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Cited by 131Genomics and Chromatin DynamicsChromatin Remodeling and CancerRNA Research and Splicing

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Paf1C regulates RNA polymerase II progression by modulating elongation rate

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Hou, Liming, Wang, Yating, Liu, Yu, Zhang, Nan, Shamovsky, Ilya, Nudler, Evgeny, Tian, Bin, Dynlacht, Brian David·2019·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1904324116PMC PMC6642404
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Abstract

Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an “accelerator” over specific early gene body regions.

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Figures

Fig. 1. Paf1 deletion through gene editing leads to increased RNA Pol II occupancy in the TSS-proximal region. (A) Schema for generating conditional Paf1-KO myoblasts using CRISPR/Cas9 gene editing. (B) Western blot showing the loss of Paf1 after Dox removal (−Dox) from 1 to 4 d. Depletion of Paf1 is accompanied by loss of H2Bub, and its level can be partially rescued by reintroducing Dox for 4 d. Note that the mobility of Dox-induced Flag-Paf1 is shifted with respect to endogenous Paf1, denoted by wild-type C2C12 (WT) in last lane. α-Tubulin was used as loading control. (C) Heatmaps show unphosphorylated Pol II occupancy over 2 kb upstream and downstream of TSS after ablation of Paf1. Nonoverlapping 5,846 expressed genes were sorted by descending levels of Paf1 occupancy (Left), and corresponding Pol II ChIP-seq read densities and log2 fold change (−Dox/+Dox) of normalized Pol II ChIP-seq read densities are shown to the Right. (D) Heatmaps were plotted as increasing gene length over regions 5 kb upstream to 100 kb downstream of the TSS. Signals are aligned according to their TSS, and TESs are indicated by dotted black lines.
Fig. 1. Paf1 deletion through gene editing leads to increased RNA Pol II occupancy in the TSS-proximal region. (A) Schema for generating conditional Paf1-KO myoblasts using CRISPR/Cas9 gene editing. (B) Western blot showing the loss of Paf1 after Dox removal (−Dox) from 1 to 4 d. Depletion of Paf1 is accompanied by loss of H2Bub, and its level can be partially rescued by reintroducing Dox for 4 d. Note that the mobility of Dox-induced Flag-Paf1 is shifted with respect to endogenous Paf1, denoted by wild-type C2C12 (WT) in last lane. α-Tubulin was used as loading control. (C) Heatmaps show unphosphorylated Pol II occupancy over 2 kb upstream and downstream of TSS after ablation of Paf1. Nonoverlapping 5,846 expressed genes were sorted by descending levels of Paf1 occupancy (Left), and corresponding Pol II ChIP-seq read densities and log2 fold change (−Dox/+Dox) of normalized Pol II ChIP-seq read densities are shown to the Right. (D) Heatmaps were plotted as increasing gene length over regions 5 kb upstream to 100 kb downstream of the TSS. Signals are aligned according to their TSS, and TESs are indicated by dotted black lines.CC BY-NC-ND · PubMed Central
Fig. 2. Paf1-deficient cells exhibit marked alterations in nucleosome occupancy over TSS-proximal regions. (A) Metaplots of MNase-seq read densities around a 2-kb window flanking the TSS of all nonoverlapping expressed genes in Paf1-KO cells with (+Dox), without (−Dox) Paf1 and rescue conditions by adding Dox back. Reexpression of Paf1 partially rescues altered nucleosome occupancy in Paf1-KO cells. RPM, reads per million. (B) Metaplots of ATAC-seq read densities around a 2-kb region flanking TSS in Paf1-depleted (siPaf1) versus control siNS cells. (C) Heatmaps of ATAC-seq reads density were plotted after ranking according to Paf1 occupancy. Log2 ratios of normalized ATAC-seq reads comparing Paf1-depleted to control cells were calculated and plotted.
Fig. 2. Paf1-deficient cells exhibit marked alterations in nucleosome occupancy over TSS-proximal regions. (A) Metaplots of MNase-seq read densities around a 2-kb window flanking the TSS of all nonoverlapping expressed genes in Paf1-KO cells with (+Dox), without (−Dox) Paf1 and rescue conditions by adding Dox back. Reexpression of Paf1 partially rescues altered nucleosome occupancy in Paf1-KO cells. RPM, reads per million. (B) Metaplots of ATAC-seq read densities around a 2-kb region flanking TSS in Paf1-depleted (siPaf1) versus control siNS cells. (C) Heatmaps of ATAC-seq reads density were plotted after ranking according to Paf1 occupancy. Log2 ratios of normalized ATAC-seq reads comparing Paf1-depleted to control cells were calculated and plotted.CC BY-NC-ND · PubMed Central
