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ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Nature Cell Biology volume 23, pages 595–607 (2021)Cite this article

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Abstract

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug–genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

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Fig. 1: RNAPII-associated ELOF1 is a putative TCR gene.
Fig. 2: ELOF1 is required for efficient transcription elongation.
Fig. 3: ELOF1 is essential for transcription recovery after UV irradiation.
Fig. 4: ELOF1 is essential for repair of transcription-blocking DNA damage.
Fig. 5: ELOF1–RNAPII interaction is required for RNAPII ubiquitylation.
Fig. 6: ELOF1 interacts with the CRL4CSA complex.
Fig. 7: CRISPR screens identify determinants of illudin S sensitivity in the absence of ELOF1 or CSB.
Fig. 8: ELOF1 protects cells against DNA damage during replication.

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Data availability

Both raw and processed ChIP-seq, Bru-seq, ATAC-seq and RNA-seq data shown in main Figs. 2–4 and Extended Data Figs. 3–9 have been deposited into the Gene Expression Omnibus (GEO) under GSE149760. The mass spectrometry proteomics data shown in main Fig. 1 and Extended Data Fig. 2 have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository54 (https://www.ebi.ac.uk/pride/) with the dataset identifier PXD024051. Additionally, publicly available reference datasets of the Hg38 genome and hg19 genome and the known Canonical gene table from the UCSC genome database (https://genome.ucsc.edu/cgi-bin/hgTables; hg38 genome, lifted-over to hg19 when needed) and gene interactions from the GeneMANIA database (https://genemania.org/) have been obtained and used in this manuscript. Published structural information has been obtained for Saccharomyces cerevisiae RAD26 bound to RNAPII https://www.rcsb.org/ PBD: 5VVS) and Komagataella pastoris ELF1 bound to RNAPII (PDB: 5XOG). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom code used for the analysis of NGS data was written in R and is available from GitHub (https://git.lumc.nl/dvandenheuvel/van-der-weegen-et-al_elof_ncb2021.git).

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Acknowledgements

We acknowledge A. Kragten, J. Balk, J. Poell, K. Kato, M. Shimada, S. Kloet, M. Paulsen, M. Vukic, D. Warmerdam, A. Ramadhin and L. Daxinger for help during this project. We also thank J. Moffat, K. Chan and A. Tong for sharing the pLCKO-TKOv3 library before publication. We thank the Amsterdam UMC NGS sequencing facilities for support. We thank P. van Veelen and A. de Ru for MS equipment maintenance. This work was funded by a LUMC Research Fellowship, a NWO-ENW-M grant (OCENW.KLEIN.090) and a NWO-VIDI grant (ALW.016.161.320) to M.S.L., a Leiden University Fund (LUF) grant to D.v.d.H. (W18355-2-EM), a KWF/Alpe Young Investigator 10701 grant to J.d.L., a CCA proof-of-concept grant to K.d.L. and R.W., an Amsterdam UMC Innovation Grant (CRISPR Expertise Center, 2019) to R.W., UM1 HG009382 and R01 CA213214 NCI grants to M.L., a KWF Young Investigator grant 11367 to R.G.-P., and an ERC starting grant 310913 to A.C.O.V. J.C.W. was supported by NIH grant HL098316 and is a Howard Hughes Medical Institute (HHMI) Investigator and an American Cancer Society Research Professor. T.E.T.M. was supported by an EMBO Long-term fellowship (ALTF 1316-2016) and a HHMI fellowship of The Jane Coffin Childs Memorial Fund for Medical Research.

