN-gamma induced inhibition of myogenesis is mediated by CIITA, which specifically interacts with myogenin. CIITA acts by both, repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. The IFN-gamma mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-gamma. We also show that CIITA is indispensible for the inhibition of myogenesis. To gain a mechanistic insight into the IFN-gamma induced repression of myogenesis, we have discovered that IFN-gamma and CIITA inhibit myogenesis by modifying gene regulation in a muscle cell subject to inflammation. We show that CIITA first interacts with JARID2, a non catalytic subunit of PRC2 complex, which induces a paused RNAPII, phosphorylated at serine 5 and then interacts with the catalytic subunit EZH2, in a JARID2 dependent manner. Our data show that both CIITA and IFN-gamma block myogenesis by the induction and recruitment of the PRC2 complex, which is normally silenced in a differentiating muscle cell. One of my dissertation aims sheds light on the silencing of CIITA in Rhabdomyosarcoma. Silencing of CIITA prevents the expression of MHC Class I and II genes. We have found that IFN-gamma signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells, but shows a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have also observed that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-gamma treatment, is blocked in both types of RMS cells. Further, our studies also impart a novel role for IFN-gamma and CIITA in inhibiting the IGF induced activation of muscle specific genes. Our data show that IFN-gamma does not block the signaling cascade of IGF. However, blocking exogenous IFN-gamma restores IGF activation of muscle specific genes. My dissertation also reveals an important role for the FACT complex in the early steps of gene activation through its histone chaperone activities that serve to open chromatin structure and facilitate transcription promoting muscle differentiation. We show that myogenin interacts with the FACT complex and the recruitment of FACT complex to muscle specific genes is dependent on myogenin. The final aim in my dissertation highlights the distinct binding profiles of the MRFs and E proteins during proliferation and differentiation. Our sequential ChIP assays show that MYOD, MYOG, and MYF5 co-occupy promoters. Taken together, my dissertation provides a comprehensive understanding of the molecular mechanisms during myogenesis and reveals the deleterious effects of chronic inflammation in skeletal muscle.
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