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

Nature Plants volume 3, pages 704–714 (2017)Cite this article

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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.

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Fig. 1: Genome-wide detection of R-loops in Arabidopsis by ssDRIP-seq.
Fig. 2: Antisense R-loops are highly enriched in the Arabidopsis genome.
Fig. 3: R-loops are enriched in both GC and AT skew regions and are anticorrelated with DNA hypermethylation.
Fig. 4: Preferential formation of R-loops in regions with various histone modifications.
Fig. 5: R-loops are strongly associated with plant-specific Pol IV and V transcription and TEs, noncoding regions and repetitive regions.

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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.).

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Author notes

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

Authors and 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

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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.

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Correspondence to Xuerui Yang or Qianwen Sun.

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Supplementary Information

Supplementary Figures 1–11

Life Sciences Reporting Summary

Supplementary Table 1

Arabidopsis public data used in this study.

Supplementary Table 2

Information for software used in this study.

Supplementary Table 3

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

Supplementary Table 4

Information for primers used in this study.

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.

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Xu, W., Xu, H., Li, K. et al. The R-loop is a common chromatin feature of the Arabidopsis genome. Nature Plants 3, 704–714 (2017). https://doi.org/10.1038/s41477-017-0004-x

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