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Identification and analysis of adenine N6-methylation sites in the rice genome

Nature Plants volume 4, pages 554–563 (2018)Cite this article

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Abstract

DNA N6-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes1,2,3,4,5,6,7,8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in transposable elements. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforce a distinct role for 6mA as a gene expression-associated epigenomic mark in eukaryotes.

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Fig. 1: Distribution of 6mA in the rice genome.
Fig. 2: 6mA in gene promoter and body regions correlates with different levels of gene expression.
Fig. 3: Comparison of 6mA distribution detected by IP–seq with two different antibodies (SYSY and Abcam) with other epigenomic marks in rice genes.
Fig. 4: 6mA levels and distribution in rice transposable elements and repeats.
Fig. 5: Osalkbh1 mutations result in increased 6mA levels.
Fig. 6: The overall structure and active site of OsALKBH1 in rice.

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Acknowledgements

We thank Y. Zhao for providing the Cas9–single gRNA system, the staff from BL17U/BL19U1/beamline of the National Centre for Protein Sciences Shanghai (NCPSS) at the Shanghai Synchrotron Radiation Facility for assistance during data collection, and the research associates at the Centre for Protein Research, Huazhong Agricultural University, for technical support. This work was supported by funds from the National Key Research and Development Program of China (2016YFD0100802), the National Science Foundation of China (grants 31730049, 31571256 and 31270787), the Program for New Century Excellent Talents in University (NCET-13-0811), the Fundamental Research Funds for the Central Universities (programme no. 2662015PY228, 2013PY063 and 2014PY017), and supported by Huazhong Agricultural University Scientific and Technological Self-innovation Foundation (programme no. 2016RC003).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China

    Chao Zhou, Changshi Wang, Hongbo Liu, Qiangwei Zhou, Qian Liu, Yan Guo, Ting Peng, Jiaming Song, Jianwei Zhang, Lingling Chen, Yu Zhao, Zhixiong Zeng & Dao-Xiu Zhou

  2. Institute of Plant Science of Paris-Saclay (IPS2), CNRS, INRA, Université Paris-sud 11, Université Paris-Saclay, Orsay, France

    Dao-Xiu Zhou

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Contributions

C.Z. performed the experiments to analyse rice genome-wide 6mA. C.W. and T.P. performed the molecular cloning and enzyme assays. H.L. conducted the 6mA mass spectrometry analysis. Q.L. and Y.Z. participated in sampling and experimentation. Y.G., T.P. and Z.Z. performed protein purification, crystallization, X-ray data acquisition and structure refinement and analysis. D.-X.Z., C.Z., Z.Z., Q.Z., J.S., J.Z. and L.C. analysed the data. D.-X.Z. coordinated and supervised the project and wrote the paper with input from C.Z. and Z.Z.

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Correspondence to Zhixiong Zeng or Dao-Xiu Zhou.

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Zhou, C., Wang, C., Liu, H. et al. Identification and analysis of adenine N6-methylation sites in the rice genome. Nature Plants 4, 554–563 (2018). https://doi.org/10.1038/s41477-018-0214-x

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