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Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

A Publisher Correction to this article was published on 23 June 2021

This article has been updated

Abstract

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR–Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

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Fig. 1: Genome-wide CRISPR–Cas9 screen identifies ELOF1 as a factor involved in the UV-induced DNA-damage response.
Fig. 2: ELOF1 is part of the elongating Pol II complex.
Fig. 3: ELOF1 is important for functional TC-NER.
Fig. 4: ELOF1 is an evolutionarily conserved TC-NER factor.
Fig. 5: Prolonged stalling of Pol II at TBLs in the absence of ELOF1.
Fig. 6: ELOF1 is crucial for proper TC-NER complex assembly.
Fig. 7: ELOF1 has an additional function apart from TC-NER.
Fig. 8: ELOF1 is important for preventing genome instability.

Data availability

All the DRB/TTchem-seq data used in this study are available under GEO accession number GSE148844. All the CPD-seq data are available under GEO accession number GSE149082. The SILAC-based quantitative interaction proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD025304. Source data are provided with this paper. Any other data are available from the corresponding author upon reasonable request.

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References

  1. 1.

    Lans, H., Hoeijmakers, J. H. J., Vermeulen, W. & Marteijn, J. A. The DNA damage response to transcription stress. Nat. Rev. Mol. Cell Biol. 20, 766–784 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Gomez-Gonzalez, B. & Aguilera, A. Transcription-mediated replication hindrance: a major driver of genome instability. Genes Dev. 33, 1008–1026 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Gaillard, H. & Aguilera, A. Transcription as a threat to genome integrity. Annu. Rev. Biochem. 85, 291–317 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Laugel, V. Cockayne syndrome: the expanding clinical and mutational spectrum. Mech. Ageing Dev. 134, 161–170 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Xu, J. et al. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair. Nature 551, 653–657 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Groisman, R. et al. CSA-dependent degradation of CSB by the ubiquitin–proteasome pathway establishes a link between complementation factors of the Cockayne syndrome. Genes Dev. 20, 1429–1434 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    van der Weegen, Y. et al. The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II. Nat. Commun. 11, 2104 (2020).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Nakazawa, Y. et al. Ubiquitination of DNA damage-stalled RNAPII promotes transcription-coupled repair. Cell 180, 1228–1244.e24 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Tufegdzic Vidakovic, A. et al. Regulation of the RNAPII pool is integral to the DNA damage response. Cell 180, 1245–1261.e21 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Okuda, M., Nakazawa, Y., Guo, C., Ogi, T. & Nishimura, Y. Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways. Nucleic Acids Res. 45, 13043–13055 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Scharer, O. D. Nucleotide excision repair in eukaryotes. Cold Spring Harb. Perspect. Biol. 5, a012609 (2013).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Yang, W. & Gao, Y. Translesion and repair DNA polymerases: diverse structure and mechanism. Annu. Rev. Biochem. 87, 239–261 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Daniels, J. P., Kelly, S., Wickstead, B. & Gull, K. Identification of a crenarchaeal orthologue of Elf1: implications for chromatin and transcription in Archaea. Biol. Direct 4, 24 (2009).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Prather, D., Krogan, N. J., Emili, A., Greenblatt, J. F. & Winston, F. Identification and characterization of Elf1, a conserved transcription elongation factor in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 10122–10135 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Joo, Y. J., Ficarro, S. B., Chun, Y., Marto, J. A. & Buratowski, S. In vitro analysis of RNA polymerase II elongation complex dynamics. Genes Dev. 33, 578–589 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ehara, H. et al. Structure of the complete elongation complex of RNA polymerase II with basal factors. Science 357, 921–924 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Mayer, A. et al. Uniform transitions of the general RNA polymerase II transcription complex. Nat. Struct. Mol. Biol. 17, 1272–1278 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Ehara, H. et al. Structural insight into nucleosome transcription by RNA polymerase II with elongation factors. Science 363, 744–747 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Steurer, B. et al. Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair. Nucleic Acids Res. 47, 3536–3549 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Steurer, B. et al. Live-cell analysis of endogenous GFP–RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II. Proc. Natl Acad. Sci. USA 115, E4368–E4376 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    van den Boom, V. et al. DNA damage stabilizes interaction of CSB with the transcription elongation machinery. J. Cell Biol. 166, 27–36 (2004).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Gregersen, L. H., Mitter, R. & Svejstrup, J. Q. Using TTchem-seq for profiling nascent transcription and measuring transcript elongation. Nat. Protoc. 15, 604–627 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Nudler, E. RNA polymerase backtracking in gene regulation and genome instability. Cell 149, 1438–1445 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Jia, N. et al. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents. Nat. Protoc. 10, 12–24 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Wienholz, F., Vermeulen, W. & Marteijn, J. A. Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair. Nucleic Acids Res. 45, e68 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Jaspers, N. G. et al. Anti-tumour compounds illudin S and irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways. DNA Repair (Amst.) 1, 1027–1038 (2002).

