Precise maps of RNA polymerase reveal how promoters direct initiation and pausing

Abstract

Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.

Fig. 1

Accumulation of Pol II at promoters, 3’ ends, 3’ splice sites, and nucleosomes. (A) Schematic of PRO-seq. (B) Average PRO-seq profile of non-overlapping genes (n=6,309) for the sense strand. Gene body regions (+1 kb from the 5’ end to −1 kb from the 3’ end) are scaled to 4 kb. Read counts are adjusted to per kilobase per million mapped (RPKM). Shaded margins surrounding the average plot show standard errors of the means. (C) High resolution PRO-seq profile from the TSS to +150 bp (n=16,746). (D) Heatmap visualization of PRO-seq profile of the annotated genes. Genes are arranged by their increasing PRO-seq density. (E) Average PRO-seq profile at 3’ splicing sites of less used and their flanking exons (n=242 each). Less used exons have RNA-seq densities less than 5% of their flanking exons (fig. S5B). (F) Average PRO-seq profile relative to the dyad centers of gene body and first nucleosomes. Region occupied by nucleosome is in grey shades.

Fig. 2

Variations of the pause sites and TSSs. (A) Examples of highly paused genes with different pausing patterns. Initiation sites from PRO-cap mapping are shown in grey. (B) Distribution of paused genes (n=3,225) by the pausing position and dispersion percentiles. Focused-proximal (Prox, n=848) and dispersed-distal (Dist, n=846) groups are indicated and axis units in base-pairs are also shown. (C) Heat map of initiation (PRO-cap) and pausing (PRO-seq) for Prox and Dist genes. (D) Association between initiation and pausing patterns. ‘TSS focusing’ in Prox vs Dist genes (left) and ‘Pausing proximity’ in focused vs dispersed initiation genes (right). The fraction of PRO-cap reads at (±1 bp) the TSS over the sum of reads around (±50 bp) the TSS represents the ‘TSS focusing’(22). Focused and dispersed initiation genes are the quartiles of the paused genes with the highest and the lowest TSS focusing respectively. Pausing Proximity Index is defined by the average of pausing position and dispersion percentiles (fig. S6A). Boxes represent 25^th, 50^th(median), and 75^th percentiles; whiskers are 5^th and 95^th percentiles. Asterisks (*) indicate p<0.001 by Kolmogorov-Smirnov(KS) test.

Fig. 3

Relationship between promoter DNA elements and Pol II pausing. (A) Frequency of TATA box and Pause Button (PB) in Prox and Dist subsets. The average frequency per gene is shown. (B) Frequency of GAGA element (lines) and GAF binding (shades) [27] in Prox and Dist subsets. (C) ‘Complex interaction model’ between DNA elements and paused Pol II. The DNA elements (blue) are at their consensus (Strong) or slightly upstream (Weak) positions and the expected changes of the pausing positions are plotted. (D) Pattern of the positional association between DNA elements and pausing positions. Pausing position percentiles are shown for gene subsets by the element positions (Cs: optimal consensus position, Up: upstream, Dn: downstream; subset information in table S2). Asterisks indicate p-values for the KS test(***: p<0.002, **: p<0.06, *: p<0.14). (E) Association of promoter DNA element strength at consensus positions with Pausing Index(6). Active genes (n=5,471) are divided into three subsets according to the distance-weighted p-values of the DNA elements to the consensus positions (table S3). Asterisks indicate p-values for the KS test (**:p<0.001,*:p<0.01)

Fig. 4

Disruption in the position of downstream DNA elements in Hsp70 promoter. (A) Structure of the Drosophila Hsp70 promoter. Mutant promoters have different length inserts at +15. (B) Initiation patterns at transgenic Hsp70 promoters. 5’ end counts of PRO-seq reads in each transgenic adult fly line are shown to compare the position and pattern of initiation. (C) Level of pausing in transgenic Hsp70 promoters. The sum of the read counts within the pausing region is normalized to the total mapped reads. (D) Positions of pausing in transgenic Hsp70 promoters.