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CK2 kinase–PRC2 signalling regulates genome-wide H3K27 trimethylation and transduces prolonged cold exposure into epigenetic cold memory in plants

Nature Plants volume 11, pages 1572–1590 (2025)Cite this article

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Abstract

Polycomb protein-mediated transcriptional repression plays a crucial role in the regulation of responses to environmental stimuli in multicellular eukaryotes, but the underlying signalling events remain elusive. During Arabidopsis vernalization, prolonged cold exposure results in the formation of a Polycomb-repressed domain at the potent floral repressor FLC to confer its stable silencing upon temperature rise or epigenetic ‘memory of prolonged cold’, enabling the plants to bloom in spring. Here we report that the evolutionarily conserved casein kinase CK2 phosphorylates and thus stabilizes histone 3 lysine-27 (H3K27) methyltransferases (PRC2 subunits) to promote H3K27 trimethylation throughout the Arabidopsis genome. We found that prolonged cold induces progressive CK2 accumulation, leading to a gradual accumulation of cellular PRC2. We further show that the cold-CK2–PRC2 signalling promotes increasing PRC2 enrichment on FLC chromatin during prolonged cold exposure as well as post-cold PRC2 spreading across FLC to establish a Polycomb-repressed domain for FLC repression in warmth. Thus, this signalling cascade transduces prolonged cold exposure, but not cold spells, into epigenetic memory of prolonged cold in warmth during vernalization. CK2 phosphorylation motifs are widely present in H3K27 methyltransferases from plants and animals. Our study reveals a new layer of control of PRC2 activity in multicellular organisms.

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Fig. 1: Phosphorylation of CLF at the conserved serine 182 is essential for its function.
Fig. 2: CK2 interacts with and phosphorylates CLF at Ser182.
Fig. 3: CK2-mediated phosphorylation of CLF and SWN stabilizes both enzymes.
Fig. 4: CK2 modulates genome-wide H3K27 trimethylation and gene expression.
Fig. 5: Prolonged cold exposure leads to cellular CK2 accumulation and consequent CLFpS182 build-up.
Fig. 6: CLFpS182 and SWNpS170 mediate cold-duration-dependent H3K27 trimethylation at the FLC locus and post-cold formation of a Polycomb-repressed domain across FLC.
Fig. 7: A working model for the cold-CK2–PRC2 signalling to transduce prolonged cold exposure into epigenetic cold memory.

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Data availability

The raw data of ChIP-seq and RNA-seq from this study have been deposited in the Genome Sequence Archive database at the National Genomics Data Center (China) with the following accession numbers: CRA015362 (ChIP-seq) and CRA015386 (RNA-seq). Additional data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank J. Goodrich (University of Edinburgh) for kindly providing the seeds of GFP:CLF and clf-81, Y. She and an in-house mass spectrometry facility for protein phosphorylation analysis, X. Luo and Y. Li for experimental assistance, Z. Shang for bioinformatic analysis and Q. Liu for critically reading this manuscript. This work is supported in part by the National Natural Science Foundation of China (grant numbers 32330007, 31830049 and 31721001 to Y.H.), Peking-Tsinghua Center for Life Sciences and Chinese Academy of Sciences.

Author information

Author notes

  1. Xiaolin Zeng

    Present address: Department of Chemistry, The University of Chicago, Chicago, IL, USA

  2. These authors contributed equally: Xiaolin Zeng, Zheng Gao, Jiabin Gu.

Authors and Affiliations

  1. Peking-Tsinghua Center for Life Sciences & State Key Laboratory of Wheat Improvement, School of Advanced Agricultural Sciences, Peking University, Beijing, China

    Xiaolin Zeng, Zheng Gao, Jiabin Gu, Yang Ou, Zhaoxu Gao & Yuehui He

  2. Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (CAS), Shanghai, China

    Xiaolin Zeng, Pengcheng Wang & Yuehui He

  3. Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, China

    Zheng Gao

  4. Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences in Weifang, Shandong, China

    Guochen Qin & Yuehui He

  5. Institute of Advanced Biotechnology and School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Pengcheng Wang

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Contributions

Y.H. conceived and designed the research. X.Z., Zheng Gao, J.G., G.Q. and Y.O. designed and conducted the experiments. X.Z., Zheng Gao, Y.H. and J.G. analysed data. Zhaoxu Gao conducted bioinformatic analysis. G.Q. and P.W. analysed the mass-spectrometry data. Y.H. wrote the paper with help from X.Z. and Zheng Gao.

