Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1

Nature Protocols volume 14, pages 1661–1685 (2019)Cite this article

Subjects

Abstract

Nascent RNA may form a three-stranded structure with DNA, called an R-loop, which has been linked to fundamental biological processes such as transcription, replication and genome instability. Here, we provide a detailed protocol for a newly developed strategy, named R-ChIP, for robust capture of R-loops genome-wide. Distinct from R-loop-mapping methods based on the monoclonal antibody S9.6, which recognizes RNA–DNA hybrid structures, R-ChIP involves expression of an exogenous catalytically inactive RNASEH1 in cells to bind RNA–DNA hybrids but not resolve them. This is followed by chromatin immunoprecipitation (ChIP) of the tagged RNASEH1 and construction of a strand-specific library for deep sequencing. It takes ~3 weeks to establish a stable cell line expressing the mutant enzyme and 5 more days to proceed with the R-ChIP protocol. In principle, R-ChIP is applicable to both cell lines and animals, as long as the catalytically inactive RNASEH1 can be expressed to study the dynamics of R-loop formation and resolution, as well as its impact on the functionality of the genome. In our recent studies with R-ChIP, we showed an intimate spatiotemporal relationship between R-loops and RNA polymerase II pausing/pause release, as well as linking augmented R-loop formation to DNA damage response induced by driver mutations of key splicing factors associated with myelodysplastic syndrome (MDS).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the R-ChIP workflow.
Fig. 2: Comparison of R-ChIP and S9.6-based methods.
Fig. 3: Expected results of R-ChIP.

Similar content being viewed by others

Data availability

R-ChIP data for HEK293T and K562 cell lines are available at the NCBI GEO repository under the accession number GSE97072. All public datasets used in this study are indicated in the corresponding figure legends.

References

  1. Aguilera, A. & Garcia-Muse, T. R loops: from transcription byproducts to threats to genome stability. Mol. Cell 46, 115–124 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Sanz, L. A. et al. Prevalent, dynamic, and conserved R-loop structures associate with specific epigenomic signatures in mammals. Mol. Cell 63, 167–178 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wahba, L., Costantino, L., Tan, F. J., Zimmer, A. & Koshland, D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation. Genes Dev. 30, 1327–1338 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chen, L. et al. R-ChIP using inactive RNase H reveals dynamic coupling of R-loops with transcriptional pausing at gene promoters. Mol. Cell 68, 745–757 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pefanis, E. et al. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161, 774–789 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Graf, M. et al. Telomere length determines TERRA and R-loop regulation through the cell cycle. Cell 170, 72–85 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Kabeche, L., Nguyen, H. D., Buisson, R. & Zou, L. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation. Science 359, 108–114 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I. & Chedin, F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814–825 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sugimoto, N. et al. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211–11216 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Duquette, M. L., Handa, P., Vincent, J. A., Taylor, A. F. & Maizels, N. Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev. 18, 1618–1629 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Roy, D., Yu, K. & Lieber, M. R. Mechanism of R-loop formation at immunoglobulin class switch sequences. Mol. Cell. Biol. 28, 50–60 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Roy, D. & Lieber, M. R. G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol. Cell. Biol. 29, 3124–3133 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Roy, D., Zhang, Z., Lu, Z., Hsieh, C. L. & Lieber, M. R. Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site. Mol. Cell. Biol. 30, 146–159 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Chedin, F. Nascent connections: R-loops and chromatin patterning. Trends Genet. 32, 828–838 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Stork, C. T. et al. Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage. Elife 5, e17548 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T. & Cimprich, K. A. Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses. Cell 170, 774–786 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, X. & Manley, J. L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122, 365–378 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Garcia-Benitez, F., Gaillard, H. & Aguilera, A. Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability. Proc. Natl. Acad. Sci. USA 114, 10942–10947 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huertas, P. & Aguilera, A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol. Cell 12, 711–721 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. El Hage, A., French, S. L., Beyer, A. L. & Tollervey, D. Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev. 24, 1546–1558 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chang, E. Y. & Stirling, P. C. Replication fork protection factors controlling R-loop bypass and suppression. Genes (Basel) 8, 33 (2017).

