Anatomy and Neuroscience - Theses
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ItemFine-tuning expression of dMYC: how the single stranded DNA binding proteins Hfp and Psi regulate dMYC expression in Drosophila( 2015)MYC proteins are critical regulators of growth and cell cycle progression essential to animal development (Lee and Quinn, 2014; Lee et al., 2012; Levens, 2010). MYC is overexpressed in approximately 70% of all cancers (Dang, 2010; 2014). Understanding the molecular mechanisms that control MYC activity is crucial to understand normal development and may lead to insights into MYC dysregulation in diseases such as cancer. This thesis investigates the regulation of MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to environmental and cellular signaling pathways to coordinately regulate MYC transcription and cell growth. Based on in vitro studies we predicted that the presence of paused, but transcriptionally active, RNA polymerase II (RNA Pol II) on the MYC promoter is required for a rapid response to cell signaling. Of particular interest to this project, David Levens' ex vivo cell culture studies have shown that in response to mitogenic signals in serum the single stranded DNA binding protein Far upstream element Binding Protein (FBP) is enriched on the promoter of the MYC oncogene. FBP binding is associated with depletion of RNA Pol II from the promoter and increased MYC transcription (Liu et al., 2006). Subsequent to the peak in MYC mRNA, FBP Interacting Repressor (FIR) is detected at the FUSE and is required for the return of MYC transcription to basal levels. Moreover, the FIR protein is required for repression of MYC transcription via interaction with the general transcription factor (TFIIH) complex (Liu et al., 2006; 2001), and is dysregulated in human colorectal cancer (Matsushita et al., 2006). This mechanism of MYC repression appears to be conserved between mammals and Drosophila, as we have demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC (dMYC) in a manner dependent on the XPB helicase subunit of the TFIIH complex (Mitchell et al., 2010). Xeroderma Pigmentosum (XP) is a rare autosomal-recessive disease characterized by pigment changes, premature ageing and in a few patients malignant tumour development (Cleaver, 2005; Oh et al., 2006). Moreover, cancer progression occurs in certain patients, but not others, with identical C terminal mutations in the XPB helicase (Oh et al., 2006). The issues of compound heterozygosity and mixed genetic backgrounds have compounded problems of resolving the disease mechanism in the small number of XPB patients. Thus mechanisms driving overproliferation and cancer associated with these XPB mutations are currently unknown (Cleaver, 2005). With Drosophila we have used single alleles and, for the first time, made clear connections between genotype and phenotype. Using Drosophila models we show that C-terminally truncated XPB/Hay alleles enhance tissue overgrowth due to reduced abundance of FIR/Hfp, to further impair transcriptionally repression of dMYC. Thus, we predict defective transcriptional repression of MYC by FIR/Hfp might provide one mechanism for spontaneous cancer progression in XP patients. Dissecting the role of the FBP protein in MYC regulation has been complicated by the fact that mammals have multiple FBP family members (FBP 1, 2 and 3), which bind highly overlapping transcriptional targets (Chung et al., 2006). Originally, based on the finding that FBP is required for activated MYC transcription in vitro (He et al., 2000; Liu et al., 2001), we predicted FBP might play a role in activating RNA pol II release and MYC transcript elongation. We used Drosophila models to test whether the sole FBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that depletion of FBP/Psi reduced dMYC mRNA levels, suggesting FBP/Psi is normally required for maintaining endogenous levels of dMYC. Moreover, ChIP for poised RNA Pol II (Ser 5) proximal to the dMYC transcription start site revealed that FBP/Psi depletion reduced RNA Pol II Ser 5 enrichment. Together these data suggest FBP/Psi is required for maintaining poised RNA Pol II on the dMYC promoter and for maintaining dMYC transcription in vivo. Based on David Levens' extensive in vitro studies, we predict that FBP/Psi and FIR/Hfp are required for “fine-tuning” signal stimulated MYC transcription. We conducted a candidate screen of known mitogenic signals to identify those capable of most strongly stimulating dMYC transcription in vivo. The Ras/ERK pathway was the most robust activator of the dMYC promoter, and was able to significantly increase dMYC mRNA abundance. Importantly, we demonstrated that Ras-stimulated dMYC transcription was significantly reduced by depletion of FBP/Psi. Thus, endogenous levels of FBP/Psi are required for maximal stimulation of dMYC transcription by mitogenic signals in vivo.