Transcription activation depends on the length of the RNA polymerase II C‐terminal domain
2021; Springer Nature; Volume: 40; Issue: 9 Linguagem: Inglês
10.15252/embj.2020107015
ISSN1460-2075
AutoresAnna Sawicka, Gabriel Villamil, Michael Lidschreiber, Xavier Darzacq, Claire Dugast‐Darzacq, Björn Schwalb, Patrick Cramer,
Tópico(s)RNA modifications and cancer
ResumoArticle8 February 2021Open Access Transparent process Transcription activation depends on the length of the RNA polymerase II C-terminal domain Anna Sawicka Anna Sawicka Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Gabriel Villamil Gabriel Villamil Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Michael Lidschreiber Michael Lidschreiber orcid.org/0000-0002-6740-2755 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Xavier Darzacq Xavier Darzacq orcid.org/0000-0003-2537-8395 Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA CIRM Center of Excellence, University of California, Berkeley, CA, USA Search for more papers by this author Claire Dugast-Darzacq Claire Dugast-Darzacq Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA CIRM Center of Excellence, University of California, Berkeley, CA, USA Search for more papers by this author Björn Schwalb Björn Schwalb Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Patrick Cramer Corresponding Author Patrick Cramer [email protected] orcid.org/0000-0001-5454-7755 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Anna Sawicka Anna Sawicka Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Gabriel Villamil Gabriel Villamil Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Michael Lidschreiber Michael Lidschreiber orcid.org/0000-0002-6740-2755 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Xavier Darzacq Xavier Darzacq orcid.org/0000-0003-2537-8395 Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA CIRM Center of Excellence, University of California, Berkeley, CA, USA Search for more papers by this author Claire Dugast-Darzacq Claire Dugast-Darzacq Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA CIRM Center of Excellence, University of California, Berkeley, CA, USA Search for more papers by this author Björn Schwalb Björn Schwalb Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Patrick Cramer Corresponding Author Patrick Cramer [email protected] orcid.org/0000-0001-5454-7755 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Author Information Anna Sawicka1, Gabriel Villamil1, Michael Lidschreiber1, Xavier Darzacq2,3, Claire Dugast-Darzacq2,3, Björn Schwalb1 and Patrick Cramer *,1 1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA 3CIRM Center of Excellence, University of California, Berkeley, CA, USA *Corresponding author (lead contact). Tel: +49 551 201 2800; E-mail: [email protected] The EMBO Journal (2021)40:e107015https://doi.org/10.15252/embj.2020107015 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation. SYNOPSIS The disordered C-terminal domain (CTD) of eukaryotic RNA polymerase II (Pol II) harbors a varying number of heptapeptide repeats across species, generally scaling with genome size. Here, experimental shortening implicates the CTD of human Pol II in transcription activation. Shortening of the CTD in human cells to half of its length does not generally alter pre-mRNA synthesis and processing. CTD shortening reduces promoter-proximal Pol II pausing. CTD shortening affects transcription of putative enhancer elements. CTD shortening delays transcription activation after external MAPK signaling stimulation. Introduction RNA polymerase II (Pol II) carries out transcription of protein-coding genes and many non-coding RNA species in eukaryotic cells (Cramer, 2019). The largest subunit of Pol II, RPB1, contains a unique C-terminal domain (CTD) that consists of heptapeptide repeats with the consensus sequence YSPTSPS (Allison et al, 1985; Corden et al, 1985). The CTD forms a long and flexible, tail-like extension from the Pol II body (Cramer et al, 2001). The CTD is essential in yeast, Drosophila, and mammalian cells (Allison et al, 1988; Bartolomei et al, 1988). In vitro, the CTD is dispensable for basal RNA synthesis (Corden, 2013), but required for activated transcription (Koleske et al, 1992). A plethora of biochemical and genetic studies have demonstrated that the CTD functions in coordinating the transcription cycle with RNA processing and other nuclear events (Phatnani & Greenleaf, 2006; Chapman et al, 2008; Buratowski, 2009; Mayer et al, 2012; Hsin & Manley, 2012; Eick & Geyer, 2013; Zaborowska et al, 2016; Harlen & Churchman, 2017; Gerber et al, 2020). The CTD recruits factors for pre-mRNA processing, in particular splicing (Fong & Bentley, 2001; David et al, 2011; Hsin & Manley, 2012). Factor