    CAS  Google Scholar 

  29. 29.

    Slyskova, J. et al. Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage. Nucleic Acids Res. 46, 9537–9549 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Veloso, A. et al. Genome-wide transcriptional effects of the anti-cancer agent camptothecin. PLoS ONE 8, e78190 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Brooks, P. J. et al. The oxidative DNA lesion 8,5′-(S)-cyclo-2′-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells. J. Biol. Chem. 275, 22355–22362 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    van Gool, A. J. et al. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6. EMBO J. 13, 5361–5369 (1994).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Mao, P., Smerdon, M. J., Roberts, S. A. & Wyrick, J. J. Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution. Proc. Natl Acad. Sci. USA 113, 9057–9062 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Duan, M., Selvam, K., Wyrick, J. J. & Mao, P. Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin. Proc. Natl Acad. Sci. USA 117, 18608–18616 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Li, S. & Smerdon, M. J. Rpb4 and Rpb9 mediate subpathways of transcription-coupled DNA repair in Saccharomyces cerevisiae. EMBO J. 21, 5921–5929 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lans, H. et al. Involvement of global genome repair, transcription coupled repair, and chromatin remodeling in UV DNA damage response changes during development. PLoS Genet. 6, e1000941 (2010).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Williamson, L. et al. UV irradiation induces a non-coding RNA that functionally opposes the protein encoded by the same gene. Cell 168, 843–855 e813 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    van der Weegen, Y. et al. ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation. Nat. Cell Biol. https://doi.org/10.1038/s41556-021-00688-9 (2021).

  39. 39.

    Schwertman, P. et al. UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair. Nat. Genet. 44, 598–602 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    He, J., Zhu, Q., Wani, G., Sharma, N. & Wani, A. A. Valosin-containing protein (VCP)/p97 segregase mediates proteolytic processing of Cockayne syndrome group B (CSB) in damaged chromatin. J. Biol. Chem. 291, 7396–7408 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Lukas, C. et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 13, 243–253 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Schoonen, P. M. et al. Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells. Nat. Commun. 8, 15981 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Olivieri, M. et al. A genetic map of the response to DNA damage in human cells. Cell 182, 481–496.e21 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Tellier, A. P., Archambault, D., Tremblay, K. D. & Mager, J. The elongation factor Elof1 is required for mammalian gastrulation. PLoS ONE 14, e0219410 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Wienholz, F. et al. FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER. Nucleic Acids Res. 47, 4011–5025 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Fei, J. & Chen, J. KIAA1530 protein is recruited by Cockayne syndrome complementation group protein A (CSA) to participate in transcription-coupled repair (TCR). J. Biol. Chem. 287, 35118–35126 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Evers, B. et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Nilson, K. A. et al. THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing. Mol. Cell 59, 576–587 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Chao, S. H. et al. Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J. Biol. Chem. 275, 28345–28348 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Weiner, A. et al. High-resolution chromatin dynamics during a yeast stress response. Mol. Cell 58, 371–386 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Yesbolatova, A., Natsume, T., Hayashi, K.-I. & Kanemaki, M. T. Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. Methods 164–165, 73–80 (2019).

