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UVR8 interacts with de novo DNA methyltransferase and suppresses DNA methylation in Arabidopsis

Nature Plants volume 7, pages 184–197 (2021)Cite this article

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Abstract

DNA methylation is an important epigenetic gene regulatory mechanism conserved in eukaryotes. Emerging evidence shows DNA methylation alterations in response to environmental cues. However, the mechanism of how cells sense these signals and reprogramme the methylation landscape is poorly understood. Here, we uncovered a connection between ultraviolet B (UVB) signalling and DNA methylation involving UVB photoreceptor (UV RESISTANCE LOCUS 8 (UVR8)) and a de novo DNA methyltransferase (DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2)) in Arabidopsis. We demonstrated that UVB acts through UVR8 to inhibit DRM2-mediated DNA methylation and transcriptional de-repression. Interestingly, DNA transposons with high DNA methylation are more sensitive to UVB irradiation. Mechanistically, UVR8 interacts with and negatively regulates DRM2 by preventing its chromatin association and inhibiting the methyltransferase activity. Collectively, this study identifies UVB as a potent inhibitor of DNA methylation and provides mechanistic insights into how signalling transduction cascades intertwine with chromatin to guide genome functions.

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Fig. 1: UVB induces DNA hypomethylation in a UVR8-dependent manner.
Fig. 2: UVB induces genome-wide CHH hypomethylation.
Fig. 3: UVB and DRM2 regulate expression of TEs.
Fig. 4: DRM2 interacts with UVR8 in vitro and in vivo.
Fig. 5: UBA domains of DRM2 mediate its interaction with UVR8.
Fig. 6: UVR8 inhibits the methyltransferase activity and chromatin association of DRM2.

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Data availability

All whole-genomic bisulfite sequencing and RNA-seq data produced during this study were deposited into the Gene Expression Omnibus under accession number GSE132944. Source data are provided with this paper.

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Acknowledgements

We thank S. Jacobsen (University of California, Los Angeles) for pSDC:GFP/cmt3 and X. Chen (University of California, Riverside) for d35S:LUC reporter lines. We thank Zhong laboratory members (A. Ding, S. Leichter and R. Scheid) for comments on the manuscript. This work was supported by the NIH (grant no. R35GM124806) and the USDA (grant no. Hatch 1012915) to X.Z. and the NIH (grant no. 1R35GM119721) to J.S. J.J. was supported by the Initiative Postdocs Supporting Program (grant no. BX201600066).

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Authors and Affiliations

  1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing, Jiangsu, China

    Jianjun Jiang & Fengquan Liu

  2. Laboratory of Genetics & Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, WI, USA

    Jianjun Jiang, Jie Liu, Dean Sanders, Shuiming Qian & Xuehua Zhong

  3. Department of Biochemistry, University of California, Riverside, CA, USA

    Wendan Ren & Jikui Song

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  1. Jianjun Jiang
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Contributions

J.J. designed and performed most experiments, analysed data, prepared figures and wrote the manuscript draft. J.L. and D.S. performed the genomic data analysis. S.Q. provided reagents and edited the manuscript. W.R. and J.S. provided recombinant DRM2 proteins. F.L. conceived the project and edited the manuscript. X.Z. conceived the project, designed experiments, analysed data and wrote the manuscript.

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Correspondence to Fengquan Liu or Xuehua Zhong.

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

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Extended data

Extended Data Fig. 1 Characterization of LUCM reporter line.

