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REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis

Abstract

RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) counteracts Polycomb-mediated gene silencing by removing methyl groups from trimethylated histone H3 lysine 27 (H3K27me3) in hundreds of genes in Arabidopsis thaliana1. Here we show that REF6 function and genome-wide targeting require its four Cys2His2 zinc fingers, which directly recognize a CTCTGYTY motif. Motifs bound by REF6 tend to cluster and reside in loci with active chromatin states. Furthermore, REF6 targets CUP-SHAPED COTYLEDON 1 (CUC1), which harbors CTCTGYTY motifs, to modulate H3K27me3 levels and activate CUC1 expression. Loss of REF6 causes CUC1 repression and defects in cotyledon separation. In contrast, REF6 does not bind CUC2, encoding a close homolog of CUC1, which lacks the CTCTGYTY motif. Collectively, these results identify a new targeting mechanism of an H3K27 demethylase to counteract Polycomb-mediated gene silencing that regulates plant development, including organ boundary formation.

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Figure 1: The C2H2-ZnF domains of REF6 are essential for REF6 function.
Figure 2: The C2H2-ZnF cluster is essential for REF6 function in H3K27me3 demethylation by regulating REF6 genomic binding.
Figure 3: The REF6 C2H2-ZnF cluster binds a specific DNA motif enriched in REF6-binding sites.
Figure 4: Distribution of CTCTGYTY motifs in the Arabidopsis genome.
Figure 5: CUC1 is a direct target of REF6.

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References

  1. Lu, F., Cui, X., Zhang, S., Jenuwein, T. & Cao, X. Arabidopsis REF6 is a histone H3 lysine 27 demethylase. Nat. Genet. 43, 715–719 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Holec, S. & Berger, F. Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 158, 35–43 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Steffen, P.A. & Ringrose, L. What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory. Nat. Rev. Mol. Cell Biol. 15, 340–356 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Lafos, M. et al. Dynamic regulation of H3K27 trimethylation during Arabidopsis differentiation. PLoS Genet. 7, e1002040 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sun, B. et al. Timing mechanism dependent on cell division is invoked by Polycomb eviction in plant stem cells. Science 343, 1248559 (2014).

    Article  PubMed  Google Scholar 

  7. Wu, M.F. et al. SWI2/SNF2 chromatin remodeling ATPases overcome Polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proc. Natl. Acad. Sci. USA 109, 3576–3581 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang, X. et al. Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 5, e129 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Hou, X. et al. Nuclear factor Y–mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis. Nat. Commun. 5, 4601 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Noh, B. et al. Divergent roles of a pair of homologous jumonji/zinc-finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16, 2601–2613 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu, X. et al. Modulation of brassinosteroid-regulated gene expression by Jumonji domain–containing proteins ELF6 and REF6 in Arabidopsis. Proc. Natl. Acad. Sci. USA 105, 7618–7623 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sun, Y. et al. Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev. Cell 19, 765–777 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yu, X. et al. A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 65, 634–646 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Lu, F., Cui, X., Zhang, S., Liu, C. & Cao, X. JMJ14 is an H3K4 demethylase regulating flowering time in Arabidopsis. Cell Res. 20, 387–390 (2010).

    Article  PubMed  Google Scholar 

  15. Larsson, A.S., Landberg, K. & Meeks-Wagner, D.R. The TERMINAL FLOWER2 (TFL2) gene controls the reproductive transition and meristem identity in Arabidopsis thaliana. Genetics 149, 597–605 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Klug, A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu. Rev. Biochem. 79, 213–231 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bailey, T.L. & Machanick, P. Inferring direct DNA binding from ChIP-seq. Nucleic Acids Res. 40, e128 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang, S. et al. C-terminal domains of a histone demethylase interact with a pair of transcription factors and mediate specific chromatin association. Cell Discovery 1, 15003 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. van Dijk, K. et al. Dynamic changes in genome-wide histone H3 lysine 4 methylation patterns in response to dehydration stress in Arabidopsis thaliana. BMC Plant Biol. 10, 238 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Coleman-Derr, D. & Zilberman, D. Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoS Genet. 8, e1002988 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sullivan, A.M. et al. Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana. Cell Rep. 8, 2015–2030 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Inagaki, S. et al. Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome. EMBO J. 29, 3496–3506 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. & Tasaka, M. Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9, 841–857 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hibara, K. et al. Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation. Plant Cell 18, 2946–2957 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Takada, S., Hibara, K., Ishida, T. & Tasaka, M. The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation. Development 128, 1127–1135 (2001).