    Article  Google Scholar 

  22. Taneja, N. et al. SNF2 family protein Fft3 suppresses nucleosome turnover to promote epigenetic inheritance and proper replication. Mol. Cell 66, 50–62 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chakraborty, P. & Grosse, F. Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes. DNA Repair (Amst.) 10, 654–665 (2011).

    Article  CAS  Google Scholar 

  24. Cristini, A., Groh, M., Kristiansen, M. S. & Gromak, N. RNA/DNA hybrid interactome identifies DXH9 as a molecular player in transcriptional termination and R-loop-associated DNA damage. Cell Rep. 23, 1891–1905 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Skourti-Stathaki, K., Proudfoot, N. J. & Gromak, N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol. Cell 42, 794–805 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Itoh, T. & Tomizawa, J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc. Natl. Acad. Sci. USA 77, 2450–2454 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Xu, B. & Clayton, D. A. RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers. EMBO J. 15, 3135–3143 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sun, Q., Csorba, T., Skourti-Stathaki, K., Proudfoot, N. J. & Dean, C. R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus. Science 340, 619–621 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Boque-Sastre, R. et al. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc. Natl. Acad. Sci. USA 112, 5785–5790 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ohle, C. et al. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell 167, 1001–1013 (2016).

    Article  CAS  PubMed  Google Scholar 

  31. Belotserkovskii, B. P., Soo Shin, J. H. & Hanawalt, P. C. Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter. Nucleic Acids Res. 45, 6589–6599 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gan, W. et al. R-loop-mediated genomic instability is caused by impairment of replication fork progression. Genes Dev. 25, 2041–2056 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, L. et al. The augmented R-loop is a unifying mechanism for myelodysplastic syndromes induced by high-risk splicing factor mutations. Mol. Cell 69, 412–425.e6 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Costantino, L. & Koshland, D. The Yin and Yang of R-loop biology. Curr. Opin. Cell Biol. 34, 39–45 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Richard, P. & Manley, J. L. R loops and links to human disease. J. Mol. Biol. 429, 3168–3180 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Powell, W. T. et al. R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation. Proc. Natl. Acad. Sci. USA 110, 13938–13943 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nadel, J. et al. RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships. Epigenetics Chromatin 8, 46 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Xu, W. et al. The R-loop is a common chromatin feature of the Arabidopsis genome. Nat. Plants 3, 704–714 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Cerritelli, S. M. & Crouch, R. J. Cloning, expression, and mapping of ribonucleases H of human and mouse related to bacterial RNase HI. Genomics 53, 300–307 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E. & Lieber, M. R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Chen, P. B., Chen, H. V., Acharya, D., Rando, O. J. & Fazzio, T. G. R loops regulate promoter-proximal chromatin architecture and cellular differentiation. Nat. Struct. Mol. Biol. 22, 999–1007 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dumelie, J. G. & Jaffrey, S. R. Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq. Elife 6, e28306 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Halasz, L. et al. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases. Genome Res. 27, 1063–1073 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vanoosthuyse, V. Strengths and weaknesses of the current strategies to map and characterize R-loops. Noncoding RNA 4, E9 (2018).

