Figure 1. c-Myc overexpression leads to increased binding at promoters of active genes.

A) Schematic of the inducible c-Myc expression system in P493-6 Burkitt's lymphoma cells. B) Western blot of relative levels of c-Myc protein (top) and actin (bottom) at 0hr, 1hr, and 24hr after induction. C) Left: Quantitative Western blot of c-Myc during induction. Increasing amounts of purified, recombinant his₆-c-Myc (left lanes) and lysates prepared from increasing numbers of cells (right lanes) shown at 0hr (top), 1hr (middle) and 24hr (bottom). Right: Coomasie stained gel of purified recombinant his₆-c-Myc. D) Images of three representative cells taken during c-Myc induction. From left to right, cells at 0hr, 1hr, and 24hr are shown. From top to bottom are images of bright field, CD9 stained, DAPI stained, c-Myc stained, and a merge of c-Myc and Dapi stained cells. E) Median c-Myc levels in the +/− 1kb region around transcription start sites (TSSs) of promoters ranked by increasing RNA Pol II occupancy at 0hr. Levels are in units of reads per million mapped reads per base pair (rpm/bp) for 0hr, 1hr, and 24hr (blue, black, red). Promoters were binned (50/bin) and a smoothing function was applied to median levels. F) Gene tracks of c-Myc binding at the NPM1 gene at 0hr (top), 1hr (middle), and 24hr (bottom). The x-axis shows genomic position. The y-axis shows signal of c-Myc binding in units of rpm/bp. G) Heatmap of c-Myc levels at TSS regions at 0hr, 1hr, and 24hr. Each row shows the +/−5kb centered on the TSS. TSSs for all actively transcribed genes in P493-6 cells are shown and ranked by c-Myc occupancy at 0hr. Color scaled intensities are in units of rpm/bp. H) Boxplots of c-Myc levels at 0hr, 1hr, and 24hr in the +/− 1kb centered on the TSS of all genes actively transcribed at the start of c-Myc induction. Units are in rpm/bp. Changes between mean c-Myc levels at promoters are significant (Welch's two-tailed t test) between 0hr and 1hr (p-value < 1e-16) and 1hr and 24hr (p-value < 1e-16). I) Venn diagram showing the overlap between sets of transcribed genes during c-Myc induction at 0hr, 1hr, and 24hr. Transcribed genes were defined as having an enriched region for either H3K4Me3 or RNA Pol II within 5kb of the annotated transcription start site (TSS). Each circle represents the set of transcribed genes at 0hr (blue), 1hr (black) or 24hr (red). See also Figure S1.