Differential Pol II pausing downstream from the EAGs is conserved between mouse and human cells and seems to be independent from the developmental stage of the cells.

<p><b>A–D</b>) Four different published Pol II ChIP-seq data sets from mES cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769-Rahl1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769-Brookes1" target="_blank">[43]</a> were used to generate average gene profiles for different forms of Pol II (Total, Ser2, Ser5 and Ser7 phosphorylated form of the CTD of the largest subunit of Pol II) for Poly(A)⁺ and core Histone genes, as indicated on the top of the figure and on the left of the panels. Y-axis represents mean tag densities.</p

Inhibition of polyadenylation increases Pol II occupancy downstream of the EAGs on poly(A)⁺ genes, but not on core histone genes.

<p>ChIP-qPCR validation of Pol II occupancy following cordycepin treatment on poly(A)⁺ genes (<b>A–C</b>) and histone gene (<b>D</b>). Pol II occupancy (Pink bars) is compared at promoters and at different distances downstream from the EAGs before and after cordycepin treatment. Values are normalized to mock controls (Blue bars) and represented as relative signal intensity values. Distances: A, B, C: +0.5–1 kb, II: +2–3 kb, III: +4–5 kb from EAGs of the indicated poly(A)⁺ genes; D: I: +0.1–0.3 kb, II: +1.5–2 kb from EAG the indicated core histone gene. Error bars represent +/− standard deviations.</p

On core histone genes Pol II occupancy downstream of the EAGs is quickly dropping.

<p>Clustering of reads obtained following anti-Mock ChIP-seq and anti-Pol II ChIP-seq on all human histone genes generates two clusters. <b>A</b>) Heatmap generated after the K-means clustering of Mock and Pol II reads in average gene body and −/+1 kb upstream and downstream of the genes. Color scale indicates the level of enrichment. <b>B</b>) Mean tag densities of Mock (Blue) and Pol II (Pink) signals in two clusters of genes (H1 = core histone; H2 = variant histone) in the average gene body and −/+1 kb upstream and downstream of the genes.</p

Pol II pausing and the corresponding transcripts downstream of the EAGs are genome-wide features of Pol II transcription termination.

<p>Chip-seq data using an anti-Pol II antibody (N-20, our study) and Gro-seq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769-Hah1" target="_blank">[42]</a> was carried out using human MCF7 cells. <b>A</b>) Mean tag densities of ChIP-seq data of Mock (Blue) and Pol II (Pink) in average genes and −/+4 kb around them are represented. Pol II enrichment density of 13787 “non-overlapping and isolated” refseq genes were calculated. TSS: transcription start site; EAG: end of annotated gene. <b>B</b>) Mean tag densities of Mock (Blue) and Pol II ChIP-seq (Pink) and Gro-seq (Green) data on average 13787 “non-overlapping and isolated” refseq genes, and −/+4 kb upstream and downstream are represented. <b>C</b>) Mean Pol II tag density from the ChIP-seq of Mock and Pol II (Blue and Pink, respectively) and Gro-seq (Green) data in the region −500 bp to +4 kb around the EAG of 500 highly expressed genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769.s002" target="_blank">Table S1</a>). Note that all the Gro-seq RNA reads map in the sense orientation when compared to the pre-mRNA.</p

Pol II pause on highly expressed genes.

<p>K-means clustering of Mock (blue) and Pol II (pink) reads on 100 highly expressed genes from MCF7 cells (for exact gene names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769.s003" target="_blank">Table S2</a>) mainly generated two distinct clusters in terms of Pol II occupancy at the corresponding EAGs. <b>A</b>) Heatmap generated after the K-means clustering of Mock (Blue) and Pol II (Pink) reads in average gene body and −/+6 kb upstream and downstream of the genes. Color scale indicates the level of enrichment. <b>B</b>) Mean tag densities of Mock (Blue) and Pol II (Pink) signals on the two clusters of genes and −/+6 kb upstream and downstream of the gene body. <b>C</b>) The locations of oligonucleotides, which were designed to validate Pol II pause profile, are represented schematically. <b>D, E</b>) ChIP-qPCR validation of the ChIP-seq data for two randomly selected genes from each cluster (as indicated). Pol II occupancy (Pink bars) compared to mock (Blue bars) on the TSS and at different distances downstream from the EAGs are represented in input %. Distances from EAGs of the indicated genes: I: +0.5–1 kb, II: +2–3 kb, III: 4–5 kb. Error bars represent +/− standard deviations.</p

Gene Ontology (GO) terms (at significant P-values), associated with the clusters represented in Figure 2.