    CAS  PubMed  Google Scholar 

  27. Vroemen, C.W., Mordhorst, A.P., Albrecht, C., Kwaaitaal, M.A. & de Vries, S.C. The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in Arabidopsis. Plant Cell 15, 1563–1577 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Crevillén, P. et al. Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state. Nature 515, 587–590 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lu, F. et al. Comparative analysis of JmjC domain–containing proteins reveals the potential histone demethylases in Arabidopsis and rice. J. Integr. Plant Biol. 50, 886–896 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Amborella Genome Project. The Amborella genome and the evolution of flowering plants. Science 342, 1241089 (2013).

  31. Najafabadi, H.S. et al. C2H2 zinc finger proteins greatly expand the human regulatory lexicon. Nat. Biotechnol. 33, 555–562 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Bowler, C. et al. Chromatin techniques for plant cells. Plant J. 39, 776–789 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  36. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Grant, C.E., Bailey, T.L. & Noble, W.S. FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017–1018 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Finnegan, E.J. & Dennis, E.S. Vernalization-induced trimethylation of histone H3 lysine 27 at FLC is not maintained in mitotically quiescent cells. Curr. Biol. 17, 1978–1983 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Lin, R. et al. Transposase-derived transcription factors regulate light signaling in Arabidopsis. Science 318, 1302–1305 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Q. Zhu for technical assistance and W. Qian for discussion. We thank the Arabidopsis Biological Resource Center for providing T-DNA insertion lines. This work was supported by the National Basic Research Program of China (grants 2013CB967300 to Xia Cui and 2013CB835200 to X. Cao), the National Natural Science Foundation of China (grants 31210103901 to X. Cao, 31271363 to Xia Cui, and 31428011 to X.Z.), and the State Key Laboratory of Plant Genomics (2014B0227-01 and 2015B0129-01). Research in the laboratory of X.Z. was supported by National Science Foundation grant 0960425. B.Z., Xiekui Cui, and X.M. were supported by the China Postdoctoral Science Foundation (2012M520020, 2014M550874, and 2014M550101, respectively).

Author information

Authors and Affiliations

Authors

Contributions

Xia Cui, F.L., Q.Q., and X. Cao conceived and designed the study. Xia Cui, F.L., and Q.Q. performed most of the experiments with help from S.Z., Y.K., Xiekui Cui, and Q.Y. High-throughput sequencing data were analyzed by B.Z. with help from L.G. and X.M. J.M. provided essential reagents for the EMSA analysis. Xia Cui, F.L., Q.Q., B.Z., X.Z., and X. Cao interpreted the data. F.L., Q.Q., B.Z., X.Z., and X. Cao wrote the manuscript.

Corresponding author

Correspondence to Xiaofeng Cao.

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Integrated supplementary information

Supplementary Figure 1 REF6 expression in transgenic lines.

(a,b) Expression of REF6 in REF6-HA ref6 and REF6ΔZnF-HA ref6 plants detected by RT–qPCR (a) and immunoblot (b). Gene expression was normalized to that of the control gene TUBULIN2 (AT5G62960). RT-qPCR was performed with four technical replicates. Data are shown as means ± s.e. (n = 4). LHP1 was used as the loading control for the immunoblot. (c) pREF6::REF6ΔZnF-HA cannot rescue the short-petiole phenotypes of ref6 mutants. Scale bar, 1 cm.

Supplementary Figure 2 H3K27me3 hypermethylation in REF6ΔZnF-HA ref6 is similar to that in ref6.