Author information

Author notes

  1. These authors contributed equally: Yana van der Weegen, Klaas de Lint.

Authors and Affiliations

  1. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

    Yana van der Weegen, Diana van den Heuvel, Marta San Martin Alonso, Daphne E. C. Boer, Noud H. M. Klaassen, Annelotte P. Wondergem, Sylvie M. Noordermeer & Martijn S. Luijsterburg

  2. Department of Clinical Genetics, Section Oncogenetics, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, The Netherlands

    Klaas de Lint, Janne J. M. van Schie, Khashayar Roohollahi, Josephine C. Dorsman, Job de Lange & Rob M. F. Wolthuis

  3. Department of Genetics, Research Institute of Environmental Medicine (RIeM), Nagoya University, Nagoya, Japan

    Yuka Nakazawa, Yuichiro Hara & Tomoo Ogi

  4. Department of Human Genetics and Molecular Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Yuka Nakazawa, Yuichiro Hara & Tomoo Ogi

  5. Howard Hughes Medical Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Tycho E. T. Mevissen & Johannes C. Walter

  6. Oncode Institute, Utrecht, The Netherlands

    Marta San Martin Alonso & Sylvie M. Noordermeer

  7. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

    Román González-Prieto & Alfred C. O. Vertegaal

  8. Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA

    Ishwarya V. Narayanan & Mats Ljungman

  9. Department of Environmental Health Sciences, Rogel Cancer Center and Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI, USA

    Mats Ljungman

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  1. Yana van der Weegen
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Contributions

Y.v.d.W. generated plasmids, U2OS and RPE1-iCas9 single KO and dKO cells, U2OS Flp-In cell-lines, performed clonogenic survivals, co-IP experiments, RRS and DRB-RRS experiments, western blot analyses, in situ PLA experiments, generated samples for pan-RNAPII ChIP-seq, ser2-RNAPII ChIP-seq, ATAC-seq, BruDRB-seq and Bru-seq, generated the figures and wrote the paper. K.d.L. optimized, performed and analysed all CRISPR screens and RNA-seq experiments, performed and analysed drug-sensitivity assays, generated gene-gene interaction network and Venn diagrams, performed γH2AX foci and DNA fibre experiments and helped write the paper. D.v.d.H. performed co-IP experiments, developed tools and analysed pan-RNAPII ChIP-seq, ser2-RNAPII ChIP-seq, ATAC-seq, BruDRB-seq, Bru-seq and helped write the paper. Y.N. performed co-IP experiments and generated TCR-seq libraries. T.E.T.M. generated recombinant xlELOF1, xlCSB and xlCRL4CSA, purified xlRNAPII and performed pull-down and in vitro ubiquitylation assays. J.J.M.v.S. created the RPE1-iCas9 cell line, performed CRISPR screens, performed γH2AX foci and DNA fibre analyses. M.S.M.A. performed and analysed DRIP–qPCR. I.V.N. analysed BruDRB-seq and Bru-seq. D.E.C.B. generated stable cell lines and performed clonogenic survival assays. R.G.-P. analysed the MS with support from A.C.O.V. N.H.M.K. generated U2OS single KO clones, Flp-In cell-lines, performed clonogenic survivals and co-IP experiments. A.P.W. performed the unscheduled DNA synthesis experiments and generated plasmids. K.R. processed and analysed the RNA-seq data, performed gene–gene interaction network analysis, and performed and analysed CRISPR screens. Y.H. analysed ser2-RNAPII ChIP-seq. J.d.L. supervised J.J.M.v.S.; J.C.D. supervised K.R.; J.C.W. supervised T.E.T.M.; S.M.N. supervised M.S.M.A.; M.L. supervised I.V.N. and analysed BruDRB-seq and Bru-seq. T.O. supervised Y.N. and Y.H. and analysed Ser2-RNAPII ChIP-seq. R.M.F.W. supervised K.d.L., co-supervised K.R. and J.J.M.v.S., conceived, coordinated and supervised the project and helped write the paper. M.S.L. supervised Y.v.d.W., D.v.d.H., D.E.C.B., N.H.M.K. and A.P.W., conceived, coordinated and supervised the project, generated all cryo-electron microscopy images and wrote the paper.

