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Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1

Abstract

To compensate for a sessile nature, plants have developed sophisticated mechanisms to sense varying environmental conditions. Phytochromes (phys) are light and temperature sensors that regulate downstream genes to render plants responsive to environmental stimuli1,2,3,4. Here, we show that phyB directly triggers the formation of a repressive chromatin loop by physically interacting with VERNALIZATION INSENSITIVE 3-LIKE1/VERNALIZATION 5 (VIL1/VRN5), a component of Polycomb Repressive Complex 2 (PRC2)5,6, in a light-dependent manner. VIL1 and phyB cooperatively contribute to the repression of growth-promoting genes through the enrichment of Histone H3 Lys27 trimethylation (H3K27me3), a repressive histone modification. In addition, phyB and VIL1 mediate the formation of a chromatin loop to facilitate the repression of ATHB2. Our findings show that phyB directly utilizes chromatin remodelling to regulate the expression of target genes in a light-dependent manner.

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Fig. 1: VIL1 and phyB synergistically inhibit hypocotyl elongation by regulating growth-promoting genes.
Fig. 2: VIL1 preferentially interacts with the active form of phyB both in vivo and in vitro.
Fig. 3: VIL1 and phyB cooperate to remodel the chromatin of the ATHB2 locus to control its expression.
Fig. 4: VIL1 and phyB cooperatively form a repressive chromatin loop to inhibit ATHB2 expression.

Data availability

The data supporting the findings of this study are available in the paper and Extended Data Figs. 110. Additional data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank R. Amasino and K. Torii for comments on the manuscript. This work was supported by NIH grant no. R01GM100108 and NSF IOS grant no. 1656764 to S.S. and NSF grant no. MCB-2014408 to E.H.

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Authors and Affiliations

Authors

Contributions

J.K., Y.B., P.K.K., B.Z., W.Z. and S.S. conceived of and implemented the method and performed the experiments and data analysis. J.K. and S.S. drafted the manuscript. S.S. and E.H. advised on the design and implementation and on the interpretation of the results and edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sibum Sung.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Eirini Kaiserli, Rongcheng Lin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 VIL1 and phyB synergistically inhibit hypocotyl elongation under various light conditions.

a, Hypocotyl length phenotype of seedlings of each genotypes grown for 7 days in long day (LD) (16 h light/8 h dark; 16 L/8D) or for 4 days in continuous white light (WLc) conditions. Scale bar: 5 mm. b - c, Quantification of hypocotyl length of seedlings grown in LD (b) or WLc (c), n = 25 seedlings. d, Hypocotyl length phenotype of seedlings of each genotype grown for 4 days in continuous red (Rc), far-red (FRc) or dark (Dc) conditions. The seedlings were treated with white light for 6 hours to induce germination before incubating under each light conditions. Scale bar: 5 mm. e - g, Quantification of hypocotyl length of seedlings grown in Rc (e), FRc (f), and Dc (g), n = 25 seedlings. In b - c and e - g, The box plot boundaries reflect the interquartile range, the horizontal line is the median and the whiskers represent 1.5X the interquartile range from the lower and upper quartiles. The letters above each box indicate statistical difference between the genotypes determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05).

Extended Data Fig. 2 VIL1 does not regulate the plant growth through PIF4.

Hypocotyl length of seedlings of each genotypes grown for 7 days at four different temperatures (12 °C, 17 °C, 22 °C, and 27 °C) in SD. b, Quantification of hypocotyl length, n = 20 seedlings. The box plot boundaries reflect the interquartile range, the horizontal line is the median and the whiskers represent 1.5X the interquartile range from the lower and upper quartiles. The letters marked as same color above each box indicate statistical difference between the genotypes at each temperature determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05). c and d, PIF4 mRNA expression in Col-0, vil1-1, phyB-9 and vil1-1phyB-9 seedlings grown for 7 days at four different temperatures in SD. Samples were collected at both ZT0 (c) and ZT6 (d). The expression levels were normalized to that of PP2A and further normalized by the transcript level of Col-0 at ZT0, at 12 °C. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The letters marked as same color above each bar indicate statistical difference between the genotypes at each temperature determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05). e, Immunoblot analysis showing that endogenous PIF4 protein levels are not changed by vil1. Proteins were extracted from seven-day-old seedlings grown in SD at ZT6. Endogenous tubulin levels were detected as controls by anti-tubulin antibody. The experiment was repeated at least two times independently with similar results.

