Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation

Abstract

It is generally assumed that DNA methylation changes at genomic regions targeted by the de novo RNA-directed DNA methylation (RdDM) pathway are unstable. Here, we show that RdDM targets in Arabidopsis can be classified into two groups on the basis of whether there is remethylation following the restoration of NRPD1 function in nrpd1 mutant plants—remethylable loci and non-remethylable loci. In contrast to the remethylable loci, the non-remethylable loci contain higher levels of the euchromatic marks of trimethylation at Lys 4 of histone H3 (H3K4me3), which interferes with the recruitment of the RdDM molecular machinery, and acetylation at Lys 18 of histone H3 (H3K18ac), which helps to recruit the DNA demethylase ROS1 to antagonize RdDM. Here, using targeted methylation erasure by CRISPR–dCas9–TET1, we demonstrate that methylated CG (mCG) and mCHG (where H represents A, C or T) are memory marks that are required for targeting the RdDM machinery to remethylable loci. Our results show that histone and DNA methylation marks are critical in determining the ability of RdDM target loci to form stable epialleles, and contribute to understanding the formation and transmission of epialleles.

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Fig. 1: Transgenerational inheritance of DNA methylation changes at RdDM target loci.
Fig. 2: DNA methylation patterns of CDs and NDs in backcrosses.
Fig. 3: Genetic and epigenetic features of CDs and NDs.
Fig. 4: Increased H3K4me3 at specific NDs in the nrpd1 mutant prevents recruitment of the RdDM machinery.
Fig. 5: ROS1 antagonizes RdDM in the process of re-establishing DNA methylation at specific ND loci.
Fig. 6: The remaining mCG and mCHG act as memory marks for the recruitment of the RdDM machinery.
Fig. 7: The behaviour of NDs in other RdDM mutants and in long-term inheritance.

Data availability

All high-throughput sequencing data reported in this paper are provided at the GEO (GSE140566). Source Data for Figs. 16 are provided with the paper.

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Acknowledgements

We thank Q. Zhang for suggestions. This research was financially supported by the Chinese Academy of Sciences, including the CAS Strategic Priority Research Program, grant number XDB27040101 (to J.-K.Z.).

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J.L., D.-L.Y. and J.-K.Z. designed the experiments. J.L., H.H., G.Z., J.P. and L.H. performed the experiments. J.L., R.L.-D., D.-L.Y., Z.L. and J.-K.Z. analysed the data and wrote the manuscript.

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Correspondence to Jian-Kang Zhu.

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Peer review information Nature Plants thanks Tzung-Fu Hsieh, Jean Molinier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Table 1

NRPD1-CD, NRPD1-ND, NRPE1-ND and RDR2-ND lists.

Supplementary Table 2

Primers used in this study.

Supplementary Table 3

Derepressed genes/TEs in the nrpd1-3 mutant.

Supplementary Table 4

CD/ND gene lists.

Supplementary Table 5

P values.

Supplementary Data

Source data for supplementary figures.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed gels.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Unprocessed gels.

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Li, J., Yang, D., Huang, H. et al. Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation. Nat. Plants 6, 661–674 (2020). https://doi.org/10.1038/s41477-020-0671-x

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