Anatomy and Neuroscience - Theses
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ItemPsi regulates transcription of the MYC oncogene in Drosophila melanogaster( 2014)The MYC oncogene drives cell growth and consequently, increased MYC is associated with most human cancers. Thus tight regulation of MYC is critical and this thesis investigates one mechanism for controlling MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to the environment in order to activate MYC transcription and cell growth. Based on in vitro studies we predict that the presence of paused, but transcriptionally active, RNA polymerase II in the MYC promoter will be required for a rapid response to the cellular environment. Of particular interest to this project, human tissue culture studies from David Levens group (NIH, Bethesda) have shown that in response to serum the single stranded DNA binding proteins FUBP and FIR are recruited to the promoter of the MYC oncogene, and are associated with release of paused RNA Polymerase II. Moreover, the FIR protein is required for repression of MYC transcription, which occurs via interaction with the general transcription factor TFIIH complex. This mechanism for MYC repression appears to be conserved between mammals and Drosophila, as the Quinn lab has demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC in a manner dependent on interaction with the TFIIH complex. However, dissecting the role of the FUBP protein (FUBP1) in MYC regulation has been complicated by the fact that mammals have multiple FUBP family members (FUBP 1,2 and 3), which bind highly overlapping transcriptional targets. Originally, based on the finding that FUBP1 binds the MYC promoter prior to FIR, we predicted FUBP1 might play a role in activating RNA Polymerase II release and MYC transcript elongation. This thesis has used the Drosophila melanogaster model system to answer the question of whether the sole FUBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that Psi knockdown significantly reduced dMYC mRNA levels, which suggests Psi may be normally required for maintaining dMYC transcription in vivo. In line with a direct role in regulating MYC transcription, Chromatin Immunoprecipitation (ChIP) revealed enrichment for Psi on the dMYC promoter. Moreover, ChIP for poised RNA Pol II (Serine 5) proximal to the dMYC transcription start site, revealed that Psi knockdown results in a reduced RNA Polymerase II Serine 5 enrichment, consistent with the reduction in dMYC mRNA in the Psi knockdown. Together the experiments detailed in this thesis provide the first evidence that MYC is regulated by RNA Polymerase II pausing in vivo. Moreover, we demonstrate that Psi, the ortholog of mammalian FUBP1, is normally required for accumulation of the activated (Ser 5 phosphorylated) RNA Polymerase II on the dMYC promoter and, thus, for the regulation dMYC transcription.