a, Luciferase images of Col-0 and three d35S:LUC reporter lines treated with DNA methylation inhibitor 5-Azacytidine (5-AzaC, 100 μM) for 7 days. LUCL and LUCH are previously reported (ref. 35) low and high LUC expressing lines, respectively. b, Copy number of 35S-LUC transgene in LUCL, LUCH, and LUCM lines revealed by qPCR of LUC using genomic DNA. Data is mean ± SD. c, McrBC-qPCR based DNA methylation assay of 35S promoter regions in LUCL, LUCH, and LUCM lines. Low amplification represents high DNA methylation level. Data is mean ± SD. **p < 0.01; ***p < 0.001 by Student’s t-test. d, Relative transcript level of LUC gene in LUCL, LUCH, and LUCM lines. Data is mean ± SD. ***p < 0.001 by Student’s t-test. e, Bisulfite sequencing of indicated regions (1 and 2) in LUCL, LUCH, and LUCM lines. f, Luciferase images of 5-d old LUCM and dd LCUM (drm1 drm2 LUCM) seedlings. g, McrBC-qPCR based DNA methylation assay of 35S promoter regions. SDC serves as a control for dd. Data is mean ± SD. ***p < 0.001 by Student’s t-test.

Extended Data Fig. 2 Flowering phenotypes of FWA transgenic plants.

a and b, The flowering time of FWA transgenic T1 plants from two replicates with replicate 1 in (a) and replicate 2 in (b). Indicated genotypes were transformed with FWA transgene and the flowering time of positive Basta-resistant T1 transformants were counted. The non-transformed plants serve as controls. The number of plants is indicated by n. dd, drm1 drm2; udd, uvr8-6 drm1 drm2; UVR8-OX, 35S:UVR8-FLAG overexpression lines (#3 and #2); UVR8W285A-OX, 35S:UVR8W285A-FLAG overexpression lines (#5 and #8). The blue dash line indicates median. Each dot represents a single plant. Significantly different (p < 0.05 by Student’s t-test) groups are labelled with different letters.

Extended Data Fig. 3 Analysis of UVB-induced differentially methylated regions.

a,b, Venn diagrams showing the overlap of CHH hypo differential methylation regions (DMRs) among drm1 drm2 (dd), cmt2-3, and UVB-induced DMRs in Col-0 (a) and UVR8-OX (b). Data of dd and cmt2-3 are from ref. 77. c, Venn diagrams showing the overlap of CHH hypo DMRs among UVB-treated Col-0 (left) or UVR8-OX (right), dd, and drm2-2 mutant. Data of drm2-2 is from ref. 78. d, Venn diagrams showing the overlap of UVB-induced CHH hypo DMRs in Col-0 with hyper DMRs in uvr8-6 mutant. e, Venn diagrams showing the overlap of UVB-induced CHH hypo DMRs in Col-0 and uvr8-6. f, The enrichment of TEs containing CHH hypo DMRs based on length. ***, p < 0.001 by Fisher’s exact test. g, The enrichment of Class II (DNA) TEs containing CHH hypo DMRs. ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant by Fisher’s exact test.

Extended Data Fig. 4 Comparison of UVB- and UVC-induced DMRs.

a, Comparison of CHH hypo DMRs by UVC treated Col-0 (compared to Col-0 without treatment) and drm1 drm2 (dd). The UVC-induced DMRs are from ref. 41 (GSE132750). b, Comparison of UVB- and UVC-induced CHH hypo DMRs in Col-0. c, Comparison of CHH hypo DMRs of UVB treated UVR8-OX (compared with Col-0,WL) and UVC. d, Overlapping of CHH hypo DMRs of UVB treated UVR8-OX (compared with UVR8-OX,WL) and UVC.

Extended Data Fig. 5 Differential expressed genes (DEG) induced by UVB.

a, Venn diagram showing the overlapping of DEGs induced by UVB in Col-0 and drm1 drm2 (dd). b, Correlation plot showing the expression level change (log2FC) of common UVB-responsive DEGs in Col-0 and dd (n = 327). c, Expression levels of marker genes, which are up-regulated by UVB, in Col-0 and dd. Data is mean with 95% confidence interval. d, Expression levels of DNA-damage induced genes in Col-0 and dd. Data is mean with 95% confidence interval. e, Expression levels of genes in UVB-signaling pathway. Data is mean with 95% confidence interval. Different letters denote significant differences (p < 0.05 by Student’s t-test) among samples. f, Venn diagram showing the overlapping of DEGs induced by long-term (10d, this study) and short-term (6 h, ref. 79) UVB treatment in Col-0. g and h, Heat map showing the expression of several UVB up-regulated (g) and down-regulated (h) DEGs in both long-term and short-term UVB treatment. i, Metaplots showing the CHH methylation level of UVB up-regulated TEs (n = 269). Data of 1 kb upstream and downstream of the TE body are shown.