    PubMed  PubMed Central  Google Scholar 

  54. 54.

    Natsume, T., Kiyomitsu, T., Saga, Y. & Kanemaki, M. T. Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15, 210–218 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔct method. Methods 25, 402–408 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Gardner, J. M. & Jaspersen, S. L. Manipulating the yeast genome: deletion, mutation, and tagging by PCR. Methods Mol. Biol. 1205, 45–78 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).

    CAS  PubMed  Google Scholar 

  61. 61.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Mao, P., Smerdon, M. J., Roberts, S. A. & Wyrick, J. J. Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer. Genome Res. 30, 12–21 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Park, D., Morris, A. R., Battenhouse, A. & Iyer, V. R. Simultaneous mapping of transcript ends at single-nucleotide resolution and identification of widespread promoter-associated non-coding RNA governed by TATA elements. Nucleic Acids Res. 42, 3736–3749 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Saldanha, A. J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Holstege, F. C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Mao, P. et al. Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity. Genome Res. 27, 1674–1684 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Hodges, A. J., Plummer, D. A. & Wyrick, J. J. NuA4 acetyltransferase is required for efficient nucleotide excision repair in yeast. DNA Repair (Amst.) 73, 91–98 (2019).

    CAS  Google Scholar 

  69. 69.

    Li, M., Ko, T. & Li, S. High-resolution digital mapping of N-methylpurines in human cells reveals modulation of their induction and repair by nearest-neighbor nucleotides. J. Biol. Chem. 290, 23148–23161 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Mukherjee, C. et al. RIF1 promotes replication fork protection and efficient restart to maintain genome stability. Nat. Commun. 10, 3287 (2019).

    PubMed  PubMed Central  Google Scholar 

  71. 71.

    Callen, E. et al. ATM prevents the persistence and propagation of chromosome breaks in lymphocytes. Cell 130, 63–75 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Cornacchia, D. et al. Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells. EMBO J. 31, 3678–3690 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    van Cuijk, L. et al. SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair. Nat. Commun. 6, 7499 (2015).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Optical Imaging Center and the Proteomics Center of the Erasmus Medical Center for support with microscopes and mass spectrometry analysis. We thank the Advanced Sequencing Facility of the Francis Crick Institute for technical assistance with DRB/TTchem-seq. We acknowledge infrastructural support from the Josephine Nefkens Precision Cancer Treatment Program. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by a grant from the Dutch Cancer Society (KWF grant 10506). This work was further funded by the Dutch organization for Scientific Research (NWO-ALW), which awarded a VIDI (864.13.004) and VICI (VI.C.182.025) grant to J.A.M. A.R.C. is supported by the Dutch Cancer Society (KWF grant 11008) and NWO VIDI (193.131). S.L. is funded by the National Science Foundation (MCB-1615550). J.J.W. is funded by the National Institute of Environmental Health Sciences (grants R01ES028698, R21ES029655 and R21ES029302). M.A.T.M.v.V. is funded by a grant from the European Research Council (ERC CoS grant 682421). H.L. is funded by The Netherlands Organization for Scientific Research (project number 711.018.007) and Cancergenomics.nl. W.V. was funded by a grant from the European Research Council (agreement 340988). J.Q.S. was supported by the Francis Crick Institute (which receives funding from Cancer Research UK (FC001166), the UK Medical Research Council (FC001166) and the Wellcome Trust (FC001166)) and by a grant from the European Research Council (agreement 693327).

