Single Molecule Analysis of Transcription in Live Cells Reveals the Gene Regulatory Function of MYC In Vivo

Abstract

We seek to determine how the MYC oncogene modulates gene transcription kinetics with single-molecule imaging techniques.Thirty-five years after the discovery of MYC and despite intensive research effort, there is still little understanding of how this transcription factor acts or why it is an oncogene. Several models have recently emerged that cast doubt on the prevailing view of MYC as a gene-specific transcription factor and instead envision its oncogenic mechanism as a global amplifier or invader of enhancer regions. We seek to evaluate these hypotheses of MYC function and shed light on the in-vivo behavior of this transcription factor by determining how MYC modulates the kinetics of transcription events in single cells using single-molecule RNA fluorescence in-situ hybridization (RNA FISH) and live cell imaging of transcription.Our initial results testing the effect of MYC overexpression on an exogenous reporter gene in U2-OS cells shows that MYC increases the saturation level of the reporter dose response, indicating that MYC is able to act as a general amplifier without the need of enhancer regions. Unexpectedly however, the positive correlation between MYC and reporter RNA levels in the population is not reflected at the single cell level. This data suggests that static measurements of RNA abundance in single cells - such FISH or RNA-Seq - needs to be interpreted within a framework that explicitly takes into account the dynamic nature of transcription events. Further, as a way to directly assess the effect of the MYC protein on the transcription behavior of reporter genes, we engineered a photo-activatable version of MYC (PA-MYC) to achieve spatial and temporal control of the transcription factor within living cells. This shall allow us to follow the direct response of the transcription in response to MYC perturbation.