Regulation of Drosophila Myc transcription by the single stranded DNA/RNA binding proteins Psi and AGO1

© 2019 Dr. Linna GuoMYC has wide-ranging functions, with potential to amplify transcriptional output to activate cell growth, metabolism and cell cycle progression, thus driving oncogenic programs. As even small increases in MYC abundance are sufficient to drive the proliferative cell growth fundamental to tumour progression, understanding the molecular mechanisms controlling MYC expression will provide insight into mechanisms of MYC dysregulation in cancer. In mammalian in vitro and ex vivo systems, the KH domain single stranded DNA/RNA binding protein FUBP1 binds the Far Upstream Sequence Element (FUSE) in the activated MYC promoter to modulate transcription. Using genetic models, this thesis demonstrates that the sole FUBP family member in Drosophila, Psi, is essential for Myc transcription, cell and tissue growth in the wing epithelium, in vivo. Psi is not only required to maintain endogenous levels of Myc mRNA abundance, but depletion results in a significant increase in RNA Pol II activity on the Myc gene. Consistent with Psi regulating Myc at the level of transcription, we demonstrate Psi interacts physically and genetically with the transcriptional Mediator complex (MED), thus providing a mechanism for integration of developmental signals for patterning Myc transcription, cell and tissue growth in the wing imaginal disc epithelium. We further report physical and genetic interaction between Psi and AGO1, the RNA binding protein component of the RNA-induced silencing complex (RISC). AGO1 loss-of-function mutations restored growth in the Psi knockdown wing, suggesting negative roles for AGO1 in Psi-dependent cell and tissue growth. AGO1 depletion was not only sufficient to increase Myc mRNA and protein abundance in the wing, but also increased Myc function i.e. transcriptional activity as measured by increased abundance of Myc targets required for ribosome biogenesis and cell growth (e.g. Ribosomal Proteins, rRNA and RNA Pol I subunits). Myc knockdown returned AGO1-depleted nucleolar compartments to the normal range, demonstrating dependency of increased growth on Myc i.e. rather than direct effects of AGO1 on ribosomal components. Interestingly, the following observations suggest AGO1 represses Myc at the level of transcription: 1) significant AGO1 enrichment on Myc promoter by ChIP, 2) physical interaction between AGO1 and MED, and 3) that AGO1 depletion activates the Myc promoter and the increased Myc mRNA requires RNA Pol II transcriptional activity. Together, these observations suggest a novel role for AGO1 as a transcriptional repressor of Myc, which underlies the tumour suppressor behaviour observed for AGO1 in the Drosophila wing