Author information

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Authors

Contributions

M.E.G. performed the majority of the experiments and generated the ELOF1 and CSB KO cell lines and the ELOF1 and RPB1 KI cell lines. D.Z. generated the CSB and UVSSA KI cells and performed live-cell imaging experiments and the TCR UDS. K.S., D.A.P., W.G., S.L. and J.J.W. performed and supervised all the S. cerevisiae experiments. B.S. and M.E.G. performed the CRISPR–Cas9 screen, and B.E. and R.B. analysed and supervised the screen. C.M., C.L. and A.R.C. performed and supervised the metaphase spread, DNA fibre analysis and 53BP1 cell cycle analysis. S.C., R.M. and J.Q.S. performed and supervised the DRB/TTchem-seq. M.v.T. performed the AlamarBlue cell-viability assay. M.v.d.W. and H.L. performed and supervised the C. elegans experiments. R.C.J. provided experimental support. Y.P.K. performed the EdU and FANCD2 foci analysis, supervised by M.A.T.M.v.V. J.H.G.L. generated images of the Pol II structure. B.G. performed the Monte-Carlo-based modelling and was supervised by A.B.H. K.B. and J.A.A.D. performed and supervised the mass spectrometry analysis. A.R. performed the UDS experiments and A.F.T. performed FACS sorting, both supervised by W.V. J.A.M. conceived and supervised the project and together with M.E.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Jurgen A. Marteijn.

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Extended data

Extended Data Fig. 1 Generation of ELOF1 knock-in and knock-out cells.

a, Brightfield images of MRC-5 cells irradiated with indicated doses of UV-C for 10 consecutive days. Images were taken every other day. Scale bar: 60 µm. b, Top 10 enriched GO terms (biological process) identified using g:Profiler of UV-sensitive genes with FDR<0.1 (n = 49). c, Schematic of the genomic ELOF1 locus. Scissors indicate target regions of the sgRNAs used to generated ELOF1 KO (−/−) cells, half arrows indicate primers used for genotyping as shown in (c). Red arrows indicate location of the qPCR primers as shown in (e). d,e, Genotyping of ELOF1 KO (−/−) cells, both originating from a single cell clone. d, Genotyping PCR of loss of exon 2 in ELOF1 −/−A cells. Assays in d, i, j and g have been performed two times with similar results. e, Top panel: Sequencing results showing frameshift mutations in the targeted genomic locus of ELOF1 −/−B. Bottom panel: Amino acid sequence of ELOF1 in ELOF1 −/−B cells. f, Relative ELOF1 levels in indicated HCT116 Wt and ELOF1 KO (−/−) cells, with ELOF1 re-expression where indicated, as determined by RT-qPCR. Relative ELOF1 mRNA expression was normalized to GAPDH signal and levels in Wt cells were set to 1. ND=not detected. Data shown in represent average ± SEM (n = 3 independent experiments. g, Immunoblot of indicated HCT116 cell lines showing CSB or ELOF1-GFP expression. Tubulin was used as loading control. h, Relative ELOF1 levels in HCT116 cells transfected with indicated siRNAs as determined by RT-qPCR. Relative ELOF1 expression was normalized to GAPDH signal and siCTRL levels were set to 1. Data shown represent average from 2 independent experiments. i, Immunoblot showing endogenous ELOF1 and XPF levels in ELOF1-mScarletI-HA KI cells (Extended Data Fig. 2a) transfected with indicated siRNAs. Tubulin was used as loading control. j, Immunoblot showing expression of Flag-tagged Wt or indicated ELOF1 mutants in HCT116 ELOF1 −/−A cells. k, Relative colony survival of CPD photolyase cells transfected with indicated siRNAs. PR indicates CPD removal by photoreactivation. Plotted curves represent averages of 2 independent experiments. Numerical data and uncropped blots are provided in source data.