<p>Gene Ontology (GO) terms (at significant P-values), associated with the clusters represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone-0038769-g002" target="_blank">Figure 2</a>.</p

Genome-wide Pol II pauses with different patterns downstream of the EAGs.

<p>Clustering of genes, which have relatively high Pol II enrichment 3′ of their EAGs and high microarray expression value (considering genes from Cluster 1 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038769#pone.0038769.s001" target="_blank">Figure S1</a>) generates four clusters: H, PA1, PA2 and PA3. Total number of non-redundant refseq genes is 3495. Number of genes (or n) in each cluster is: Cluster H, n = 39; Cluster PA1, n = 74; Cluster PA2, n = 492 and Cluster PA3, n = 2890. <b>A</b>) Heatmap generated after K-means clustering of Mock and Pol II reads in the regions −/+4 kb upstream and downstream of the EAG. Color scale indicates the level of enrichment. <b>B</b>) Mean tag densities of Mock (Blue) and Pol II (Pink) on genes −/+4 kb upstream and downstream of the EAG in each cluster. <b>C</b>) The distribution of the expression levels of genes belonging to the H, PA1, PA2 and PA3 clusters (see A and B) is displayed by Whisker plot. The plots represent relative mRNA expression level of each cluster. The median is indicated with a horizontal line in each box showing that genes in Cluster PA1 have higher relative mRNA expression level than Cluster PA2 and PA3. <b>D</b>) ChIP-qPCR validation of the ChIP-seq data on two randomly selected genes (refseq ids and gene names are given) from each cluster. Pol II occupancy (Pink bars) compared to the mock (Blue bars) at different distances downstream from the EAGs are represented in input %. Distances from EAGs on the indicated genes: Cluster H: I: +0.1–0.3 kb, II: +1.5–2 kb; Cluster PA 1–3: I: +0.5–1 kb, II: +2–3 kb, III: +4–5 kb. Error bars represent +/− standard deviations. <b>E</b>) The locations of oligonucleotides, which were used to validate Pol II pause profiles, are represented schematically.</p

Amongst intronless genes only histone genes have narrow Pol II pause peaks downstream of the EAGs.

<p>Clustering of Mock and Pol II reads on all human intronless genes generates two clusters. <b>A</b>) Heatmap generated after the K-means clustering of Mock and Pol II reads in average gene body and −/+6 kb upstream and downstream of the genes. Color scale indicates the level of enrichment. <b>B</b>) Mean tag densities of Mock (Blue) and Pol II (Pink) in two clusters (Core histones and Other intronless genes) −/+6 kb upstream and downstream of the gene body.</p

In the absence of CSB UVB does not inhibit Pol II and TFIIH binding at promoters of group A genes.

<p>MCF7 cells were transfected with either scrambled siRNA or siRNAs targeting CSB (ERCC6) (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen.1004483.s004" target="_blank">Figure S4</a>), 72 hours following siRNA transfection cells were treated with UVB, or not. ChIP was carried out 3 hours following UVB treatment of MCF7 cells as well as on non-treated cells. Q-PCRs were carried out to monitor Pol II (dark blue bars) (in A), and p62 (light blue bars) (in B) occupancy on the promoters of genes selected from group A (<i>ubc, rplp1</i>), group B (<i>p21, wdr24</i>) and on a negative control (<i>intergenic</i>) region in the presence or absence of CSB, as indicated. Control ChIPs (NoAb, red bars) was carried out with Sepharose G beads only. The occupancy values at the promoters are represented in input %. Error bars represent +/− standard deviations in two biological replicates.</p

The protein level of Pol II and GTF subunits does not change after UVB treatment.

<p>(A) Western blot assays were carried out to measure the levels of total Pol II at the indicated time points after UVB treatment by using the N-20 antibody. (B) The different IIA and IIO forms of Pol II were densitometrically quantified in each time points from four independent experiments and are represented in a bar chart, where red bars indicate Pol IIA and the blue bars indicate Pol IIO forms. In each lane Pol IIA+IIO signals are taken as 100%. Error bars represent +/− standard deviation of the four independent experiments. (C) The phosphorylation levels of Pol II Rpb1-CTD Ser2, Ser5 and Ser7 were analyzed by western blot by using the indicated antibodies, respectively. (D) The levels of the indicted GTF subunits such as TFIIB, TBP and TFIIH/CDK7 were also tested in the above prepared extracts, as indicated. Tubulin-α was used as a loading control.</p