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DREAM complex suppresses DNA methylation maintenance genes and precludes DNA hypermethylation

Nature Plants volume 6, pages 942–956 (2020)Cite this article

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Abstract

The DNA methyltransferases MET1 and CMT3 are known to be responsible for maintenance of DNA methylation at symmetric CG and CHG sites, respectively, in Arabidopsis thaliana. However, it is unknown how the expression of methyltransferase genes is regulated in different cell states and whether change in expression affects DNA methylation at the whole-genome level. Using a reverse genetic screen, we identified TCX5, a tesmin/TSO1-like CXC domain-containing protein, and demonstrated that it is a transcriptional repressor of genes required for maintenance of DNA methylation, which include MET1, CMT3, DDM1, KYP and VIMs. TCX5 functions redundantly with its paralogue TCX6 in repressing the expression of these genes. In the tcx5 tcx6 double mutant, expression of these genes is markedly increased, thereby leading to markedly increased DNA methylation at CHG sites and, to a lesser extent, at CG sites at the whole-genome level. Furthermore, our whole-genome DNA methylation analysis indicated that the CG and CHG methylation level is lower in differentiated quiescent cells than in dividing cells in the wild type but is comparable in the tcx5/6 mutant, suggesting that TCX5/6 are required for maintenance of the difference in DNA methylation between the two cell types. We identified TCX5/6-containing multi-subunit complexes, which are known as DREAM in other eukaryotes, and demonstrated that the Arabidopsis DREAM components function as a whole to preclude DNA hypermethylation. Given that the DREAM complexes are conserved from plants to animals, the preclusion of DNA hypermethylation by DREAM complexes may represent a conserved mechanism in eukaryotes.

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Fig. 1: TCX5 and TCX6 mediate DNA demethylation and anti-silencing.
Fig. 2: TCX5 and TCX6 prevent excessive cell proliferation and thereby maintain the proper size of plant organs.
Fig. 3: The Arabidopsis DREAM complex functions in DNA demethylation and anti-silencing.
Fig. 4: TCX5 and TCX6 function as transcriptional repressors of cell cycle genes and methylation maintenance genes.
Fig. 5: TCX5 and TCX6 preclude DNA hypermethylation at the whole-genome level.
Fig. 6: TCX5 and TCX6 preclude DNA hypermethylation and transcriptional silencing of TEs and promoter-methylated genes in ddm1 and cmt3 mutant backgrounds.
Fig. 7: Effect of tcx5/6 on DNA methylation at the whole-genome level in shoot apical meristems and mature leaves.
Fig. 8: Effect of tcx5/6 on the expression of methylation maintenance genes in mature leaves and shoot apical meristems.

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Data availability

Raw RNA–seq, ChIP–seq and bisulfite sequencing data have been deposited in the GEO database (accession no. GSE137754).

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Acknowledgements

This work was supported by the National Key Research and Development Programme of China (no. 2016YFA0500801) from the Chinese Ministry of Science and Technology, to X.-J.H.

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  1. These authors contributed equally: Yong-Qiang Ning, Na Liu.

Authors and Affiliations

  1. National Institute of Biological Sciences, Beijing, China

    Yong-Qiang Ning, Na Liu, Ke-Ke Lan, Yin-Na Su, Lin Li, She Chen & Xin-Jian He

  2. The College of Life Sciences, Northwest University, Xi’an, China

    Yong-Qiang Ning

  3. College of Life Sciences, Beijing Normal University, Beijing, China

    Na Liu

  4. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    She Chen & Xin-Jian He

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Contributions

Y.-Q.N., N.L., K.-K.L., L.L. and S.C. performed the experiments. Y.-N.S. performed bioinformatics analyses. Y.-Q.N., N.L and X.-J.H. conceived the study, designed the experiments and wrote the manuscript.

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Correspondence to Xin-Jian He.

