Abstract
Promoter-proximal pausing of RNA polymerase (Pol) II is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here, we analyzed transcription in species across the tree of life. Unicellular eukaryotes display an accumulation of Pol II near transcription start sites, which we propose transitioned to the longer-lived, focused pause observed in metazoans. This transition coincided with the evolution of new subunits in the negative elongation factor (NELF) and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat-shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.
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Data availability
Tables in CSV format can be downloaded from GitHub (https://github.com/alexachivu/PauseEvolution_prj). Data generated in this study were deposited to the Gene Expression Omnibus under accession number GSE223913. Source data are provided with this paper.
Code availability
Custom code for analyzing sequencing data can be found on GitHub (https://github.com/alexachivu/PauseEvolution_prj/).
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Acknowledgements
We thank members of the C.G.D. and J.T.L. labs for valuable discussions and suggestions throughout the life of this project and M. A. Subirats for preparing samples from C. owczarzaki, C. fragrantissima and S. arctica. We acknowledge the Fundación Pública Galega Centro Tecnolóxico de Supercomputación de Galicia for access to the FinisTerraeIII supercomputer and V. Shabardina for facilitating access. Work in this publication was primarily supported by a grant from the National Aeronautics and Space Administration exobiology program (17-EXO-17-2-0112). Additional funding was also available from the National Human Genome Research Institute (R01-HG010346 and R01-HG009309) to C.G.D., the National Institute of General Medical Sciences (R01 GM147731) to I.L.B. and C.G.D., and the National Institutes of Health (NIH; RM1-GM139738) to J.T.L. A.A. was supported by the NIH (T32GM007739 and F30HD103398). M.M.L. was supported by an Ayuda Juan de la Cierva Incorporación postdoctoral fellowship (IJC2018-036657-I) from the Spanish Ministry of Science and Innovation. Work in A.K.H.’s lab was supported by the NIH (R01HD094868, R01DK127821, R01HD086478 and P30CA008748). Work in I.R.-T.’s lab was supported by a European Research Council Consolidator Grant (ERC-2012-Co-616960). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Some of the figures in this manuscript were created using BioRender.com.
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J.J.L., C.G.D. and A.G.C. designed the study. E.J.R., A.A. and G.C. performed the experimental research. M.M.L., A.G.C., W.W., J.J.L. and C.G.D. interpreted the protein sequence comparisons across the tree of life. B.A.B., A.G.C., G.B., W.W., J.J.L., S.P.C., J.T.L. and C.G.D. analyzed and interpreted the sequencing data. A.G.C., J.J.L., J.T.L. and C.G.D. wrote the manuscript. A.A., A.C.V., J.J.S., A.H.W., C.A.M., I.L.B., I.R.T., A.K.H. and R.B. collected the cells or provided the samples for experimental research. All authors were involved in revisions and approved the final manuscript.
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Extended data
Extended Data Fig. 1 TSS reannotation.
PRO-seq (blue) and PRO-cap24 (red) metaprofiles for S. Pombe10 (a), S. cerevisiae10 (b), D. Melanogaster24 (c), and H. Sapiens98 (d). Upper panels use published gene annotations, lower panels use reannotated genes. Note the relative depletion of PRO-cap reads upstream and downstream of the TSS and more focused pause in PRO-seq signal of D. melanogaster and H. sapiens in re annotated panels.
Extended Data Fig. 2 Clustering species by their pausing index values.
Density maps of pausing indexes for each species. The plots are split into four quantiles and colored accordingly.
Extended Data Fig. 3 Association of NELF and HEXIM subunits with pausing.
Box and whiskers plots show pausing index values in each species. Samples are clustered by the presence (n = 12) or absence (n = 8) of any NELF subunits (a), and presence () or absence () of HEXIM subunits (b). A two-sided Mann-Whitney test was used to compute p-values. Boxplot whiskers extend from the first/third quartile to the minima/maxima, ignoring outliers, while horizontal lines denote the median.(c) Box and whiskers plot of pausing indexes in each species. Boxes are clustered by which NELF subunits are present in each species for which PRO-seq (n = 11), GRO-seq (n = 2), and ChRO-seq (n = 6), were used in this project. Boxplot whiskers extend from the first/third quartile to the minima/maxima, ignoring outliers, while horizontal lines denote the median.
Extended Data Fig. 4 Pause motif search.
(a) Box and whiskers plots depict enrichment of motif scores for the pause button in each species. Samples are clustered by the presence or absence of NELF subunits (None: n = 8, NELF-B or C/D: n = 3, All, or lacking NELF-E: n = 9). A two-sided Mann-Whitney test was used to compute p-values. Boxplot whiskers extend from the first/third quartile to the minima/maxima, ignoring outliers, while horizontal lines denote the median. (b) Metaprofile plot of reads mapping to maternal and paternal alleles for genes with stronger pause motif on the maternal (left) or paternal (right) alleles. (c) Scatter plot of enrichment motif score of Initiator sequence plotted against the mean pausing index per species (R = 0.105, p = 0.661). Each dot is colored by the number of NELF subunits found in each sample. We fit a linear regression to derive the R2 and the p-value. A 95% confidence interval around this regression line is shown. (d) Box and whiskers plots depict enrichment of motif scores for the initiator motif in each species. Samples are clustered by the presence or absence of NELF subunits (None: n = 8, NELF-B or C/D: n = 3, All, or lacking NELF-E: n = 9). A two-sided Mann-Whitney test was used to compute p-values. Boxplot whiskers extend from the first/third quartile to the minima/maxima, ignoring outliers, while horizontal lines denote the median.