Fig. 3. Ser5 phosphorylation of Pol II accumulates in early gene bodies after Paf1 depletion. (A and B) Heatmaps of Ser5 phosphorylated Pol II (Ser5P) ChIP-seq densities were plotted based on Paf1 occupancy (A) or gene length (C) as described in the legend to Fig. 1 C and D, respectively. (C) A metagene plot of normalized Ser5P ChIP-seq densities over regions 5 kb upstream to 100 kb downstream of the TSS (+Dox versus −Dox). (D) Profiles for individual genes of various lengths (short versus long) are shown. (E) Proteomic analysis of chromatin-associated proteins interacting with Paf1. Purple arrows indicate associations identified in this study, red arrows indicate associations identified in one MS experiment, and black arrows indicate previously published interactions.
Fig. 3. Ser5 phosphorylation of Pol II accumulates in early gene bodies after Paf1 depletion. (A and B) Heatmaps of Ser5 phosphorylated Pol II (Ser5P) ChIP-seq densities were plotted based on Paf1 occupancy (A) or gene length (C) as described in the legend to Fig. 1 C and D, respectively. (C) A metagene plot of normalized Ser5P ChIP-seq densities over regions 5 kb upstream to 100 kb downstream of the TSS (+Dox versus −Dox). (D) Profiles for individual genes of various lengths (short versus long) are shown. (E) Proteomic analysis of chromatin-associated proteins interacting with Paf1. Purple arrows indicate associations identified in this study, red arrows indicate associations identified in one MS experiment, and black arrows indicate previously published interactions.CC BY-NC-ND · PubMed Central
Fig. 4. Paf1-depleted cells exhibit reductions in H2Bub deposition over early gene body regions. (A) Heatmaps showing log2 ratio of H2Bub ChIP-seq densities at promoter-proximal regions (TSS −2 kb to TSS +2 kb) in Paf1-KO cells ranked according to decreasing Paf1 occupancy. (B) Heatmaps showing log2 ratio of H2Bub ChIP-seq densities were plotted based on increasing gene length. (C and D) Metagene plots of H2Bub ChIP-seq read densities in Paf1-KO cells with (+) or without (−) Dox treatment in different groups. Genes were grouped according to expression level (C; high [FPKM > 30]) or low (0 < FPKM < 30) expression) or length (D; long [>20 kb], intermediate [10 kb < gene length< 20 kb], and short [<5 kb]), as indicated. (E) H2Bub ChIP-seq for individual short or long genes are shown. Zoomed-in shaded rectangular area denotes region with decreased H2Bub levels. (F) A metagene plot of normalized H2Bub ChIP-seq densities over regions 5 kb upstream to 100 kb downstream of the TSS (+Dox versus −Dox). (G) Scatterplots of log2 fold changes of normalized Ser5P occupancy versus log2 fold changes of normalized H2Bub in 2 regions (TSS to +20 kb and +20 kb to TES) in Paf1-KO cells.
Fig. 4. Paf1-depleted cells exhibit reductions in H2Bub deposition over early gene body regions. (A) Heatmaps showing log2 ratio of H2Bub ChIP-seq densities at promoter-proximal regions (TSS −2 kb to TSS +2 kb) in Paf1-KO cells ranked according to decreasing Paf1 occupancy. (B) Heatmaps showing log2 ratio of H2Bub ChIP-seq densities were plotted based on increasing gene length. (C and D) Metagene plots of H2Bub ChIP-seq read densities in Paf1-KO cells with (+) or without (−) Dox treatment in different groups. Genes were grouped according to expression level (C; high [FPKM > 30]) or low (0 < FPKM < 30) expression) or length (D; long [>20 kb], intermediate [10 kb < gene length< 20 kb], and short [<5 kb]), as indicated. (E) H2Bub ChIP-seq for individual short or long genes are shown. Zoomed-in shaded rectangular area denotes region with decreased H2Bub levels. (F) A metagene plot of normalized H2Bub ChIP-seq densities over regions 5 kb upstream to 100 kb downstream of the TSS (+Dox versus −Dox). (G) Scatterplots of log2 fold changes of normalized Ser5P occupancy versus log2 fold changes of normalized H2Bub in 2 regions (TSS to +20 kb and +20 kb to TES) in Paf1-KO cells.CC BY-NC-ND · PubMed Central
Fig. 5. Paf1 loss dramatically hampers transcriptional elongation. (A) Heatmaps showing log2 fold change (−Dox/+Dox) of normalized Proseq read densities for genes in both forward (n = 3,023) and reverse (n = 2,745) strands within 2-kb regions flanking the TSS before or after Paf1 depletion in Paf1-KO cell. Both sense and antisense directions for both strands are indicated. (B) Metagene analysis of Proseq data for both sense and antisense directions in regions within 2 kb of the TSS. (C) Analysis as in A, but plotted as a function of gene length in Paf1-KO cells expressing (+Dox) or lacking (−Dox) Paf1 expression. (D) Metagene plot of mean log2 ratios shows that the increase in Proseq reads correlates with the accumulation of Ser5P, RNA-seq reads, and the reduction of H2Bub over an early ∼20- to 30-kb gene body window. Data in SI Appendix, Fig. S7E were replotted here for comparison. (E) Metagene plots of Proseq data at each time interval after DRB treatment (0, 5, 15, and 30 