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. 2012;7(6):e38769.
doi: 10.1371/journal.pone.0038769. Epub 2012 Jun 11.

RNA Polymerase II Pausing Downstream of Core Histone Genes Is Different From Genes Producing Polyadenylated Transcripts

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Free PMC article

RNA Polymerase II Pausing Downstream of Core Histone Genes Is Different From Genes Producing Polyadenylated Transcripts

Krishanpal Anamika et al. PLoS One. .
Free PMC article

Abstract

Recent genome-wide chromatin immunoprecipitation coupled high throughput sequencing (ChIP-seq) analyses performed in various eukaryotic organisms, analysed RNA Polymerase II (Pol II) pausing around the transcription start sites of genes. In this study we have further investigated genome-wide binding of Pol II downstream of the 3' end of the annotated genes (EAGs) by ChIP-seq in human cells. At almost all expressed genes we observed Pol II occupancy downstream of the EAGs suggesting that Pol II pausing 3' from the transcription units is a rather common phenomenon. Downstream of EAGs Pol II transcripts can also be detected by global run-on and sequencing, suggesting the presence of functionally active Pol II. Based on Pol II occupancy downstream of EAGs we could distinguish distinct clusters of Pol II pause patterns. On core histone genes, coding for non-polyadenylated transcripts, Pol II occupancy is quickly dropping after the EAG. In contrast, on genes, whose transcripts undergo polyA tail addition [poly(A)(+)], Pol II occupancy downstream of the EAGs can be detected up to 4-6 kb. Inhibition of polyadenylation significantly increased Pol II occupancy downstream of EAGs at poly(A)(+) genes, but not at the EAGs of core histone genes. The differential genome-wide Pol II occupancy profiles 3' of the EAGs have also been confirmed in mouse embryonic stem (mES) cells, indicating that Pol II pauses genome-wide downstream of the EAGs in mammalian cells. Moreover, in mES cells the sharp drop of Pol II signal at the EAG of core histone genes seems to be independent of the phosphorylation status of the C-terminal domain of the large subunit of Pol II. Thus, our study uncovers a potential link between different mRNA 3' end processing mechanisms and consequent Pol II transcription termination processes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pol II pausing and the corresponding transcripts downstream of the EAGs are genome-wide features of Pol II transcription termination.
Chip-seq data using an anti-Pol II antibody (N-20, our study) and Gro-seq was carried out using human MCF7 cells. A) 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) 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. C) 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 (Table S1). Note that all the Gro-seq RNA reads map in the sense orientation when compared to the pre-mRNA.
Figure 2
Figure 2. Genome-wide Pol II pauses with different patterns downstream of the EAGs.
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 Figure S1) 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. A) 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) Mean tag densities of Mock (Blue) and Pol II (Pink) on genes −/+4 kb upstream and downstream of the EAG in each cluster. C) 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. D) 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. E) The locations of oligonucleotides, which were used to validate Pol II pause profiles, are represented schematically.
Figure 3
Figure 3. Pol II pause on highly expressed genes.
K-means clustering of Mock (blue) and Pol II (pink) reads on 100 highly expressed genes from MCF7 cells (for exact gene names see Table S2) mainly generated two distinct clusters in terms of Pol II occupancy at the corresponding EAGs. A) 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) 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. C) The locations of oligonucleotides, which were designed to validate Pol II pause profile, are represented schematically. D, E) 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.
Figure 4
Figure 4. On core histone genes Pol II occupancy downstream of the EAGs is quickly dropping.
Clustering of reads obtained following anti-Mock ChIP-seq and anti-Pol II ChIP-seq on all human histone genes generates two clusters. A) 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) 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.
Figure 5
Figure 5. Amongst intronless genes only histone genes have narrow Pol II pause peaks downstream of the EAGs.
Clustering of Mock and Pol II reads on all human intronless genes generates two clusters. A) 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) 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.
Figure 6
Figure 6. 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.
A–D) Four different published Pol II ChIP-seq data sets from mES cells , 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.
Figure 7
Figure 7. Inhibition of polyadenylation increases Pol II occupancy downstream of the EAGs on poly(A)+ genes, but not on core histone genes.
ChIP-qPCR validation of Pol II occupancy following cordycepin treatment on poly(A)+ genes (A–C) and histone gene (D). 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.

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References

    1. Orphanides G, Lagrange T, Reinberg D. The general transcription factors of RNA polymerase II. Genes Dev. 1996;10:2683. - PubMed
    1. Ptashne M, Gann A. Transcriptional activation by recruitment. Nature. 1997;386:577. - PubMed
    1. Hochheimer A, Tjian R. Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes Dev. 2003;17:1320. - PubMed
    1. Nechaev S, Fargo DC, dos Santos G, Liu L, Gao Y. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science. 2010;327:338. - PMC - PubMed
    1. Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S. c-Myc regulates transcriptional pause release. Cell. 2010;141:445. - PMC - PubMed

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