binding to the CTD relies on interactions with short regions in the CTD comprising only 1–3 heptapeptide repeats (Meinhart et al, 2005). The length of the CTD differs strongly between species. CTD length appears to scale with genome size and to correlate inversely with gene density (Stiller & Hall, 2002; Quintero-Cadena et al, 2020). Genetic studies revealed the minimal number of CTD repeats that supports viability of various species (Bartolomei et al, 1988; West & Corden, 1995; Schwer et al, 2012; Lu et al, 2019). In the yeast Saccharomyces cerevisiae, only eight out of the 26 CTD repeats are required for viability, although 13 repeats are needed to overcome temperature sensitivity (Nonet & Young, 1989). The human CTD contains 52 repeats, but human cells expressing Pol II with only 25 CTD repeats grow normally (Boehning et al, 2018). Based on available literature, it remains poorly understood why a certain CTD length is required for Pol II function in various species. CTD length correlates with the distance between gene promoters and their regulatory enhancer elements, suggesting that the CTD facilitates promoter-enhancer contacts (Allen & Taatjes, 2015). Consistent with this model, CTD shortening can reduce expression of genes that are controlled by enhancers (Allison & Ingles, 1989; Scafe et al, 1990; Gerber et al, 1995; Aristizabal et al, 2013). Recent studies suggested that the CTD is involved in the formation of promoter-enhancer contacts that rely on protein clustering driven by liquid–liquid phase separation (LLPS) (Sabari et al, 2018; Nair et al, 2019). Indeed, the CTD can undergo LLPS in vitro (Boehning et al, 2018) and can be incorporated into LLPS droplets of transcriptional activators (Kwon et al, 2014; Burke et al, 2015). The CTD is also important for Pol II clustering in vivo, as shortening of the human CTD from 52 to 25 repeats decreases the number and size of Pol II clusters in the human cell nucleus (Boehning et al, 2018). Here, we systematically investigate the functional consequences of shortening the length of the CTD in human cells. We use a human cell line that expresses Pol II with a CTD truncated from 52 to 25 repeats (Boehning et al, 2018). Multiomics analysis of this cell line shows that CTD shortening does not generally alter RNA synthesis and processing, but that rapid RNA synthesis changes in response to an external signal are delayed and compromised. These results reveal that the normal CTD length is required for efficient transcription activation in human cells and are consistent with the model that the CTD is critical for rapid Pol II recruitment to genes upon their activation. Results CTD shortening hardly alters the human transcriptome To investigate the functional consequences of CTD length shortening in human cells, we used U2OS osteosarcoma cells carrying an α-amanitin-resistant variant of RPB1 (Meininghaus et al, 2000) with 52 CTD repeats ("RPB1-52R", corresponding to wild-type) or with 25 CTD repeats ("RPB1-25R") (Boehning et al, 2018). To characterize RNA metabolism genome-wide, we subjected both cell lines to transient transcriptome analysis (TT-seq) (Fig 1A and B, Materials and Methods). TT-seq uses a short pulse of metabolic RNA labeling with 4-thiouridine and subsequent sequencing of the labeled, newly synthesized RNA fragments (Schwalb et al, 2016). Together with standard RNA-seq and with the use of kinetic modeling (Sun et al, 2012), TT-seq can provide RNA synthesis and degradation rates. Figure 1. CTD shortening hardly changes the transcriptome and RNA metabolism in human cells Schematic representation of the CTD variants expressed in U2OS cells used in this study. Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control. Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control, and the data were normalized using spike-in counts. 16,214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted P value < 0.05, log2 fold change ≥ 1), and 704 genes were significantly downregulated (adjusted P value < 0.05, log2 fold change ≤ 1). Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12,014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12,014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12,014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Download figure Download PowerPoint We collected TT-seq and RNA-seq data for two biological replicates in both RPB1-52R and RPB1-25R cells (Fig EV1A and B). We used RNA spike-in probes to enable detection of global changes (Materials and Methods). We also confirmed that expression of the RPB1 mutation that confers α-amanitin resistance does not alter RNA levels (Fig EV1C). Comparison of the data from the two different cell lines shows that CTD shortening does not lead to global changes in RNA levels for RefSeq genes (Fig 1C). Only ~ 7% of all expressed genes were differentially expressed (Padj < 0.05 with fold change > 2 (upregulated genes) or < 0.5 (downregulated genes)) (Fig 1C). Downregulated genes were enriched in factors regulating mesenchymal cell proliferation (Fig EV1D), in agreement with slightly reduced cell proliferation (Fig EV1E). We also did not observe changes in the RNA synthesis of transposable elements (Fig EV1F). Consistent with these findings, mRNA synthesis and degradation rates as well as mRNA half-lives were also essentially unchanged (Fig 1D–F). In conclusion, CTD shortening does generally not alter the transcriptome or RNA metabolism in human cells. Click here to expand this figure. Figure EV1. The transcriptome and RNA metabolism in human cells hardly change upon CTD shortening Spearman correlation of read counts in RefSeq genes in biological replicates of TT-seq experiments. Spearman correlation of read counts in RefSeq genes in biological replicates of total RNA- seq experiments. MA plot showing changes in RNA synthesis in TT-seq datasets upon expression of the α-amanitin-resistant full-length CTD variant in U2OS cells in steady state conditions. Wild-type U2OS cells are used as a control, and the data were normalized using spike-in counts. 16,214 expressed genes annotated in RefSeq were analyzed, 7 genes were significantly upregulated (adjusted P value < 0.05, log2 fold change ≥ 1, depicted in red) and 5 genes were significantly downregulated (adjusted P value < 0.05, log2 fold change ≤ 1, depicted in red). Gene ontology analysis of significantly downregulated genes (adjusted P value < 0.05, log2 fold change ≤ 1) upon CTD shortening. GO categories with FDR≤ 0.05 are shown. Proliferation curve generated for cells expressing the α-amanitin-resistant RPB1-52R and RPB1-25R CTD variants, as well as wild-type U2OS cells. Error bars show standard deviation of 3 biological replicates. Statistical significance was estimated using two-way ANOVA, followed by the Tukey multiple comparisons test. The difference in growth rate between RPB1-52R and RPB1-25R cells is significant (adjusted P value = 0.0014322). RNA synthesis of transposable elements in TT-seq datasets upon expression RPB1-25R CTD variant in U2OS cells in steady state conditions. Wild-type U2OS cells are used as a control and the data were normalized using spike-in counts. 1,026 expressed transposable elements were analyzed. Differentially expressed transposons are in red. Five were significantly upregulated (adjusted P value < 0.05, log2 fold change ≥ 1) and 9 were significantly downregulated (adjusted P value < 0.05, log2 fold change ≤ 1). Download figure Download PowerPoint CTD shortening hardly affects pre-mRNA splicing In order to reveal possible consequences of CTD shortening for pre-mRNA splicing kinetics, we used our TT-seq datasets and quantified sequencing reads that were derived from unspliced transcripts ("unspliced reads"). These reads either span exon–intron junctions (5′ splice sites, 5′SSs) or intron–exon junctions (3′ splice sites, 3′SSs). To avoid confounding effects of alternative splicing, we used only major mRNA isoforms that constitute at least 70% of total RNA-seq expression for a given gene (Materials and Methods). Using this criterion, we identified a total of 6,260 major RNA isoforms in RPB1-52R and in RPB1-25R cells, containing 24,393 5′SSs and 24,995 3′SSs. We then computed a "splicing ratio" that we defined as the ratio of spliced (exon–exon) reads over the sum of spliced and unspliced reads. CTD shortening did increase the splicing ratio only very slightly (Fig 2A), and this was independent of the position of the intron within the transcript (Fig 2B–D). Figure 2. CTD shortening hardly alters pre-mRNA splicing kinetics and mRNA isoforms A. Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads for all constitutive 5′SSs and 3′SSs detected in major RNA isoforms in RPB1-52R and RPB1-25R cells. P value was calculated using Mann–Whitney U-test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. B–D. Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads in RPB1-52R and RPB1-25R cells for all constitutive 5′SSs and 3′SSs detected in major RNA isoforms based on intron position in the transcript: (B) first intron junction, (C) intermediate intron junctions and (D) last intron junctions. P values were calculated using Mann–Whitney U-test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. E. Differential mRNA isoform usage upon CTD shortening. Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1-52R and RPB1-25R cells using long-read sequencing. 47,416 isoforms were detected, RPB1-52R condition was used as a control. 