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Correspondence to Yuehui He.

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Extended data

Extended Data Fig. 1 Analysis of the phosphorylation of S182 on CLF.

a, Tandem MS spectrum of the phosphopeptide IYYDQTGGEALICpSDSEEEAIDDEEEKRDFLEPEDYIIR showing the phosphorylation of S182 on the functional GFP:CLF protein extracted from Arabidopsis seedlings. b, List of phosphorylation sites in the GFP:CLF protein identified by LC-MS/MS. c, Functionality analysis of CLF:Flag, driven by a native promoter region of CLF and introduced into clf-29, a null mutant with phenotypes less severe than clf-81 that bears a point mutation in the catalytic domain39. Dots represent individual measurements (n = 12), and middle lines for medians. P values are derived from one-way ANOVA. d, Phos-tag SDS-PAGE analysis of CLF phosphorylation. Total proteins extracted from the cold-exposed seedlings of CLF:Flag (#1), were treated with calf intestinal alkaline phosphatase (CIP), followed by separation in an SDS-PAGE gel containing 50 μM Zn2+-Phos-tag. Immunoblotting was conducted using anti-Flag. At the bottom is a normal SDS-PAGE blot, serving as a control. e, Treatment of CLFpS182:Flag (anti-Flag immunoprecipitations) with CIP. ACTIN serves an endogenous control. The experiments in (d,e) were conducted at twice independently with similar results.

Source data

Extended Data Fig. 2 Functional analysis of pS182 on CLF and conservation of the CK2 phosphorylation sites among CLF homologs in plants and animals.

a, Phenotypes of the indicated lines grown in LDs. Shown are T1 (the first generation) transgenic seedlings expressing CLFpro-CLF, CLFS182A or CLFS182T. Scale bars, 1 cm. b,c, Analysis of FLC (b) and FT (c) expression in the indicated seedlings. Two independent lines for each transgene were examined. Transcript levels were quantified by real-time PCR (qPCR), and then normalized to the constitutively-expressed PP2A. Data are presented as mean values ± s.d. of three biological replicates, and P values are derived from one-way ANOVA. d,e, Levels of H3K27me3 on total histones in the indicated seedlings, as revealed by immunoblotting with anti-H3K27me3. Representative protein blots are shown in (d), whereas relative levels of H3K27me3 are shown in (e). The levels of H3 serve as loading controls. Relative fold changes over WT (set as 1.0) are presented. Data are presented as mean values ± s.d. of three biological replicates, and P values are derived from one-way ANOVA. f, Sequence alignment of CLF homologs from land plants. The conserved Ser182 of CLF is indicated by an asterisk. g, Schematic illustration of a CK2 phosphorylation motif conserved among EZH1 homologs in animals. h, Sequence alignment of animal EZH1 homologs. Accession numbers: human EZH1, NP_001982.2; mouse EZH1, NP_001390750; chicken EZH1, XP_040509295; frog EZH1, XP_031750333; zebrafish EZH1, NP_001035072; fruitfly E(z), NP_001261682.

Source data

Extended Data Fig. 3 Analysis of ck2 mutants and transgene functionality.

a-c, Schematic illustrations of the mutations in ck2-b1, ck2-b2 (a), ck2-b3 (b) and ck2-b4 (c) mutants. Boxes for exons; A of the start codon ATG as +1. Dashes indicate the deleted DNA base pairs, while red nucleotides denote insertions. d, Knockout of CK2-B1 expression in the ck2b quadruple mutant of ck2-b1/b2-1/b3-1/b4-1, as revealed by RT-PCR. The constitutively-expressed TUBULIN2 (TUB2) serves as a loading control. The experiment was conducted twice independently with similar results. e, Flowering times of the indicated mutants grown in LDs. f,g, Functionality analysis of Flag-tagged CK2-B3 (f) and CK2-A3 (g) transgenes. These transgenes driven by respective native promoters, were introduced into ck2-a1a2a3 or ck2-b1b2b3. Two independent homozygous transgenic lines were scored for flowering times. h, Functionality analysis of CLF:HA, driven by a native promoter region of CLF and introduced into clf-29. Two independent homozygous transgenic lines were scored for flowering times. i, FLC expression in the seedlings of WT and ck2-a1a2a3. Data are presented as mean values ± s.d. of three biological replicates. Dots in (e-h) represent individual measurements (n = 12), and middle lines for medians. All data in (e-i) were analyzed by two-tailed t test.