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Extended data

Extended Data Fig. 1 The enlarged-organ phenotypes of the tcx5/6 mutant were restored by co-expression of the TCX5-Flag and TCX6-Myc transgenes.

a, Determination of the expression of the TCX5-Flag and TCX6-Myc transgenes in the complementation lines T2-1 and T2-2 by immunoblotting. The expression level of the histone H3 is shown as a loading control. The experiment was performed twice and produced similar results. b–e, The size of cotyledons (b) (n = 14), rosette leaves (c) (n = 10), petals (d) (n = 15), and seeds (e) (n = 5) was measured in WT, tcx5/6, and two independent transgenic plants co-expressing TCX5-Flag and TCX6-Myc in the tcx5/6 mutant background. Values are means ± SD.

Extended Data Fig. 2 The effect of aly1, aly2, and aly3 on DNA methylation.

The gDNA from the indicated genotypes was cleaved by the DNA methylation-specific DNA restriction enzyme McrBC followed by amplification. The unmethylated actin gene ACT7 was used as an internal control. This experiment was independently repeated three times with similar results.

Extended Data Fig. 3 The effect of tcx5/6 on the expression and DNA methylation of ROS1.

a, Browser view of RNA-seq and bisulfite-seq signals on the DNA demethylase gene ROS1 in the wild type and the tcx5/6 mutant. CG, CHG, and CHH methylation are independently shown. The scale of normalized reads is shown for the RNA-seq data. The scale of DNA methylation ratio is shown for the bisulfite-seq data. b, The expression level of ROS1 in the wild-type Col-0 and the tcx5/6 mutant as determined by two independent replicates of RNA-seq. The y-axis indicates Fragments Per Kilobase Million (FPKM). P was determined by glmFit in edgeR. c, The DNA methylation level of the methylated ROS1 promoter region as determined by bisulfite-seq.

Extended Data Fig. 4 TCX5 and TCX6 function as transcriptional repressors of genes involved in DNA replication and cell division.

a, Browser view of TCX5-Flag ChIP-seq signals and RNA-seq signals on the genes involved in DNA replication and cell division. The scales of normalized reads in the RNA-seq and ChIP-seq data are shown. b, Confirmation of the effect of tcx5/6 on the expression of indicated genes by RT-PCR. Values are means ± SD of three biological replicates. c, Confirmation of the enrichment of TCX5-Myc in the promoter regions of indicated genes by ChIP followed by PCR. Values are means ± SD of three biological replicates.

Extended Data Fig. 5 The association of the DREAM complex components TCX6, ALY3, MSI1, and LIN37B with chromatin.

The association of TCX6, ALY3, MSI1, and LIN37B with chromatin were determined by ChIP-qPCR in transgenic plants harboring TCX6-Myc, ALY3-Flag, MSI1-Flag, and LIN37B-Myc, respectively. The association of these DREAM complex components with the transcription start site of indicated cell cycle genes and methylation maintenance genes was detected. Shown are mean values of two independent biological replicates.

Extended Data Fig. 6 Determination of the expression levels of methylation maintenance genes in the DREAM complex mutants.

The expression levels of MET1, DDM1, CMT3, KYP, and VIM1 in the DREAM complex mutants aly2/3, lin37a/b, and my3r1 were determined by RT-PCR. Values are means ± SD of three biological replicates. P value was determined by student’s t test with 2 tails.

Extended Data Fig. 7 The DNA methylation levels of indicated loci in the WT and tcx5/6 mutant plants.

a, Browser view of CG, CHG, and CHH methylation signals at indicated loci as determined by the whole-genome DNA methylation analysis. Twelve-day-old seedlings were subjected to the whole-genome DNA methylation analysis. The scale of the ratio of DNA methylation is shown. b, DNA methylation levels of indicated loci at CG, CHG, and CHH sites as determined by the whole-genome DNA methylation analysis.

Extended Data Fig. 8 The DNA methylation and expression levels of TEs showing lower expression in the tcx5/6;ddm1 mutant than in the ddm1 mutant.

a, The DNA methylation levels of indicated TEs at CG, CHG, and CHH sites as determined by the whole-genome DNA methylation analysis. The raw data of the DNA methylation levels are shown in Data set 8. b, The expression levels of indicated TEs as determined by RT-PCR. Shown are mean values of two independent biological replicates.