335 isoforms show differential expression (P value < 0.05, Fischer's exact test). F. Alternative splicing events [in %] detected in 335 differentially expressed isoforms upon CTD shortening in U2OS cells. Download figure Download PowerPoint To investigate whether CTD shortening affects alternative splicing (Cramer et al, 1999), we isolated chromatin-associated RNA, which is enriched in nascent pre-mRNAs (Bhatt et al, 2012). We directly sequenced these long RNAs with the Oxford Nanopore technology from both types of cells (Materials and Methods). In these data, only 335 out of 47,416 detected mRNA isoforms were differentially expressed (Fig 2E), and the majority of these showed intron retention (Fig 2F). Together, these results show that CTD shortening in human cells does hardly alter pre-mRNA splicing and alternative splicing. CTD shortening slightly alters promoter-proximal pausing of Pol II To investigate whether CTD shortening affects promoter-proximal pausing, we used a previously developed multiomics approach that provides kinetic insights (Gressel et al, 2017, 2019). Briefly, this approach uses kinetic modeling to fit TT-seq data and Pol II occupancy data and estimate the productive initiation frequency and the duration of Pol II pausing in the promoter-proximal region. To map occupancy with transcriptionally engaged Pol II over the genome, we performed precision run-on sequencing (PRO-seq) (Mahat et al, 2016) in RPB1-52R and RPB1-25R cells for two biological replicates (Fig EV2A and B). Spike-in probes were derived from Drosophila RNAs and used for quantification (Materials and Methods). Click here to expand this figure. Figure EV2. CTD shortening leads to minor changes in Pol II pausing at steady state A, B. Spearman correlation of read counts in the first constitutive exon of genes encoding major isoforms in biological replicates of PRO-seq data. C, D. Scatter plot of pausing duration (d) and productive initiation frequencies (I) in genes encoding major isoforms in RPB1-52R and RPB1-25R cells. The gray-shaded area depicts impossible combinations of I and d according to published kinetic theory (Ehrensberger et al, 2013). E, F. Histogram of distances from the PAS to the transcription termination site (TTS) of genes encoding major isoforms. Download figure Download PowerPoint Analysis of the PRO-seq data showed that CTD shortening slightly decreased Pol II occupancy in the promoter-proximal window downstream of the transcription start site (TSS) (Fig 3A), whereas the position of the Pol II occupancy peak remained unchanged (Fig 3B and C). To investigate the reasons for the decrease in Pol II occupancy, we calculated the productive transcription initiation frequency (I) and the duration of Pol II pausing (d) by combining TT-seq with PRO-seq occupancy data as described (Gressel et al, 2017). I was computed from TT-seq coverage over non-first constitutive exons, whereas d was obtained as a ratio of PRO-seq signal over I in the promoter-proximal window (see Materials and Methods). Whereas I was essentially unchanged, d was slightly decreased (Figs 3D–F, and EV2C and D). These results show that CTD shortening does not substantially alter Pol II pausing behavior under steady state conditions except for a slight decrease in pause duration. Figure 3. CTD shortening leads to minor changes in Pol II pausing at steady state A. CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. B, C. Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp. D. Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. E. Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. P value = 0.13 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. F. Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. P value < 2.2e-16 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. G. Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. H. Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4,480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal, and shaded area refer to 95% bootstrap confidence intervals. I. Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal, and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. J. Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. P value < 2.2e-16 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. K. Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1,958 genes encoding major isoforms. P value = 0.8 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Download figure Download PowerPoint CTD shortening does not alter Pol II velocity and termination TT-seq metagene profiles over gene bodies were unaltered upon CTD shortening (Fig 3G), suggesting that Pol II elongation velocity is largely unchanged. To investigate this, we estimated Pol II velocity within expressed genes from our data as described previously (Gressel et al, 2017). We found that Pol II elongation velocity is unchanged upon CTD shortening (Fig 3H). We then plotted TT-seq data around the polyadenylation site (PAS) and found no changes upon CTD shortening (Fig 3I). Poly-A tail length as measured by Oxford Nanopore sequencing was not substantially altered either, indicating that 3′ end processing was normal (Fig 3J). We also called transcription termination sites downstream of the PAS as described (Schwalb et al, 2016) and also did not find any changes upon CTD shortening (Figs 3K and EV2E and F). In summary, these results indicate that Pol II elongation and termination do not show any obvious changes upon CTD shortening in human cells under steady state conditions. CTD shortening reduces the number of transcribed putative enhancers To investigate whether CTD shortening affects transcription of enhancers, we annotated putative enhancer RNAs (eRNAs) based on our TT-seq data with the use of a previously established strategy (Schwalb et al, 2016; Michel et al, 2017; Zacher et al, 2017). We annotated 2,954 putative eRNAs in RBP1-52R cells, but found only 1,779 enhancers in RBP1-25R cells (Fig 4A). The lower number of annotated eRNAs in cells expressing Pol II with a shorter CTD did not arise from technical limitations because the TT-seq samples were sequenced to the same depth of over 100 M reads (Table EV1). On average, putative eRNAs were also slightly shorter and showed slightly lower synthesis rates (Fig 4B and C). Putative enhancers were enriched in binding sites for members of the Activator Protein-1 (AP-1) transcription factor family, including FOS, JUN, and ATF (Fig EV3A). These factors function in bone development and regeneration (Ohta et al, 1991; Wagner, 2002) and in osteosarcoma tumorigenesis (Leaner et al, 2009), consistent with cell-type specificity criteria for enhancers (Shlyueva et al, 2014; Heinz et al, 2015). Figure 4. CTD shortening in human cells alters transcribed putative enhancers Pie chart showing numbers of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. Box plots showing RNA synthesis levels (RPKs) of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. P value = 9.27e-103 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. Box plots showing lengths of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. P value = 0.000519 (Mann–Whitney U-test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. CTD shortening in human cells alters transcribed putative enhancers Transcription factor-binding sites enriches in the area of ± 50 bp from the TSS of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. P values were determined by the hypergeometric test. Bar plots showing statistics of our enhancer-promoter pairing strategy. The left bar plot shows a fraction of annotated putative enhancers that could be paired to promoters using our strategy. 2,490 putative enhancers in RPB1-52R cells (out of all 2,954 enhancers annotated in RPB1-52R cells) could be paired with promoters using our strategy (84.29% of all the putative enhancers we annotated in RPB1-52R could be paired to promoters). In RPB1-25R cells, 83.83% of annotated putative enhancers could be paired to promoters. The right panel shows a fraction of active promoters in U2OS cells that could be paired to putative enhancers using our strategy. There are 17,319 active promoters in U2OS cells (gray bar). In RPB1-52R cells, 1,755 of active promoters could be paired with putative enhancers (which constitute 10.13% of all active promoters in RPB1-52R cells). In the case of RPB1-25R cells, 1,241 of all active promoters could be paired with putative enhancers (which constitutes 7.17% of all active promoters). Bar plot showing a number of putative enhancers paired per gene in RPB1-52R and RPB1-25R cells. P value = 0.019 (χ2 test). MA plot showing changes in putative eRNAs synthesis upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data were normalized using spike-in counts. 4,335 putative eRNAs were analyzed, 117 putative eRNAs were significantly upregulated (adjusted P value < 0.05, log2 fold change ≥ 1, depicted in red) and 154 putative eRNAs were significantly downregulated (adjusted P value < 0.05, log2 fold change ≤ 1, depicted in red). Boxplot showing changes in putative eRNA synthesis if paired with genes that were significantly upregulated (adjusted P value < 0.05, log2 fold change ≥ 1) (left box) or significantly downregulated (adjusted P value < 0.05, log2 fold change ≤ 1) (right box) upon CTD shortening. Box limits are the first and third quartiles, the band inside the box is
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