Article

The R-loop is a common chromatin feature of the Arabidopsis genome

Received:
Accepted:
Published online:

Abstract

R-loops are functional structures in chromatin comprising one single-stranded DNA and a DNA:RNA hybrid. Here, we report ssDRIP-seq, a single-strand DNA ligation-based library preparation technique for genome-wide identification of R-loops. When applied in Arabidopsis, ssDRIP-seq exhibits high efficiency, low bias and strand specificity. We found that Arabidopsis R-loops are enriched by both AT and GC skews, and are formed in the sense and antisense orientations. R-loops are strongly enriched in gene promoters and gene bodies, and are highly associated with noncoding RNA and repetitive genomic regions. Furthermore, R-loops are negatively correlated with CG DNA hypermethylation, and are prevalent in regions with multiple chromatin modifications, showing strong correlations with the activated and repressed gene loci. Our analyses indicate that R-loops are common features in the Arabidopsis genome and suggest that the R-loops play diverse roles in genome organization and gene regulation, thereby providing insights into plant nuclear genome formation and function.

  • Subscribe to Nature Plants for full access:

    $62

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Shaw, N. N. & Arya, D. P. Recognition of the unique structure of DNA:RNA hybrids. Biochimie 90, 1026–1039 (2008).

  2. 2.

    Skourti-Stathaki, K. & Proudfoot, N. J. A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev. 28, 1384–1396 (2014).

  3. 3.

    Santos-Pereira, J. M. & Aguilera, A. R loops: new modulators of genome dynamics and function. Nat. Rev. Genet. 16, 583–597 (2015).

  4. 4.

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

  5. 5.

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

  6. 6.

    Boguslawski, S. J. et al. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J. Immunol. Methods 89, 123–130 (1986).

  7. 7.

    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).

  8. 8.

    Ginno, P. A., Lim, Y. W., Lott, P. L., Korf, I. & Chedin, F. GC skew at the 5' and 3' ends of human genes links R-loop formation to epigenetic regulation and transcription termination. Genome Res. 23, 1590–1600 (2013).

  9. 9.

    Chan, Y. A. et al. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLoS Genet. 10, e1004288 (2014).

  10. 10.

    El Hage, A., Webb, S., Kerr, A. & Tollervey, D. Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet. 10, e1004716 (2014).

  11. 11.

    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).

  12. 12.

    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).

  13. 13.

    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).

  14. 14.

    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).

  15. 15.

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

  16. 16.

    Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

  17. 17.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  18. 18.

    Bailey, T. L. DREME: motif discovery in transcription factor ChIP-seq data. Bioinformatics 27, 1653–1659 (2011).

  19. 19.

    Hetzel, J., Duttke, S. H., Benner, C. & Chory, J. Nascent RNA sequencing reveals distinct features in plant transcription. Proc. Natl Acad. Sci. USA 113, 12316–12321 (2016).

  20. 20.

    Lim, Y. W., Sanz, L. A., Xu, X., Hartono, S. R. & Chedin, F. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutieres syndrome. eL ife 4, e08007 (2015).

  21. 21.

    Matzke, M. A., Kanno, T. & Matzke, A. J. RNA-Directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu. Rev. Plant Biol. 66, 243–267 (2015).

  22. 22.

    Shafiq, S. et al. DNA topoisomerase 1 prevents R-loop accumulation to modulate auxin-regulated root development in rice. Mol. Plant 10, 821–833 (2017).

  23. 23.

    Bagchi, D. N. & Iyer, V. R. The determinants of directionality in transcriptional initiation. Trends Genet. 32, 322–333 (2016).

  24. 24.

    Wu, X. & Sharp, P. A. Divergent transcription: a driving force for new gene origination? Cell 155, 990–996 (2013).

  25. 25.

    Mellor, J., Woloszczuk, R. & Howe, F. S. The interleaved genome. Trends Genet. 32, 57–71 (2016).

  26. 26.

    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).

  27. 27.

    Skourti-Stathaki, K., Kamieniarz-Gdula, K. & Proudfoot, N. J. R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014).

  28. 28.

    Pontes, O. et al. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126, 79–92 (2006).