Corresponding authors

Correspondence to Rob M. F. Wolthuis or Martijn S. Luijsterburg.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Dong Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Human ELOF1 protects cells against transcription stress.

a, Front-view and side-view of the yeast orthologue of CSB, Saccharomyces cerevisiae RAD26 (purple), bound to RNAPII (grey) (PDB: 5VVS). b, Schematic representation of the CRISPR/Cas9 screen in RPE1-iCas9 cells in the presence of Illudin S (IC60; 25 nM). c, Network analysis of highly significant hits representing genes that promote Illudin S toxicity. Grey lines reflect known protein-protein interactions (Cytoscape, BioGRID). d, Sanger sequencing of the indicated RPE1-iCas9 single ELOF1-KO clones. e, 72 h drug sensitivity assay of indicated RPE1-iCas9 KO clones. The experiment has been performed twice and each symbol represents the median of 6 technical replicates of an independent experiment. UT, untreated f, Clonogenic Illudin S survival of the indicated RPE1-iCas9 cells. The experiment has been performed three times (except for ELOF1-KO 2-12 where the experiment has been done twice) and each symbol represents the mean of 2 technical replicates of an independent experiment. Numerical data are provided in Source data extended data Fig. 1.

Source data

Extended Data Fig. 2 Human ELOF1 and yeast ELF1 show similar RNAPII-binding modes.

a, Sanger sequencing of the indicated U2OS (FRT) ELOF1-KO clone. b, Clonogenic Illudin S survival of U2OS (FRT) WT, CSB-KO, ELOF1-KO, and ELOF1-KO complemented with ELOF1WT-GFP. The experiment has been performed twice and each symbol represents the mean of 2 technical replicates of an independent experiment. c, Alignment of human ELOF1, Xenopus leavis ELOF1, and Saccharomyces cerevisiae ELF1. Conserved residues are indicated in yellow, zinc-finger cysteines in magenta, residues involved in the RPB1 or RPB2 interaction in green. Note that the C-terminus of Saccharomyces cerevisiae ELF1 (83–145) is absent in human ELOF1 and Xenopus leavis ELOF1. (d) Co-immunoprecipitation (IP) of ELOF1WT-GFP and ELOF1S72K/D73K-GFP on the combined soluble and chromatin fraction. Data shown represent 4 independent experiments. (e) Volcano plot depicting the statistical differences between 4 replicates of the MS analysis on ELOF1WT-GFP pull-down in mock treated and UV-irradiated samples. The fold change (log2) is plotted on the x-axis and the significance (t-test -log10(P value)) is plotted on the y-axis. RNAPII subunits are indicated in red, elongation factors are indicated in blue, GFP is indicated in green, and PAF1 subunits are indicated in purple. (f) Clonogenic Illudin S survival of U2OS (FRT) WT, ELOF1-KO, and TY1-tagged ELOF1 rescue cell lines. The experiment has been performed twice and each symbol represents the mean of 2 technical replicates of an independent experiment. Uncropped blots and numerical data are provided in Source data extended data Fig. 2.

Source data

Extended Data Fig. 3 ELOF1 promotes transcription elongation.

a, Calculated speed of RNAPII, quantified by wave-front analyses, in WT (red) or ELOF1-KO (blue) cells 30 min (n=3 experiments) or 60 min (n=2 experiments) after DRB release. Data represents the median per condition (black line) and median within individual replicates (black circles). b, Metaplots of BrU signal (nascent transcription) from 2 kb before the TSS to 2 kb after the TTS in 767 genes between 25–50 kb (upper), 562 genes between 50–100 kb (middle), or 400 genes of >100 kb in WT (red) or ELOF1-KO (blue) cells. Profiles are normalized to 100% at promoter-proximal BrU peaks instead of area under the curve for better comparison of transcription profiles. Profiles are averages of 2 independent replicates. c, Heatmaps of ATAC-seq data around the TSS (−5 kb until +5 kb) of 3,000 genes of 3–100 kb in unirradiated RPE1-iCas9 cells (WT or ELOF1-KO). Numerical data are provided in Source data extended data Fig. 3.