Extended Data Fig. 6 DRM2 interacts with UVR8 and its active form UVR8W285A.

a, Heatmap showing the NSAF score (an indicator of normalized spectral abundance factor) of UVR8 and DRM2 in various immunoprecipitation-mass spectrometry (IP-MS) experiments. The IP-MS data of DRM2 is from ref. 34, DRM3 and NRPE1 are from ref. 80, CMT3 is from ref. 81, DRD1 and DMS3 are from ref. 82, MORC6 is from ref. 69, IDN2 is from ref. 83, HDA9 and PWR are from ref. 84, HOS15 is from ref. 85, HD2C is from ref. 86, EBS is from ref. 87. b, Coomassie bright blue staining of non-boiled GST-UVR8 proteins on SDS-PAGE. The GST-UVR8W285A proteins serve as control for monomer. c, Co-immunoprecipitation of UVR8 and DRM2 with FLAG beads from N. benthamiana leaves co-expressing UVR8-HA and DRM2-FLAG. d, Co-immunoprecipitation of UVR8 and DRM2 with FLAG beads from transgenic Arabidopsis plants co-expressing UVR8-HA and 3F9M-DRM2. UVR8-HA in Col-0 serves as a control. e, Immunoblots showing protein levels with or without UVB treatment. The ±UVB set-up is the same as that in Fig. 4g. Actin serves as an internal control. f, Split luciferase assay showing the interaction between DRM2 and UVR8W285A. The indicated constructs were co-expressed in N. benthamiana leaves and imaged after spraying with the luciferin. nLuc- and cLuc-only vectors serve as negative controls. Two biological replicates are shown. g, Bimolecular fluorescence complementation (BIFC) assays in N. benthamiana leaves showing the interaction between DRM2 and different forms of UVR8. Scale bar, 10 μm. h, BIFC assays co-expressing indicated proteins in N. benthamiana leaves. Scale bar, 10 μm.

Extended Data Fig. 7 DRM2 interacts with both the core domain and the C-terminus of UVR8.

a, Bimolecular fluorescence complementation (BIFC) assays in N. benthamiana leaves. nYFP-fused full length UVR8, UVR8 core domain (UVR8N396), and C-terminus (UVR8C44) were co-expressed with DRM2-cYFP. The arrow indicates the nuclei showing nuclear bodies. Scale bar, 10 μm. b, Magnified images showing the interaction of UVR8-DRM2, UVR8N396-DRM2 in nuclear bodies. Scale bar, 10 μm.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–5, Tables 1–3, descriptions for Supplementary Data 1–3, and references.

Reporting Summary

Supplementary Data 1

This table includes the lists of proteins identified in two biological replicates (Experiment 1 and Experiment 2). The transgenic lines used are 3F9M-DRM2 (3xFLAG-9xMyc-BLRP-DRM2). The DRM2-9NK is 3F9M-DRM2 carrying a mutation in BLRP motif.

Supplementary Data 2

UVB-induced CHH DMRs.

Supplementary Data 3

This table lists the expression data of all genes in RNA-seq. The expression data are the average of two biological experiments. Significance is defined by Q value ≤ 0.05 by Cufflinks.

Source data

Source Data Fig. 1

Full scans of western blots.

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Jiang, J., Liu, J., Sanders, D. et al. UVR8 interacts with de novo DNA methyltransferase and suppresses DNA methylation in Arabidopsis. Nat. Plants 7, 184–197 (2021). https://doi.org/10.1038/s41477-020-00843-4

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