Extended Data Fig. 9 The effect of tcx5/6 on DNA methylation and transcriptional silencing of SDC and FWA.

a, Browser views of RNA-seq and DNA methylation signals of SDC and FWA as determined by RNA-seq and whole-genome DNA methylation analyses, respectively. The scale of normalized reads is shown for the RNA-seq data. The scale of the ratio of DNA methylation is shown for the bisulfite-seq data. b, CG, CHG, and CHH methylation levels of the methylated SDC promoter and the methylated region over the transcription start site of FWA. The DNA methylation levels were calculated based on the whole-genome DNA methylation analysis. c, The developmental defects of the ddm1 mutant are partially restored by tcx5/6 in the tcx5/6;ddm1 mutant. Images are bolting plants and 3-week-old seedlings. The chart shown in the right indicates the weight of rosette leaves of the WT and indicated mutants (n = 8).

Extended Data Fig. 10 The effect of tcx5/6 on the expression of cell cycle genes in mature leaves and shoot apical meristems.

a, Browser view of RNA-seq signals of indicated cell cycle genes in mature leaves (ML) and shoot apical meristems (SAM) from the wild type and tcx5/6 mutant plants. The scale of normalized RNA-seq reads is shown. b, The effect of tcx5/6 on the expression of the cell cycle genes in ML and SAM is shown by the value of log2(fold change between the expression in tcx5/6 and WT).

Supplementary information

Supplementary Information

Supplementary Figs. 1–18.

Reporting Summary

Supplementary Dataset 1

Transfer DNA mutants used for reverse genetic screening.

Supplementary Dataset 2

Proteins co-precipitated with TCX5, ALY3, LIN37B, RBR1 and MSI1 as determined by mass spectrometry.

Supplementary Dataset 3

Components of DREAM and DREAM-LIKE complexes in different species.

Supplementary Dataset 4

A summary of RNA-seq, ChIP–seq and whole-genome bisulfite–seq data shown in this study.

Supplementary Dataset 5

Up- and downregulated TEs and genes in the tcx5/6 mutant relative to WT from seedlings.

Supplementary Dataset 6

Full list of genes overlapping with TCX5 ChIP–seq peaks.

Supplementary Dataset 7

Expression of RdDM-related genes in WT and the tcx5/6 mutant as determined by RNA-seq.

Supplementary Dataset 8

Full list of hyper-DMRs identified in the tcx5/6 mutant.

Supplementary Dataset 9

RNA-seq and DNA methylation levels of ddm1-upregulated TEs in WT and indicated mutants.

Supplementary Dataset 10

RNA-seq and DNA methylation levels of cmt3-upregulated TEs in WT and indicated mutants.

Supplementary Dataset 11

RNA-seq and DNA methylation levels of ddm1-upregulated genes in WT and indicated mutants.

Supplementary Dataset 12

RNA-seq and DNA methylation levels of cmt3-upregulated genes in WT and indicated mutants.

Supplementary Dataset 13

Full list of hyper- and hypo-DMRs in SAM relative to ML at CX, CG, CHG and CHH sites from WT.

Supplementary Dataset 14

Full list of hyper- and hypo-DMRs in the tcx5/6 mutant relative to WT from ML at CX, CG, CHG and CHH sites.

Supplementary Dataset 15

Full list of hyper- and hypo-DMRs in the tcx5/6 mutant relative to WT from SAM at CX, CG, CHG and CHH sites.

Supplementary Dataset 16

Up- and downregulated genes in SAM relative to ML in WT.

Supplementary Dataset 17

Up- and downregulated genes in the tcx5/6 mutant relative to WT from ML.

Supplementary Dataset 18

Up- and downregulated genes in the tcx5/6 mutant relative to WT from SAM.

Supplementary Dataset 19

DNA oligos used in this study.

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Ning, YQ., Liu, N., Lan, KK. et al. DREAM complex suppresses DNA methylation maintenance genes and precludes DNA hypermethylation. Nat. Plants 6, 942–956 (2020). https://doi.org/10.1038/s41477-020-0710-7

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