Source data

Extended Data Fig. 3 VIL1 inhibits the plant growth by repressing growth-promoting genes.

a - c, mRNA expression of ATHB2 (a), HFR1 (b) and PIL1 (c) in Col-0, vil1-1, phyB-9 and vil1-1phyB-9 seedlings grown for 7 days at four different temperatures in SD. Samples were collected at ZT6. The expression levels were normalized to that of PP2A and further normalized by the transcript level of Col-0 at ZT0, at 12 °C shown in Fig. 1. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The letters marked as same color above each bar indicate statistical difference between the genotypes at each temperature determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05).

Extended Data Fig. 4 The expression of growth-promoting genes is dependent on PIF4.

a - c, mRNA expression of ATHB2 (a), HFR1 (b) and PIL1 (c) in Col-0 and pif4-2 seedlings grown for 7 days at 22 °C in SD. Samples were collected both at ZT0 and at ZT6. The asterisks indicate significant difference (P < 0.05). d to f, mRNA expression of ATHB2 (d), HFR1 (e), and PIL1 (f) in Col-0, vil1-1, phyB-9, vil1-1 phyB-9, pif4-2, vil1-1 pif4-2, and vil1-1 phyB-9 pif4-2 seedlings grown for 7 days at 22 °C in SD. Samples were collected at ZT0. The expression levels were normalized to that of PP2A and further normalized by the expression level of Col-0. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The letters above each bar indicate statistical difference between the genotypes determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05).

Extended Data Fig. 5 VIL1 alone does not regulate the expression of growth-promoting, hormone signaling genes.

a - d, mRNA expression of YUC8 (a, b) and IAA29 (c, d) in Col-0, vil1-1, phyB-9 and vil1-1phyB-9 seedlings grown for 7 days at four different temperatures in SD. Samples were collected at ZT0 (a, c) and ZT6 (b, d) respectively. The expression levels were normalized to that of PP2A and further normalized by the transcript level of Col-0 at ZT0, at 12 °C. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The letters marked as same color above each bar indicate statistical difference between the genotypes at each temperature determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05).

Extended Data Fig. 6 VIL1 protein does not interact with PIF4 protein in vitro.

In vitro pull-down assay showing that MBP-VIL1 protein does not interact with GST-PIF4 proteins. MBP, MBP-VIL1 and MBP-SPA1 proteins were expressed in E. coli and purified using amylose-agarose beads. GST-PIF4 was also expressed in E. coli. Amylose-agarose bound MBP, MBP-VIL1 and MBP-SPA1 were used to pull-down different amount of purified GST-PIF4 protein. Pull-down samples were loaded 10% SDS-PAGE. GST-PIF4 was detected using anti-GST antibody, while the bait protein was visualized following Coomassie blue staining. The asterisks indicate each of MBP, MBP-VIL1 or MBP-SPA1 protein. The experiment was repeated at least two times independently with similar results.

Source data

Extended Data Fig. 7 VIL1 and phyB directly regulate the expression of HFR1 and PIL1 by inducing H3K27me3 on their loci.

a and c, Hypocotyl length phenotype of VIL1 complementation lines (pVIL1:VIL1-myc/vil1-1 and pVIL1:VIL1-Flag/vil1-1) and another allele of vil1 mutant (vil1-2). The seedlings were grown for 7 days at 22 °C in SD. A scale bar = 5 mm. b and d, Quantification data of hypocotyl length shown in a, n = 25 seedlings. The box plot boundaries reflect the interquartile range, the horizontal line is the median and the whiskers represent 1.5X the interquartile range from the lower and upper quartiles. The letters above each box indicate statistical difference between the genotypes determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05). e, ATHB2, HFR1 and PIL1 loci, G-box elements, ChIP-qPCR amplicons. The red vertical bars indicate G-box regions. RE1, RE2, RE, P1, and P2 indicate ChIP-qPCR amplicons. f, ChIP assay showing the relative enrichments of VIL1-myc in phyB-9 on ATHB2, HFR1, and PIL1 loci by comparing to enrichments of VIL1-myc at ZT0 and at ZT6. The ChIP assay used anti-myc antibody on seven days old seedlings grown at 22 °C in SD. The immunoprecipitated DNA relative to input was further normalized to that of 5S rDNA. Error bars: ± s.d., n = 3, biological replicates. The asterisks indicate statistical difference in a two-tailed student t-test (P < 0.05). g, H3K27me3 levels in Col-0, vil1-1, phyB-9, vil1-1phyB-9, and pif4-2 at ZT6. This ChIP assay used anti-H3K27me3 antibody and anti-H3 antibody. The y axis indicates H3K27me3 enrichment relative to H3 enrichment (normalized by that of 5S rDNA). Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The asterisks indicate statistical differences in a two-tailed student t-test (P < 0.05). n.s. indicates not significant.

