Fig. 4

High RNAP2 TSSR density triggers increased RNAP2 gene body density. a, b Scheme to visualize a gene’s RNAP2 density by its TSSR and gene body density and a third variable, such as gene expression. PI is shown by the gene’s position along indicated diagonals. a Schematic plot of one gene, colored by its expression level. b All genes in GM12878. A smoothed spline mean trend line between RNAP2 TSSR and gene body density showed a biphasic slope, with an inflection point (pink arrow) and higher slope at high RNAP2 TSSR density. The steeper portion of the trend line suggested that high TSSR RNAP2 density enhanced RNAP2 gene body density. c Mean trend lines for five different cell types. A biphasic trend line with an inflection point at high RNAP2 TSSR density was a common feature of all cell lines. d Highly expressed genes (FPKM >1000) were over-represented among genes on the steep portion of the mean trend line, compared to all genes. e Functional annotations associated with genes on the steep portion of the mean trend line were consistently enriched for multiple GO biological process terms across several cell types. f Promoter density of CCNT2, a pause-release protein, correlated with TSSR RNAP2 density in K562 cells. g Inhibition of pause-release abrogated enhanced RNAP2 gene body density at high RNAP2 TSSR density. Treatment with pause-release inhibitor FP flattened the steeper portion of the mean trend line observed in untreated cells in both IMR90 and mES cells. h Modulation of pause-release regulators NELFA and BRD4 did not abrogate the inflection point. NELFA was knocked down with siRNA and BRD4 was antagonized with the small molecule JQ1. Neither treatment abolished the inflection point or the steeper portion of the mean trend line