Characterisation of the Myc negative autoregulation mechanism

grantor: University of TorontoThe c-myc proto-oncogene encodes a highly conserved nuclear phosphoprotein central to the regulation of cellular proliferation, differentiation and apoptosis; however, the molecular mechanisms of c-Myc protein activity are not well understood. Structurally, c-Myc resembles a transcription factor and when in a complex with the Max protein, Myc binds to DNA E-box elements and activates transcription. Recent evidence also supports an important biological role for Myc in the downregulation of specific gene transcription. We have previously reported one physiological example of Myc transcriptional repression, Myc negative autoregulation, whereby Myc protein, in combination with additional cellular trans-acting factors, suppresses transcription initiation from the c-myc promoter. In this thesis, I address how dysfunction within this homeostatic feedback mechanism may contribute toward c-Myc oncogenic activation and tumourigenesis, and characterise essential cis and trans components of the Myc negative autoregulatory pathway. I have demonstrated that within the tumourigenic L929 cell line, c-myc activation involves dysfunction of Myc negative autoregulation as well as other regulatory mechanisms. Furthermore, this activation accompanies abrogation of the Myc-induced apoptosis pathway, illustrating two cooperative events which can contribute to tumour progression. Somatic cell hybridisation of L929 cells to Rat-1 fibroblasts competent for Myc autosuppression and apoptosis results in partial complementation of both pathways, indicating that dysfunction of these events likely results from multiple genetic lesions. To identify cis and trans components of the c-myc promoter which respond to Myc negative autoregulation, I conducted detailed analysis of the human c-myc promoter in Rat-1 fibroblasts. I have described a 142 bp fragment of the c-myc promoter which is suppressed fourfold relative to basal levels in the presence of ectopic Myc expression. I have further characterised the essential cis components by constructing mutations in known transcription control elements within this minimal promoter fragment. In addition, by stably expressing MycEG and MaxEG protein dimerisation mutants in Rat-1 cells, I have shown that Myc-Max heterodimerisation is obligatory for Myc autosuppression. From these results I suggest two models to describe the molecular events participating in the Myc negative autoregulation pathway.Ph.D