Figure 1. Broad Pol II pausing upon BRD4 depletion but not bromodomain inhibition.

A. Schematic of the updated BRD4-IAA7 degron line. The F-box protein AtAFB2 was integrated into the C terminus of the BRD4 locus under the control of an independent promoter. Western blot showing the acute depletion of BRD4 by auxin (500 uM) or dBET6 (250 nM) treatment but not JQ1 (1 uM) in the BRD4-IAA7 degron line. Cells were treated with auxin for 2 or 3h, or with JQ1 or dBET6 for 3h. These treatment concentrations/durations are relevant to all of Figure 1.

B. Genome browser track examples of total Pol II ChIP-seq signal at the representative genes BRD2, PRSS22 and HSPA8 upon auxin treatment (0, 3 or 5h), JQ1 or dBET6 treatment (3h).

C. Heatmap showing the genome-wide Pol II occupancy profile upon auxin treatment and the profile of fold change in Pol II occupancy compared to control, in which the differential between promoter (high occupancy, red) and gene body (low occupancy, blue) indicates paused Pol II. The gene list (N=6481) in this analysis is used throughout the study (except for Figures 3H–J).

D. Estimated Cumulative Density Function (ECDF) of pause release ratios (PRR, reported as log2 fold change) for auxin treatment vs untreated control. The PRR is the ratio of Pol II occupancy within gene bodies to its occupancy at promoters. A leftward shift of the curve indicates an increase in the frequency and/or duration of promoter-proximal pausing, while a rightward shift indicates reduced pausing and/or more efficient release into gene bodies.

E. Heatmap showing the genome-wide Pol II occupancy profile upon JQ1 or dBET6 treatment and the profile of fold change in occupancy vs control.

F. ECDF of the log2PRR upon JQ1 or dBET6 treatment.

G. Boxplot of the log2PRR for auxin, JQ1 and dBET6 treatment.

H. PCA analysis of the log2PRR for auxin, JQ1 and dBET6 treatment.