  29. 29.

    Wendte, J. M. & Pikaard, C. S. The RNAs of RNA-directed DNA methylation. Biochim. Biophys. Acta 1860, 140–148 (2017).

  30. 30.

    Nakama, M., Kawakami, K., Kajitani, T., Urano, T. & Murakami, Y. DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation. Genes Cells 17, 218–233 (2012).

  31. 31.

    Lahmy, S. et al. Evidence for ARGONAUTE4-DNA interactions in RNA-directed DNA methylation in plants. Genes Dev. 30, 2565–2570 (2016).

  32. 32.

    Haag, J. R. et al. In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol. Cell 48, 811–818 (2012).

  33. 33.

    Shi, Y. & Berg, J. M. Specific DNA-RNA hybrid binding by zinc finger proteins. Science 268, 282–284 (1995).

  34. 34.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  35. 35.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  36. 36.

    Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  37. 37.

    Ramirez, F., Dundar, F., Diehl, S., Gruning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).

  38. 38.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  39. 39.

    Yang, Y. et al. SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis. Nucleic Acids Res. 45, 181–197 (2017).

  40. 40.

    Jenjaroenpun, P., Wongsurawat, T., Sutheeworapong, S. & Kuznetsov, V. A. R-loopDB: a database for R-loop forming sequences (RLFS) and R-loops. Nucleic Acids Res. 45, D119–D127 (2017).

  41. 41.

    Stroud, H., Greenberg, M. V., Feng, S., Bernatavichute, Y. V. & Jacobsen, S. E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).

  42. 42.

    Luo, C. et al. Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production. Plant J. 73, 77–90 (2013).

  43. 43.

    Gruntman, E. et al. Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics 9, 371 (2008).

Download references

Acknowledgements

We thank all members of the Sun and Yang labs for the useful discussion and suggestions, and we appreciate F. Chédin for sharing the detailed DRIP-seq protocol. We also appreciate the suggestions from three anonymous reviewers for improving the quality of this manuscript. This work was supported by grants from the National Key R&D Program (2016YFA0500800 to the Sun Lab), the National Natural Science Foundation of China (91540109 and 31671381 to X.Y.), the Tsinghua University Initiative Scientific Research Program (to the Sun Lab), the Tsinghua-Peking Joint Center for Life Sciences and the 1000 Young Talent Program of China (to Q.S. and X.Y.).

Author information

Author notes

  1. H. Xu and K. Li contributed equally to this work.

Affiliations

  1. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China

    • Wei Xu
    • , Hui Xu
    • , Yingxu Fan
    • , Yang Liu
    • , Xuerui Yang
    •  & Qianwen Sun
  2. Center for Plant Biology, Tsinghua University, Beijing, 100084, China

    • Wei Xu
    • , Kuan Li
    • , Yingxu Fan
    •  & Qianwen Sun
  3. MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    • Hui Xu
    • , Yang Liu
    •  & Xuerui Yang
  4. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    • Kuan Li

Authors

  1. Search for Wei Xu in:

  2. Search for Hui Xu in:

  3. Search for Kuan Li in:

  4. Search for Yingxu Fan in:

  5. Search for Yang Liu in:

  6. Search for Xuerui Yang in:

  7. Search for Qianwen Sun in:

Contributions

Q.S. initiated the project, and supervised the data analysis together with X.Y.; Q.S. and W.X. developed the ssDRIP-seq method and W.X. performed all experiments; W.X., H.X., K.L., Y.L. and Y.F. analysed the data; W.X., H.X., X.Y. and Q.S. prepared the figures with help from K.L. and Y.L.; and Q.S. wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xuerui Yang or Qianwen Sun.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Figures 1–11

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Arabidopsis public data used in this study.

  4. Supplementary Table 2

    Information for software used in this study.

  5. Supplementary Table 3

    Detailed information of R-loop peaks and their overlap with various genome features.

  6. Supplementary Table 4

    Information for primers used in this study.

  7. Supplementary Dataset 1

    Identified R-loop motifs by HOMER. The files include HOMER Known Motif Enrichment Results, forward peak motifs, reverse peak motifs, and unstranded peak motifs.