A Living, Single Cell View of Myc's Effects on Transcription

Abstract

How does c-MYC act on transcription events in single living cells to cause a global amplification of gene expression? To answer this question, we employ two recently-developed imaging techniques: 1) single-molecule FISH to quantify RNA in fixed cells, and 2) live cell imaging with the MS2-PP7 stem loop system to observe real-time RNA production at a given gene locus. Our results from both the exogenous reporter and endogenous gene show that MYC increases the duration of transcription events— this results in greater gene expression, but also an increase in its heterogeneity. These findings provide living, single cell evidence of MYC as a global amplifier of gene expression, and suggests the mechanism is by stabilizing the active period of a gene. We speculate that the added consequence of MYC's propensity to increase gene expression heterogeneity could drive cells to transiently populate pathological states and phenotypes that may ultimately make them susceptible to cancer. In addition, we seek to assess the direct effects of MYC on gene transcription. We engineered a photo-inducible version of MYC (Pi-MYC) to achieve spatial and temporal control of MYC within living cells. We demonstrate that Pi-MYC has oncogenic capability similar to wildtype MYC by coordinating with mutant RAS to transform mouse fibroblasts and inducing them to exhibit anchorage-independent growth. This tool shall allow us to follow the direct activity of genes in response to MYC perturbation.