Source data

Extended Data Fig. 4 ELOF1 promotes genome-wide transcription recovery.

a, Quantification of 5-EU levels of the indicated RPE1-iCas9 cells normalized to mock-treated levels for each cell line. Cells were either mock-treated or UV-irradiated (3 h or 24 h; 12 J m-2). The experiment has been performed twice and each black circle represents the median of 2 technical replicates of an independent experiment, >80 cells collected per technical replicate. The black line represents the median of all the cells collected. b, Quantification of 5-EU levels normalized to the baseline level before DRB treatment for each condition. The experiment has been performed twice and each black circle represents the median of 2 technical replicates of an independent experiment, >80 cells collected per technical replicate. The black line represents the median of all the cells collected. c, Western blot analysis of RPE1-iCas9 ELOF1-KO cells complemented with GFP-tagged versions of ELOF1. Data shown represent 3 independent experiments. d, Metaplots of BrU signal (nascent transcription) in 767 genes between 25–50 kb, or in 561 genes between 50–100 kb in WT (upper) or ELOF1-KO (lower) cells after mock treatment (red), or 3 h (blue), 8 h (black), and 24 h (green) after UV irradiation (9 J m-2). Profiles are averages of 2 independent replicates. Uncropped blots and numerical data are provided in Source data extended data Fig. 4.

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Extended Data Fig. 5 Global gene-expression changes in response to UV irradiation.

Volcano plots of RNA-seq in the indicated RPE1-iCas9 cell lines depicting the downregulation (blue) or upregulation (red) of gene expression in response to UV irradiation (24 h; 9 J m-2). The fold change (log2) is plotted on the x-axis and the significance (-log10 P value) is plotted on the y-axis. a, WT, (b) CSB-KO, (c) ELOF1-KO. Only genes indicating at least 2 counts per million (CPM) in at least 33% of samples were included in the analysis. FDR-adjusted P values < 0.05 were considered significant. Two short UV-response genes (ATF3, CDKN1A) are highlighted. d, Box plot depicting the gene length of the 650 most significantly downregulated genes (in blue) or the 650 most significantly upregulated genes (in red). The horizontal line represents the median (center); Upper Bound: gene length scores larger than 75% of all data points; Lower Bound: gene lengths scores shorter than 75% of all data points. Points above and below the box represent the outliers.

Extended Data Fig. 6 Genome-wide redistribution of RNAPII in response to UV irradiation.

a, Heatmaps of pan-RNAPII ChIP-seq data around the TSS of 3,000 genes of 3–100 kb, ranked according to RNAPII signal in mock-treated WT cells. Heatmaps of the same genes are shown after UV irradiation (9 J m-2) in WT or ELOF1-KO cells. b, Averaged metaplots of pan-RNAPII ChIP-seq of 3,000 genes of 3–100 kb from the TSS until the TTS in the indicated RPE1-iCas9 cells after mock-treatment (red) or at 8 h (blue) after UV irradiation (9 J m-2). (c) As in b showing the area around the TTS (−2 kb until +2 kb).

Extended Data Fig. 7 Metaplots of TCR-seq using a Ser2-RNAPII antibody.

a, Individual metaplots (two replicates for each condition) of ser2-RNAPII TCR-seq of 3,000 genes for 3–100 kb from the TSS until the TTS (−5 kb and +5 kb, respectively) in the indicated RPE1-iCas9 cells after mock-treatment or at 1 h, 4 h, or 8 h after UV irradiation (9 J m-2). The coding (non-transcribed) strand in shown in red, while the template (transcribed) strand is shown in blue.

Extended Data Fig. 8 Histogram plots of TCR-seq using a Ser2-RNAPII antibody.

a, Frequency distribution plots of the gene-by-gene ser2-RNAPII strand-specificity index (SSI). SSIs below −0.1 or above 0.1 are presented in red. A unimodal distribution indicates no strand-bias (and thus no DNA damage in the template strand), while a trimodal distribution reflects a strand-bias caused by DNA damage in the template strand.