Source data

Extended Data Fig. 2 ELOF1 is part of the Pol II complex.

a, Left panel: Schematic of the genomic locus of ELOF1 for generating ELOF1-mScarletI-HA KI cell line. Half arrows indicate primer locations. Right panel: Genotyping PCR and immunoblot for ELOF1-KI cell line (top). LaminB1 was used as loading control (bottom). b, Immunoblot of HCT116 GFP-RPB1 KI. Tubulin was used as loading control. Immunoblots have been performed two times with similar results. c, Histograms showing intensities of GFP and mScarletI measured over the indicated dotted line in HCT116 double KI cells. d, Native immunoprecipitation of P-Ser2-modified Pol II in HCT116 cells followed by immunoblotting for indicated proteins. Cells were harvested 1 hour after mock treatement or irradiation with 16 J/m2 UV-C. IgG was used as binding control. IP has been performed two times with similar results. e, Interaction heat map based on the SILAC ratios of MRC-5 GFP-RPB1-interacting proteins as determined by quantitative interaction proteomics. Average SILAC ratios of duplicate experiments are plotted and represent RPB1-interactors relative to empty beads. SILAC ratio >1 indicates increase in interaction. * indicates proteins quantified in one experiment. f, Top 10 enriched GO terms (biological processes) identified using g:Profiler of 55 proteins identified as ELOF1 interactor with an average SILAC ratio of 2.5 or higher. Uncropped blots are provided in source data.

Extended Data Fig. 3 ELOF1 stimulates transcription elongation.

a, Browser tracks from DRB/TTchem-seq experiment at ATM, DAP and SCAF4. Results are shown 10, 20, 30 or 40 minutes after DRB release. b, Metagene profiles of DRB/TTchem-seq in HCT116 Wt or indicated KO (−/−) cells, with ELOF1 re-expression where indicated, 10, 20, 30, or 40 minutes after DRB release for 50-100Kb long genes. c, Transcription levels as determined by relative EU incorporation in HCT116 cells transfected with indicated siRNAs. Red lines indicate average integrated density ± SEM. siCTRL n = 247, siSPT4 n = 272, siSPT5 n = 288, siCSB n = 286, siELOF1 #1 n = 285 cells analyzed across 3 (siELOF1 and siCSB) and 4 (siCTRL) independent experiments. d, Representative images of EU incorporation in HCT116 cells transfected with indicated siRNAs. Scale bar: 20 µm. e, Immunoblot for indicated proteins in HCT116 cells transfected with indicated siRNAs. Tubulin was used as loading control. Experiment has has been performed two times with similar results. f, Images of HCT116 Wt and ELOF1 −/−A cells transfected with indicated siRNAs, stained with coomassie blue 10 days after transfection. Cell growth experiment has been executed two times with similar results. Numerical data and uncropped blots are provided in source data.

Source data

Extended Data Fig. 4 Role of ELOF1 during TC-NER and protection against different DNA damaging agents.

a,b, Representative immunofluorescence images of EU incorporation in (a) indicated HCT116 cells, or b, HCT116 cells transfected with indicated siRNAs, 2 or 18 hours after 8 J/m2 UV-C or mock treatment (NT). Scale bar: 20 µm. c, Transcription restart after UV damage as determined by relative EU incorporation in HCT116 cells transfected with indicated siRNAs, 2 or 18 hours after 8 J/m2 UV-C or mock treatment (NT). Relative integrated density of UV-irradiated samples are normalized to mock-treated and set to 100%. Red lines indicate average integrated density of, respectively n = 492, 485, 435, 487, 461, 395, 352, 399, 380, 644, 575, 512 cells collected from three independent experiments. d, Representative immunofluorescence images of amplified EdU signal in XP186LV fibroblasts (XP-C) transfected with indicated siRNAs, 7 hours after exposure to 8 J/m2 UV-C. Scale bar: 20 µm. TCR-UDS has been performed three times with similar results. e, Relative ELOF1 mRNA levels in XP186LV fibroblasts (XP-C) following transfection with indicated siRNAs determined by RT-qPCR. ELOF1 expression was normalized to GAPDH expression and siCTRL levels were set to 1. Data shown represent average of 2 independent experiments. f, Representative fluorescence images of EdU incorporation 3 hours after irradiation with 16 J/m2 UV-C in C5RO (hTert) cells transfected with indicated siRNAs. Scale bar: 20 µm. (g) as in (f) in C5RO (hTert) cells. Data shown represent average of 2 independent experiments. h–k, Relative colony survival of indicated HCT116 Wt and KO (−/−) cells, with ELOF1 re-expression where indicated, continuously exposed to indicated concentrations of (h) camptothecin (CPT) or (i) potassium bromate (KBrO3), or irradiated with indicated doses of (j) ionizing radiation (IR), or exposed to indicated concentrations of (k) hydroxyurea (HU) or (l) aphidicolin. Plotted curves represent averages ± SEM. CPT: n = 2; KBrO3: n = 3; IR: n = 5; HU: n = 3, others n = 4 independent experiments. m, Relative colony survival of HCT116 cells transfected with indicated siRNAs following exposure to indicated doses of UV-C. Plotted curves represent averages ± SEM. siCTRL, siSPT4 and siSPT5 n = 4, siELOF1 #1, siCSB, siXPF n = 2 independent experiments. *P ≤ 0.05, ****p ≤ 0.0001 analyzed by two-sided unpaired T-test in (c) and one-sided unpaired T-test in (h-m). Numerical data are provided in source data.