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Extended Data Fig. 4 Characterization of CLFpS182 phosphorylation.

a, Purification of recombinant CK2α3 and CK2β3 from E. coli. Affinity-purified MBP:His:CK2α3 and GST:CK2β3 were separated by SDS-PAGE, followed by staining with Coomassie blue R250. MBP, maltose-binding protein; GST for glutathione s-transferase. The experiment was conducted at least twice independently with similar results. b, Tandem MS spectrum of the phosphopeptide IYYDQTGGEALICpSDSEEEAIDDEEEKR showing the phosphorylation of S182 on CLF. A CLF fragment (aa 111-451) was phosphorylated in vitro by the reconstituted CK2 holoenzyme of CK2α3 and CK2β3, followed by tandem MS. c, Confocal fluorescent imaging of CLF:GFP and CLFS182A:GFP root tips. Root tips of 7-d-old seedlings were imaged; scale bar for 20 μm. d, Expression levels of CLF or CLF transgenes at a seedling stage. Transcript levels were quantified by qPCR and normalized to PP2A. Data are presented as mean values ± s.d. of three biological replicates. e, Functionality analysis of Flag:CLF. 15 plants per genotype were scored, and T1 plants of Flag:CLF (in clf-29) were examined. Data are presented as mean values ± s.d., and P values are derived from one-way ANOVA. f, Levels of Flag:CLF and Flag:CLFS182A in transgenic seedlings. Randomly-chosen T1 seedings were examined by immunoblotting. ACTIN serves as a loading control. The experiment was conducted twice independently with similar results. g, Relative expression of CLF and SWN in the seedlings of WT and ck2b-quad. Transcripts were quantified by qPCR and normalized to TUB2. Relative expression to WT is presented. Data are presented as mean values ± s.d. of three biological replicates.

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Extended Data Fig. 5 CK2 phosphorylates the S170 on SWN for its stabilization.

a, SWNpro-SWN:9xHA is fully functional. The transgene was introduced into clf swn by genetic transformation. Seedlings were grown on half-strength MS media under a long-day (LD) condition. b, Co-immunoprecipitation of SWN with CK2β3. SWN:HA was immunoprecipitated by anti-Flag recognizing CK2β3:Flag, from the F1 seedlings of a SWN:9xHA line crossed to the CK2-B3:Flag line. The experiment was conducted twice independently with similar results. c, Confirmation of the phosphorylation of Ser170 on the SWN:HA protein in the indicated seedlings by an antibody elicited by the phosphorylated antigen of a short polypeptide conserved in CLF and SWN. SWN:HA was immunoprecipitated with anti-HA beads. Part of the samples were dephosphorylated with calf intestinal alkaline phosphatase (CIP), followed by western blotting. ACTIN serves as a loading control for total protein used in each anti-HA immunoprecipitation. The experiment was conducted twice independently with similar results. d,e, Immunoblotting analysis of the degradation of total SWN:HA (phosphorylated and unphosphorylated at S170) following cycloheximide (CHX) treatment. A SWN:HA line (in ck2b quad) was crossed to WT and ck2b-quad, and the resulting F1 seedlings were treated by the translation inhibitor CHX, followed by immunoblotting. Representative blots were shown in (d), while relative levels of total SWN:HA over the ‘0 h ck2b+/- sample’ (set as 1.0) are shown in (e). Data are presented as mean values ± s.d. of three biological replicates, and P values are derived from one-way ANOVA.

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Extended Data Fig. 6 Ser170 phosphorylation is essential for SWN function.

a,b, Phenotypes of swn-7, clf-29, lines of the SWN transgene in clf swn, lines of SWNS170A in clf swn, lines of SWNS170D in clf swn, and lines of SWNS170E in clf swn. Seedlings were grown on half-strength MS media for 9 d (a) and 17 d (b) under a LD condition. After germination, the clf swn mutant exhibited stunted growth and failed to continue developing. Scale bars, 2.5 mm.