Source data

Extended Data Fig. 8 VIL1 protein is stabilized by light and VIL1 enrichment is dependent on phyB.

a and b, Diurnally regulated VIL1-myc protein levels (a) and VIL1 mRNA levels (b). a, The seedlings of pVIL1:VIL1-myc/vil1-1 transgenic plants grown at 22 °C in SD for 7 days were collected at eight different time points (ZT0 to ZT20). The VIL1-myc protein levels were detected by anti-myc antibody. Endogenous tubulin levels were detected as controls by anti-tubulin antibody. b, VIL1 mRNA levels in Col-0 seedlings grown at 22 °C in SD for 7 days. Samples were collected at eight different time points (ZT0 to ZT20). The expression levels are normalized to that of PP2A. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). c, VIL1-myc protein levels were stabilized in light. Four-day-old dark grown seedlings were either kept in the dark or treated with continuous red-light (10.7 µmol m-2 s-1) for the period indicated in the panel. Anti-myc and anti-RPT5 antibodies were used to detect VIL1-myc and RPT5 (control), respectively. d, Unchanged VIL1-myc protein levels in phyB-9 mutants. Proteins were extracted in the seedlings grown for 7 days at 22 °C in SD at ZT6. In a, c and d, all experiments were repeated at least two times independently with similar results. e, FLC locus with ChIP-qPCR amplicons (P1 and P2). f, Similar enrichments of VIL1 on FLC locus between at ZT0 and at ZT6. Error bars: ± s.d., n = 3, biological replicates. g, ChIP assay showing that relative enrichments of VIL1-myc on ATHB2 are reduced in phyB-9 at ZT6. Error bars: ± s.d. n = 3, biological replicates. f and g, The ChIP assay used anti-myc antibody on seven days old seedlings grown at 22 °C in SD. The immunoprecipitated DNA relative to input was further normalized to that of 5S rDNA. In f and g, the asterisks indicate statistical difference in a two-tailed student t-test (***P < 0.0005, *P < 0.05), n.s. indicates not significant.

Extended Data Fig. 9 PRC2 targets the growth-promoting genes.

The IGV views showing the enrichments of CLF-GFP and SWN-GFP, and H3K27me3 in WT, clf, swn and clf swn double mutants on ATHB2, HFR1 and PIL1 loci.

Extended Data Fig. 10 phyB coordinates with PRC2 through VIL1 to regulate its target genes.

a, Hypocotyl length of seedlings of each genotypes grown for 7 days at four different temperatures (12 °C, 17 °C, 22 °C, and 27 °C) in SD. Scale bar: 5 mm. b, Quantification of hypocotyl length, n = 20 seedlings. c, Hypocotyl length of seedlings of Col-0, vil1-1, vrn2, phyB-9, vil1-1 phyB-9, and vrn2 phyB-9 grown for 7 days at 22 °C in SD, Scale bar: 5 mm, n = 20 seedlings. In b and c, the box plot boundaries reflect the interquartile range, the horizontal line is the median and the whiskers represent 1.5X the interquartile range from the lower and upper quartiles. The letters marked as same color above each box indicate statistical difference between the genotypes at each temperature determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05). d and e, Relative interaction frequency (RIF) in 3C assay between the Anchor primer and a series of F(forward) primers (d), or between the Anchor primer and a series of R(reverse) primers (e) in Col-0, and the vrn2 mutant at ZT6. The enrichment of each region was normalized to that of PP2A. These values were further normalized to the enrichment of each region in pATHB2:ATHB2-Flag plasmid DNAs. Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). f and g, Model: A novel phyB regulatory module. f, phyB inhibits PIF4 by promoting its protein degradation and repression of DNA binding activity to repress the plant growth under the light. Our model shows a novel regulatory module in which the active form of phyB directly interacts with VIL1 to mediate chromatin remodeling at the growth-promoting genes by recruiting PRC2. Chromatin remodeling by the phyB-VIL1 module ensures the repression of growth-promoting genes, which otherwise can be activated by PIF4. g, On ATHB2 locus, active phyB forms a repressive chromatin loop with VIL1-PRC2. The presence of both phyB and VIL1 is important for the loop formation and for repressive H3K27me3 mark on this locus to fully inhibit ATHB2 expression. However, since neither phyB nor VIL1 can directly bind to DNA, it is possible that unknown DNA-binding protein (X) may be involved in phyB-VIL1 association with the target chromatin.

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Kim, J., Bordiya, Y., Kathare, P.K. et al. Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1. Nat. Plants 7, 1213–1219 (2021). https://doi.org/10.1038/s41477-021-00986-y

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