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Extended Data Fig. 5 Role of yeast elf1 in TC-NER.

a, Indicated mutant yeast strains were serially tenfold diluted, spotted, and exposed to indicated UV-C doses. Spot assay has been performed three times with similar results. b, Schematic showing the CPD-seq method. Isolated DNA is sonicated and adaptors are ligated. CPDs are cleaved by T4 endonuclease V and APE1 nuclease to generate 3’ ends. Following denaturing of the DNA, ends are ligated to a second adaptor that allows CPD sequencing. c, Gene plot analysis of CPD-seq data for ~4500 yeast genes, ordered by transcription frequency66. Plots depict unrepaired CPDs following 2-hour repair relative to no repair for both the transcribed strand (TS) and non-transcribed strand (NTS). Each row represents approximately 10 genes. TSS=transcription start site, TTS=transcription termination site. d, Left panel: Representative gel of bulk repair of UV-induced CPD lesions in Wt and elf1∆ mutant yeast measured by T4 endonuclease V digestion and alkaline gel electrophoresis of genomic DNA isolated from UV-irradiated yeast (100 J/m2 UV-C light) after the indicated time. Right panel: Quantification of CPD repair from n = 3 WT and n = 4 elf1∆ experiments ±SEM. *P ≤ 0.05 analyzed by unpaired two-sided t-test. e, Single nucleotide resolution analysis of CPD-seq data downstream of the TSS of ~5200 yeast genes. Plots depict fraction of unrepaired CPDs following 2-hour repair relative to no repair for both TS and NTS. Nucleosome positioning data51 is shown for reference. f, Controls for UV spotting assays shown in Fig. 4d. g, Image showing repair of CPDs in the TS of the RPB2 gene for indicated yeast strains. The image was generated by converting sequencing reads aligned to RPB2 into bands. U: unirradiated cells. Nucleotide positions relative to TSS (+1) are indicated on the left. h, Left: Relative percentage of CPDs remaining within 54 bp downstream of the TSS of the RPB2 gene. Right: Relative percentage of CPDs remaining in the downstream region (69-353 bp) of the RPB2 gene. Data are presented as mean values from all CPD sites within the indicated regions (0-54 and 69-353 bp) of the RPB2 gene ±SD from one single experiment, error bars are shown for most relevant strains. n = 8 sites (left panel), and n = 73 (right panel). i, Representation of the C. elegans elof-1 genomic organization, depicting the 180 bp emc203 deletion allele generated with CRISPR-Cas9. Shaded boxes: exons, black: coding sequences. Numerical data are provided in source data .