Extended Data Fig. 5 nelfe-FKBP12 homozygous cell line generation.
a) Schematic of CRISPR design to add the FBBP12 tag at the nelfe locus. b) PCR validation of CRISPR insertion of the FKBP12 tag. c) Microscopy images evaluating the degradation efficiency before (top) and after a 30 min treatment with 500 nM dTAG-13 (bottom) in the edited and unedited cell lines. Hoechst was used as a nuclear control, while anti-HA antibodies measure the added tag, and anti-NELFE measures the NELF-E protein level. Arrows point out the presence of Feeder cells. d) Degradation efficiency of NELF-E as measured by western blotting. b-Actin was used as a loading control, while anti-HA measures the level of NELFE-HA protein. Input denotes the relative amount of total protein loaded.
Extended Data Fig. 6 nelfb and nelfe-FKBP12 homozygous cell line validation.
a) Western blot of whole cells following NELF-E degradation with 500 nM dTAG-13 for 0 to 24 h of treatment. b) Western blot validation of chromatin fraction vs nuclear soluble fractionation. c) Western blot of NELF-B and -E proteins after degradation of either protein for 1 h. Both nuclear-soluble and chromatin-bound proteins were analyzed. d) Quantification of western blot signal in (c) for NELF-E after degradation of NELF-B, and vice-versa (n = 3 biological replicates).
Extended Data Fig. 7 Effect of NELF-B and NELF-E degradation on Pol II distribution.
a) WashU browser shots at the Nanog gene locus before and after NELF-B and -E degradation. b) Heat maps of spike-in normalized PRO-seq signal. c) Heatmaps of log2 fold changes of normalized PRO-seq signal relative to untreated controls. All heat maps are centered on active TSSs in mESCs. d) Metaprofiles of cluster 3 genes at each dTAG time point, the recovery of pause-like behavior between 30 and 60 min, and the proto-pause observed in S. pombe for reference. The proto-pause region is shaded in the two rightmost panels.
Extended Data Fig. 8 Characterization of transcription recovery clusters after NELF-B degradation.
a) Bar plots depict the percentage of transcribed enhancers and gene promoters in each cluster defined in Fig. 5e (cluster 1: n = 8,030, cluster 2: n = 16,078, cluster 3: n = 3,792). b) Enrichment profiles of the pause motif published previously49 plotted in a 1 kb window centered on TSSs found in each of the clusters defined in Fig. 5e. c) Violin plots depict log10 transformed initiation (right) or pause release (left) rates in each cluster. A two-sided Mann-Whitney test was used to compute p-values, where n.s. defines non-significant p-values, and (***) p-values < 2.2e-16. d) Plots depict the enrichment of the TATA box, Initiator, MTE, and DPE sequence motifs in each cluster in Fig. 3e. e) Meta profiles depict the enrichment of TBP, TAF-12, TFIIA, TFIIB, H3K9ac, and Med1 per cluster. ChIP-seq data from ref.66.
Extended Data Fig. 9 Pol II trickles into gene bodies effect after NELF-B degradation.
Heatmaps of log2 fold changes in PRO-seq signal in NELF-B tagged cell lines at all TSSs, Clusters 1, 2, and 3 (in this order from top to bottom rows). The heatmaps depict log2 fold change relative for the following comparisons (from left to right columns): untreated PRO-seq signal, log2 fold change for 30 min/0 min, 60 min/0 min, and 60 min/30 min of dTAG-13 treatment.
Extended Data Fig. 10 Correlations between heat shock PRO-seq data.
a) Principal component analysis (PCA) of non-heat shock (NHS), dTAG-13 treatment (dTAG), heat shock (HS), and a pre-treatment of dTAG-13 followed by heat shock (HS+dTAG). b) WashU browser shots at the heat-triggered genes (Hist1h3b, Hsp1h1, Hist1h3b) in the NELF-B (a) and NELF-E (b) edited cell lines. c) Heatmaps of log2 fold changes in PRO-seq signal in NELF-B (left) and NELF-E tagged (right) cell lines. The heatmap rows depict fold changes relative to NHS for the following treatments: dTAG-13 treatment alone, HS alone, and dual treatment of dTAG-13 and HS.
Supplementary information
Supplementary Information
Supplementary Figs. 1–15.
Supplementary Data 1
Two subdirectories. ‘Westerns’ contains raw western blots in .scn format (visualize with Fiji/ImageJ). ‘Phylogenies’ contains alignments and phylogenies for relevant proteins.
Source data
Source Data Fig
. 1 Source Data Fig. 2 Sequence, domain and sequence alignment data (.fasta, .phy, .hmm and .pfam formats), phylogenetic trees (.iqtree, .contree and .treefile) and an extended description of the methods used to generate the presence/absence table in Fig. 1d. Sequence data and an extended description of methods used to assess NELF presence for Fig. 2d.
Source Data Fig. 4
Raw western blot scan files (.scn format), which can be opened and quantified in ImageJ.
Source Data Extended Data Fig. 1
Metadata including calculated pausing indices, protocol and number of NELF subunits present for species included in main figures.
Source Data Extended Data Fig. 2
Quantification of NELF-B upon degradation of NELF-E and vice versa.
Source Data Extended Data Fig. 3
Conservation of key proteins.
Source Data Extended Data Fig. 4
Metadata, sequencing quality control, methods notes and accession numbers.
Source Data Extended Data Fig. 5
Raw western blot scan files (.scn format), which can be opened and quantified in ImageJ.
Source Data Extended Data Fig. 6
Raw western blot scan files (.scn format), which can be opened and quantified in ImageJ.
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Chivu, A.G., Basso, B.A., Abuhashem, A. et al. Evolution of promoter-proximal pausing enabled a new layer of transcription control. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01718-y
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DOI: https://doi.org/10.1038/s41594-025-01718-y