Extended Data Fig. 7 CK2 modulates genome-wide H3K27 trimethylation and gene expression in Arabidopsis.

a, Principle component (PC) analysis of WT and ck2b-quad transcriptomes at a seedling stage. Data are plotted using two PCs. Three biological replicates for each genotype were performed. b, Gene Ontology (biological processes) enrichment analysis of DEGs in ck2b (relative to WT). The abscissa represents the proportion of enriched genes in each GO term, and the size of the circle indicates the number of enriched genes. P values are adjusted using the Benjamini-Hochberg procedure. c, Heatmaps showing H3K27me3 distribution at individual loci in WT and ck2b-quad seedlings. TSS for transcription start site, TES for transcription end site, and scales on the right represent RPM values. d,e, Correlation analysis of biological replicates of H3K27me3 ChIP-seq experiments using WT (d) and ck2b-quad (e) seedlings. Three biological replicates were performed for each genotype. Values on both x and y axes are read coverages. f, Venn diagram showing overlapping of the protein-coding genes upregulated in ck2b-quad, list of genes upregulated in clf-29 swn-4, and the protein-coding loci bearing H3K27me3. Notably, the list of genes upregulated in clf swn is from the published study49. g, Levels of H3K27me3 across the FLC locus in the seedlings of WT and clf-29. Levels of the FLC fragments immunoprecipitated by anti-H3K27me3 were quantified by qPCR and normalized to input DNA. Data are presented as mean values ± s.d. of three biological replicates.

Extended Data Fig. 8 Expression of CK2 subunit genes and CLF over the course of prolonged cold exposure.

a-h, Expression of CK2-A1 (a), CK2-A2 (b), CK2-A3 (c), CK2-A4 (d), CK2-B1 (e), CK2-B2 (f), CK2-B3 (g) and CK2-B4 (h) in FRI-Col seedlings over the course of 35-d cold exposure. i, CLF expression over the course of 35-d cold exposure. a-i, Transcripts were quantified by qPCR and normalized to PP2A. Relative expression to pre-cold /no cold is presented, and data are presented as mean values ± s.d. of three biological replicates. j, Examination of CK2α3:Flag abundances following the co-treatment of CX-4945 with the protease inhibitor MG132. ACTIN serves as a loading control. The experiment was conducted twice independently with similar results.

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Extended Data Fig. 9 Characterization of CK2B function in prolonged cold-induced H3K27 trimethylation.

a, Analysis of SWN:HA levels in the seedlings of ck2b+/- and ck2b-quad over the course of 35-d cold treatment. ACTIN serves as an endogenous control. The experiment was conducted twice independently with similar results. b,c, Levels of H3K27me3 on total histones in the seedlings of WT and ck2b-quad over the course of 35-d cold exposure, as revealed by immunoblotting with anti-H3K27me3. Representative protein blots are shown in (b), whereas relative levels of H3K27me3 are shown in (c). H3 serves as an endogenous control. The levels of H3K27me3 were normalized to H3; data are presented as mean values ± s.d. of three biological replicates, and P values are derived from one-way ANOVA.

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Extended Data Fig. 10 Characterization of the roles of CLF, SWN and CK2 in vernalization.

a, SWN expression in the indicated seedlings. Transcripts were quantified by qPCR and normalized to PP2A. Data are presented as mean values ± s.d. of three biological replicates. b, Phenotypes of the indicated lines grown in LDs. c, ChIP analysis of H3K27me3 enrichment at FLC upon 35-d cold exposure (vernalization) in the indicated seedlings. Levels of FLC fragments were quantified by qPCR and normalized to total input DNA. Data are presented as mean values ± s.d. of three biological replicates. d, Flowering times of the indicated lines (grown in LDs) following vernalization (35-d cold treatment). e, Total leaf number at flowering of the transgenic lines of FRI clf-29 expressing CLFpro-CLF or CLFS182A. Plants were grown in LDs. f, FRI ck2b is partially vernalization-insensitive. FRI-Col and FRI ck2b seedlings were exposed to cold for 35 d. Dots in (d-f) represent individual measurements (n = 12), and middle lines for medians. The data in (c) and (d) were analyzed by one-way ANOVA and two-tailed t test, respectively.

Supplementary information

Reporting Summary

Supplementary Table 1

List of genes misregulated in ck2b-quad.

Supplementary Table 2

List of H3K27me3-bearing genes upregulated in ck2b-quad.

Supplementary Table 3

List of primers used in this study.

Source data

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Zeng, X., Gao, Z., Gu, J. et al. CK2 kinase–PRC2 signalling regulates genome-wide H3K27 trimethylation and transduces prolonged cold exposure into epigenetic cold memory in plants. Nat. Plants 11, 1572–1590 (2025). https://doi.org/10.1038/s41477-025-02054-1

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