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Extended Data Fig. 6 Effects of ELOF1 deficiency on Pol II elongation speed and complex composition.

a, Relative ELOF1 mRNA levels in GFP-RPB1 KI cells transfected with indicated siRNAs as determined by RT-qPCR. ELOF1 expression was normalized to GAPDH signal and levels of control cells were set to 1. Data shown represent average of 2 independent experiments. b, FRAP analysis of GFP-RPB1 mobility after depletion of indicated factors. Mock-treated curves corresponding to Fig. 4a. siCTRL n = 28, siELOF1 #1 n = 20, siCSB NT n = 14 cells analyzed across 4, 3 and 3 independent experiments respectively. c, Left panel: Residence time of elongating Pol II or right panel: relative fraction size of promoter-bound or elongating Pol II as determined by Monte-Carlo-based modeling of RPB1 mobility as shown in (a). d, Native immunoprecipitation of Pol II in Wt and ELOF −/−A cells followed by immunoblotting for indicated proteins. Cells were harvested 1 hour after mock treatment or irradiation with 16 J/m2 UV-C. MG132: treatment with 50 µM proteasome inhibitor MG132, 1 hour before UV irradiation. e, Native immunoprecipitation of Pol II in Wt and ELOF −/−A cells followed by immunoblotting for indicated proteins. Cells were harvested 1 hour after mock treatment or irradiation with 16 J/m2 UV-C. IP experiments depicted in d and e were executed two times with similar results. Numerical data and uncropped blots are provided in source data.

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Extended Data Fig. 7 ELOF1 KO impairs recruitment of UVSSA but not CSB.

a, Left panel: Schematic of the genomic locus of CSB and used strategy for generating the homozygous CSB-mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for CSB-KI cell line. b, Left panel: Schematic of the genomic locus of UVSSA and used strategy for generating the homozygous UVSSA- mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for UVSSA-KI cell line. Experiments depicted in a and b were performed two times with similar results. c, Left panel: CSB mobility was determined by FRAP analysis of CSB-mScarletI after the indicated treatments. THZ1: 1 hour treatment (2 µM) before UV-C irradiation (4 J/m2) or mock treatment. Right panel: Relative immobile fraction of CSB as determined by FRAP analysis. Plotted values represent mean ± SEM and are normalized to mock treated. NT n = 32; UV n = 28; THZ1 n = 15; THZ1+UV n = 18 cells analyzed across 2 independent experiments. d, Same as C but for UVSSA-mScarletI. NT n = 10; UV n = 16; THZ1 n = 16; THZ1+UV n = 17 cells analyzed across 2 independent experiments. e,f, FRAP analyses of CSB-mScarletI (e) or UVSSA-mScarletI (f) mobility after transfection with indicated siRNAs in individual graphs. Cells were mock treated (NT) or analyzed directly (UV) or 5 hours (5hr UV) after irradiation with 4 J/m2 UV-C. g, Relative fluorescence intensity of UVSSA in UVSSA-KI cells transfected with indicated siRNAs as determined by live-cell imaging. Plotted values represent mean ± SEM. siCTRL NT n = 30, UV+5h UV n = 21; siELOF1 #1 NT n = 38, UV n = 34, 5h UV n = 16; siELOF1 #2 NT+UV n = 19, 5h UV n = 16 cells analyzed across 4 independent experiments for siCTRL and 3 for siELOF1 and siCSB. h, FRAP analysis of CSB in CSB-KI cells transfected with indicated siRNAs 2 hours after UV. VCPi: VCP inhibitor (5 µM) was directly added after UV-C (4 J/m2). i, Immunoblot of chromatin fraction of indicated cell lines 1 hour after 12 J/m2 UV-C or mock treatment. NAEi = 1 hour treatment with NEDDylation inhibitor (10 µM). SSRP1 is shown as loading control. j, Immunoblot of chromatin fraction of HCT116 cells transfected with indicated siRNAs 1 hour after 12 J/m2 UV-C or mock treatment. SSRP1 is shown as loading control. Immunoblots depicted in i and j were executed two times with similar results. Numerical data and uncropped blots are provided in source data.