    PubMed  Google Scholar 

  45. Konig, F., Schubert, T. & Langst, G. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences. PLoS ONE 12, e0178875 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Hartono, S. R. et al. The affinity of the S9.6 antibody for double-stranded RNAs impacts the accurate mapping of R-loops in fission yeast. J. Mol. Biol. 430, 272–284 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Phillips, D. D. et al. The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6. J. Mol. Recognit. 26, 376–381 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li, X. et al. GRID-seq reveals the global RNA-chromatin interactome. Nat. Biotechnol. 35, 940–950 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bell, J. C. et al. Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts. Elife 7, e27024 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Schmitz, K. M., Mayer, C., Postepska, A. & Grummt, I. Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev 24, 2264–2269 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bacolla, A., Wang, G. & Vasquez, K. M. New perspectives on DNA and RNA triplexes as effectors of biological activity. PLoS Genet. 11, e1005696 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Mondal, T. et al. MEG3 long noncoding RNA regulates the TGF-beta pathway genes through formation of RNA-DNA triplex structures. Nat. Commun. 6, 7743 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. O’Leary, V. B. et al. PARTICLE, a triplex-forming long ncRNA, regulates locus-specific methylation in response to low-dose irradiation. Cell Rep. 11, 474–485 (2015).

    Article  PubMed  Google Scholar 

  54. Postepska-Igielska, A. et al. LncRNA Khps1 regulates expression of the proto-oncogene SPHK1 via triplex-mediated changes in chromatin structure. Mol. Cell 60, 626–636 (2015).

    Article  CAS  PubMed  Google Scholar 

  55. Li, Y., Syed, J. & Sugiyama, H. RNA-DNA triplex formation by long noncoding RNAs. Cell Chem. Biol. 23, 1325–1333 (2016).

    Article  CAS  PubMed  Google Scholar 

  56. Nowotny, M. et al. Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD. EMBO J. 27, 1172–1181 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Nguyen, H. D. et al. Functions of replication protein A as a sensor of R loops and a regulator of RNaseH1. Mol. Cell 65, 832–847 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Bhatia, V. et al. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature 511, 362–365 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Legros, P., Malapert, A., Niinuma, S., Bernard, P. & Vanoosthuyse, V. RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes. PLoS Genet. 10, e1004794 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Tresini, M. et al. The core spliceosome as target and effector of non-canonical ATM signalling. Nature 523, 53–58 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Rhee, H. S. & Pugh, B. F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147, 1408–1419 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Rotem, A. et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Li, Q., Brown, J. B., Huang, H. & Bickel, P. J. Measuring reproducibility of high-throughput experiments. Ann. Appl. Stat. 5, 1752–1779 (2011).

    Article  Google Scholar 

  65. Landt, S. G. et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22, 1813–1831 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Core, L. J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to members of the Fu lab for cooperation, reagent sharing and insightful discussion that improved this work. We thank T. Hishida of the J.C. Izpisua Belmonte lab (Gene Expression Laboratories, Salk Institute for Biological Studies) for providing the pPyCAG plasmid and W. Li for critical comments on the manuscript. This work was supported by NIH grants (GM049369, GM052872, HG004659 and DK098808) to X.-D.F. and Start-up funds of Wuhan University (1304/413100052 and 1304/413100072) to L.C. We thank S. Dowdy (University of California, San Diego) for providing HEK293T cells

Author information

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA

    Jia-Yu Chen, Xuan Zhang, Xiang-Dong Fu & Liang Chen

  2. Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of China

    Liang Chen

Authors
  1. Jia-Yu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang-Dong Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.C. and X.-D.F. conceived the idea; L.C. and J.-Y.C. codeveloped the R-ChIP method and data analysis pipeline; L.C. and X.Z. performed the experiments. J.-Y.C. performed bioinformatics analysis with assistance from L.C.; L.C., J.-Y.C. and X.-D.F. wrote the manuscript.

Corresponding authors

Correspondence to Xiang-Dong Fu or Liang Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Chen, L. et al. Mol. Cell 68, 745–757 (2017): https://doi.org/10.1016/j.molcel.2017.10.008

Chen, L. et al. Mol. Cell 69, 412–425.e6 (2018): https://doi.org/10.1016/j.molcel.2017.12.029

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, JY., Zhang, X., Fu, XD. et al. R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1. Nat Protoc 14, 1661–1685 (2019). https://doi.org/10.1038/s41596-019-0154-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0154-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research