Figure 3. c-Myc overexpression leads to transcriptional amplification.

A) Boxplots of the fold changes in levels of H3K4Me3 (green) and Cdk9 (pink) between 24hr and 0hr at the top 2,000 core promoters with the highest increased c-My occupancy during c-Myc induction (left), the top 2,000 enhancers with the highest increased c-Myc occupancy during c-Myc induction (center), and 2,000 enhancers without increased c- Myc occupancy during c-Myc induction (right). The differences in the levels of changes between H3K4Me3 and Cdk9 were significant (Welch's two-tailed t test) at core promoters (p-value < 2.2e-16) and at enhancers with increased c-Myc occupancy (p-value = 6.48e-07) and not significant at enhancers without increased c-Myc occupancy (p-value = 0.06). B) Western blots of RNA Pol II at 0hr, 1hr, and 24hr using antibodies specific to various forms of the enzyme. From top to bottom: RNA Pol II Ser2P specific (H5, Covance), RNA Pol II Ser2P specific (A300-654A, Bethyl), RNA Pol II Ser5P specific (H14, Covance), hypophosphorylated RNA Pol II specific (8WG16, Bethyl), total RNA Pol II (N-20, Santa Cruz). For RNA Pol II Ser2P and Ser5P antibodies, the ratio of signals vs. 0hr is displayed for each timepoint below the blot. C) Bar graph of mean +/− SEM RNA Pol II enrichment ratio between elongating and initiating regions during c-Myc induction at 0hr, 1hr, and 24hr (left, center, right) for the top 5,000 genes ranked by RNA Pol II occupancy at 0hr. The y-axis shows the ratio between the fold enrichment overbackground of RNA Pol II in the elongating region versus the fold enrichment over background of RNA Pol II in the promoter region. Changes between 0hr, 1hr, and 24hr are significant (Welch's two-tailed t test, p-value < 1e-16). D) Empirical cumulative distribution plots of RNA Pol II traveling ratios (TR) for 1,000 transcribed genes (Rahl et al., 2010). Genes were randomly selected from the pool of genes containing higher than background levels of RNA Pol II at the promoter and gene body at 0hr, 1hr, and 24hrs. Differences in the TR distribution at 0hr and 24hr are significant (Welch's two-tailed t test, p-value = 4.5e-5). E) Left: Bar graph showing quantification of total RNA levels for cells at 0hr, 1hr, and 24hr. Units are in ng of total RNA per 1,000 cells and represented as mean +/− SEM. Right: 5% TBE urea gel of ethidium bromide stained total RNA extracted from equivalent numbers of cells at 0hr, 1hr, and 24hr. Bands corresponding to the 5.8S rRNA subunit, 5S rRNA subunit, and tRNA are labeled. F) Boxplot of transcripts/cell estimations from NanoString nCounter gene expression assays for active (right) or silent (left) genes at 0hr, 1hr, and 24hr. 755 active genes (expressed > 1 transcripts/cell) at 0hr are shown (left, red). 514 silent genes (expressed < 0.5 transcripts/cell) at 0hr are shown (right, black). The number of genes with increased expression between 0hr and 1hr are significant (Wilcoxon rank sum test) for active genes (p-value < 2.2e-16) and non significant for silent genes (p-value = 0.997). See also Figure S3