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Extended Data Fig. 8 The additional role of ELOF1 in preventing transcription-mediated replication hindrance.

a, Left panel: Representative immunofluorescence images of EU incorporation in indicated HCT116 cells, untreated, or 2 or 22 hours after a 2-hour exposure to 10 µg/ml mitomycin C. Scale bar: 20 µm. Right panel: Transcription restart after mitomycin C as determined by relative EU incorporation in the indicated HCT116 cells. Mitomycin C-treated samples are normalized to mock treated levels and set to 100%. Red lines indicate average integrated density ± SEM of, respectively, n = 1008, 1008, 727, 938, 960, 715, 1133, 1162, 784, 884, 616, 461, 978, 1013, 693, 221, 220, 206 cells collected from four independent experiments. b, Relative colony survival of indicated cell lines with siRNA transfection following exposure to indicated doses of UV-C. Plotted curves represent averages ± SEM, n = 3 independent experiments. c, Percentage of RNA synthesis in untreated HCT116 cells and cells treated with 0.1 µM flavopiridol for 2 hours as determined by FACS-based quantification of EU pulse labeling. Experiment has been performed two times with similar results. d, Fork progression measured by tract lengths of CldU (red) in μm is depicted for indicated HCT116 cells, untreated or after 15 minutes 0.1 µM flavopiridol treatment. Tracts of respectively n = 300, 300, 304 cells collected from two independent experiments. e, As (d) but after treatment for 1 hour with 25 nM MMC. Tracts of, respectively n = 406, 422, 408, 406 cells collected from two independent experiments. f, Representative immunofluorescence images of 53BP1 foci in indicated HCT116 cells, untreated or 6, 24 or 48 hours after exposure to 8 J/m2 UV-C. Scale bar: 20 µm. g, Number of 53BP1 foci as determined in (f) quantified by Cellprofiler and plotted against normalized integrated intensity of DAPI, respectively, n = 10494, 7870, 13916, 16647, 9539, 8313, 8610, 8817, 11253, 10950, 10314, 10494 cells collected from two independent experiments. h, Number of FANCD2 foci per mitosis in prometaphase (left) or anaphase (right) in indicated HCT116 cells in untreated conditions or 48 hours after 4J/m2 UV-C. n = 90 cells collected from 3 independent experiments for all conditions. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001 analyzed by two-sided unpaired T-test (a), one-sided unpaired T-test (b), Kruskal-Wallis test for multiple comparisons (d,e), and two-sided unpaired t-test (Mann-Whitney) (h). Numerical data are provided in source data.

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Extended Data Fig. 9 Pol II-ELOF1 complex structure together with CSB.

a, S.cerevisiae Pol II (5vvr.pdb) with Rpb1 in green, Rpb2 in cyan, DNA in orange and Rad26 (CSB) in pink. The P.pastoris Pol II in complex with elongation factors (5xog.pdb) was superimposed onto this structure (Rpb1 subunits aligned onto each other), and all subunits except Elf1 (ELOF1; purple) were omitted for clarity. Conserved lysine K1246 (K1268 in mammalian Pol II) is indicated in dark red. b, Close up of Elf1 (ELOF1) binding region.

Supplementary information

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Supplementary Table 1

Table showing negatively regulated genes from the CRISPR–Cas9 screen resulting from MaGecK analysis of the change in abundance of sgRNAs in UV-treated over mock-treated samples.

Supplementary Table 2

Table with SILAC ratios and peptide numbers as determined using quantitative interaction proteomics. Each tab represents a different experiment as indicated.

Supplementary Table 3

Table with information on used antibodies.

Supplementary Table 4

Table with primer information.

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Geijer, M.E., Zhou, D., Selvam, K. et al. Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability. Nat Cell Biol 23, 608–619 (2021). https://doi.org/10.1038/s41556-021-00692-z

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