Extended Data Fig. 9 Validation of TCR-seq with a pan-RNAPII antibody.

a, Individual metaplots (two replicates for each condition) of pan-RNAPII TCR-seq of 3,000 genes of 3–100 kb from the TSS until the TTS (−5 kb and +5 kb, respectively) in the indicated RPE1-iCas9 cells after mock-treatment or at 1 h, 4 h, or 8 h after UV irradiation (9 J m-2). The coding (non-transcribed) strand in shown in red, while the template (transcribed) strand is shown in blue. b, Frequency distribution plots of the gene-by-gene pan-RNAPII strand-specificity index (SSI). SSIs below −0.1 or above 0.1 are presented in red. A unimodal distribution indicates no strand-bias (and thus no DNA damage in the template strand), while a trimodal distribution reflects a strand-bias caused by DNA damage in the template strand.

Extended Data Fig. 10 ELOF1 is not involved in global genome repair.

a-b, Unscheduled DNA synthesis (UDS) in the indicated RPE1-iCas9 cells following local UV irradiation (30 J m-2; 1 h). DNA damage was identified by CPD staining. a, Representative images (Scale bar, 10 µm) and (b) quantification of EdU levels normalized to WT cells. The experiment has been performed twice and each black circle represents the median of 2 technical replicates of an independent experiment, >80 cells collected per technical replicate. The black line represents the median of all the cells collected. c, Endogenous RNAPIIo Co-IP on U2OS (FRT) ELOF1-KO cells complemented with ELOF1WT-GFP after knockdown of CSB (siCSB) or as a control luciferase (siLUC). Data shown represent 2 independent experiments. d, Western blot analysis of CSA protein levels in the indicated RPE1-iCas9 cells after mock treatment, or 7 h, 24 h, and 48 h after UV irradiation (9 J m-2; n=2). e, Quantification of 5-EU levels of the indicated RPE1-iCas9 cells normalized to mock-treated levels for each cell line. Cells were either mock-treated or UV-irradiated (3 h or 24 h; 9 J m-2). 10 µM FT671 (USP7 inhibitor) was added to the indicated cells 24 h prior to UV irradiation. The experiment has been performed three times and each black circle represents the median of 2 technical replicates of an independent experiment, >50 cells collected per technical replicate. The black line represents the median of all the cells collected. f, Western blot analysis of CSA protein levels from e. g, GST pull-down of immobilized recombinant Xenopus laevis (xl) ELOF1 incubated with recombinant xlRAD23B. Data shown represent 2 independent experiments. h, In vitro ubiquitylation of recombinant xlELOF1 and xlCSB with recombinant xlCRL4CSA, E1, E2, ubiquitin, and ATP. In vitro ubiquitylation reactions were stopped at the indicated times. Data shown represent 3 independent experiments. (i) Representative images of staining with TY1 antibodies in U2OS (FRT) WT cells and ELOF1-KO cells complemented with TY1-tagged ELOF1. Data shown represent 2 independent experiments. j, Representative image of staining with TY1 and RBX1 antibodies at 1 h after local UV irradiation (50 J m-2) in U2OS (FRT) ELOF1-KO cells complemented with TY1-tagged ELOF1. Data shown represent 2 independent experiments. k, Results of mining a recent CRISPR screen repository27. Shown are the Z-scores for the indicated sgRNAs (targeting ELOF1 (blue), CSA (orange), CSB (green), UVSSA (black) after exposure to the indicated genotoxic agents. Uncropped blots and numerical data are provided in Source data extended data fig. 10.

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Supplementary information

Reporting Summary

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Supplementary Tables

Supplementary Table 1: Cell lines. Supplementary. Table 2: sgRNAs. Supplementary Table 3: Plasmids. Supplementary Table 4: Primers. Supplementary Table 5: Antibodies. Supplementary Table 6: Sequence depth. Supplementary Table 7: Read counts (no mismatches allowed) per pLCKO-TKOv3 guide for the four CRISPR screens presented. (A) General information for the screens. (B) Raw read counts of the CRISPR screens.

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van der Weegen, Y., de Lint, K., van den Heuvel, D. et al. ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation. Nat Cell Biol 23, 595–607 (2021). https://doi.org/10.1038/s41556-021-00688-9

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