min) over the entire gene body plus 5-kb flanking regions upstream of TSS and downstream of TES. RPM, reads per million. (F) Proseq data are shown for two individual genes before (+Dox) and after (−Dox) Paf1 ablation at each time point after DRB treatment (0, 5, 15, and 30 min). Note that the “travel distance” of engaged Pol II after DRB treatment was affected by Paf1 ablation (+Dox versus −Dox). (G) Heatmaps show the log2 fold change of normalized Proseq signal in DRB treatment time points (5 min, 15 min, and 30 min) compared with untreated control (0 min) in +Dox and −Dox conditions. Note that the red color indicates the “travel distance” of engaged Pol II after DRB treatment. (H) Elongation rates were calculated using a HMM method. Boxplots show the distribution of elongation rates in cells expressing (blue) or lacking (red) Paf1 at indicated time intervals before and after Paf1 removal. “+” sign in the boxplot denotes the mean of elongation rates. **P < 0.01 by Wilcoxon rank-sum test. Note the compressed distribution of elongation rates after Paf1 removal.
Fig. 5. Paf1 loss dramatically hampers transcriptional elongation. (A) Heatmaps showing log2 fold change (−Dox/+Dox) of normalized Proseq read densities for genes in both forward (n = 3,023) and reverse (n = 2,745) strands within 2-kb regions flanking the TSS before or after Paf1 depletion in Paf1-KO cell. Both sense and antisense directions for both strands are indicated. (B) Metagene analysis of Proseq data for both sense and antisense directions in regions within 2 kb of the TSS. (C) Analysis as in A, but plotted as a function of gene length in Paf1-KO cells expressing (+Dox) or lacking (−Dox) Paf1 expression. (D) Metagene plot of mean log2 ratios shows that the increase in Proseq reads correlates with the accumulation of Ser5P, RNA-seq reads, and the reduction of H2Bub over an early ∼20- to 30-kb gene body window. Data in SI Appendix, Fig. S7E were replotted here for comparison. (E) Metagene plots of Proseq data at each time interval after DRB treatment (0, 5, 15, and 30 min) over the entire gene body plus 5-kb flanking regions upstream of TSS and downstream of TES. RPM, reads per million. (F) Proseq data are shown for two individual genes before (+Dox) and after (−Dox) Paf1 ablation at each time point after DRB treatment (0, 5, 15, and 30 min). Note that the “travel distance” of engaged Pol II after DRB treatment was affected by Paf1 ablation (+Dox versus −Dox). (G) Heatmaps show the log2 fold change of normalized Proseq signal in DRB treatment time points (5 min, 15 min, and 30 min) compared with untreated control (0 min) in +Dox and −Dox conditions. Note that the red color indicates the “travel distance” of engaged Pol II after DRB treatment. (H) Elongation rates were calculated using a HMM method. Boxplots show the distribution of elongation rates in cells expressing (blue) or lacking (red) Paf1 at indicated time intervals before and after Paf1 removal. “+” sign in the boxplot denotes the mean of elongation rates. **P < 0.01 by Wilcoxon rank-sum test. Note the compressed distribution of elongation rates after Paf1 removal.CC BY-NC-ND · PubMed Central
Fig. 6. Model indicating roles for Paf1 in controlling productive transcription elongation over a specialized window downstream of the TSS.
Fig. 6. Model indicating roles for Paf1 in controlling productive transcription elongation over a specialized window downstream of the TSS.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Hou, L., Wang, Y., Liu, Y., Zhang, N., Shamovsky, I., Nudler, E., Tian, B., & Dynlacht, B. D.
Affiliations
  • NYU Langone Health (US)
  • Rutgers New Jersey Medical School (US)
  • New York University (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Dynlacht, Brian David
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2019-06-28
Volume
116
Issue
29
Pages
14583-14592
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1904324116
PMC
PMC6642404
OpenAlex
W2955819370
Cited by
129
References
53
Citations by year
2026: 11 · 2025: 13 · 2024: 20 · 2023: 21 · 2022: 18 · 2021: 22 · 2020: 22 · 2019: 2
FWCI
5.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
NIH (R01GM122395, R01GM126891)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
Paf1C, RNA polymerase II, transcription elongation, elongation rate, Ser5 phosphorylation, histone H2B ubiquitylation, nascent RNA, early termination, Spt5, Spt6, FACT complex, gene body, conditional gene deletion
MeSH terms
Animals, Carrier Proteins, Cell Line, Histones, Promoter Regions, Genetic, RNA Polymerase II, Myoblasts, RNA, Small Interfering, Mice, Ubiquitination, Gene Knockout Techniques, Transcription Elongation, Genetic, Transcription Termination, Genetic, CRISPR-Cas Systems

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