(a) Number of genes showing H3K27me3 hypermethylation in ref6 and REF6ΔZnF-HA ref6 plants. (b) Diagram showing the comparison of H3K27me3-hypermethylated genes in ref6-1 and ref6-3 (ref. 1). The greater number of H3K27me3-hypermethylated genes called in ref6-1 compared to ref6-3 is mainly due to greater sequencing depth in ChIP-seq experiments. (c) Density profile of H3K27me3 in 1,000 randomly selected genes. The H3K27me3 signal was summarized in fixed 1-kb regions around the transcriptional start site (TSS), transcriptional termination site (TTS), and center of the gene.

Supplementary Figure 3 The C2H2-ZnF domains are dispensable for the H3K27me3 and H3K27me2 demethylase activity of REF6.

(a) Overexpression of REF6ΔZnF-YFP-HA reduced the levels of H3K27me3 and H3K27me2 in vivo. REF6ΔZnF-YFP-HA fusion protein was transiently expressed in tobacco, and nuclei were isolated for immunostaining. More than 25 pairs of non-transfected nuclei versus transfected nuclei in the same field of view were analyzed. Arrows indicate transfected nuclei. Scale bars, 2 μm. (b) Quantitative analysis of a. Data are shown as means ± s.e. (n = 20). WT, wild type (non-transfected nuclei). (c) REF6ΔZnFox plants showed similar phenotypes to REF6ox plants. The plants showed upward-curling leaves with deeper serrations in the margin, early flowering, and terminal flower phenotypes of various degrees. Scale bar, 1 cm. (d) Activation of Polycomb target genes in REF6ΔZnFox plants. Expression of Polycomb target genes was analyzed by RT–qPCR. Gene expression is normalized to that of the control gene TUBULIN2 (AT5G62960). RT-qPCR was performed with four technical replicates. Data are shown as means ± s.e. (n = 4). (e) H3K27me3 and H3K27me2 levels are reduced in REF6ΔZnFox plants. H3 lysine methylation status is shown in two REF6ΔZnFox lines and one REF6ox line as detected by immunoblotting with the antibodies specified on the right. Immunoblotting with antibody to H3 showed equal loading.

Supplementary Figure 4 REF6 direct targets identified by ChIP-seq with antibody to HA.

(a) Density profile of REF6 binding signals in 1,000 randomly selected genes. REF6 binding signal was summarized in fixed 1-kb regions around the transcriptional start site (TSS), transcriptional termination site (TTS), and center of the gene. (b) Number of genes showing enrichment in REF6-HA ref6 and REF6ΔZnF-HA ref6 plants by ChIP-seq using antibody to HA.

Supplementary Figure 5 Genome Browser view of ChIP-seq data and ChIP–qPCR validation.

(a,b) ChIP-seq data and ChIP–qPCR validation for NAC004 (a) and SUS3 (b) loci. ChIP–qPCR used another biological replicate of samples. Data from ChIP–qPCR analysis are shown as assay-site fold enrichment of the signal from immunoprecipitation over the background. Col was used as the negative-control sample. ChIP–qPCR was performed with four technical replicates. Data are shown as means ± s.e. (n = 4). (c,d) ChIP-seq data at another two genes, HB23 (AT1G26960) (c) and CER3 (AT5G57800) (d). Regions validated by ChIP–qPCR in a and b are marked by black lines on top of the gene model. The locations of CTCTGYTY motifs are indicated by blue bars above the gene models. NA, not analyzed.

Supplementary Figure 6 EMSA showing that the CTCTGYTY motif but not the flanking sequence is important for protein–DNA interaction.

(a,d) Sequences of 50-bp DNA fragments of the NAC04 (a) and CUC1 (d) loci, containing one and two CTCTGTTT motifs, respectively, and their mutant versions used in b, c, and e. WT, wild type. (b) The flanking sequence has minimal effect on DNA–protein interaction. (c) GST-REF6C interacts with probes containing the other three variants of the CTCTGYTY motif. (e) EMSA showing that one CTCTGTTT motif is sufficient for DNA–protein interaction.

Supplementary Figure 7 Occupancy profiles of REF6 binding and hypermethylated H3K27me3 sites around peak summits in response to the number of motifs within REF6 binding peaks.

Supplementary Figure 8 REF6 binds CUC1 and CUC3 in shoot apical tissues.

(a,b) The CTCTGYTY motifs at the CUC1 (a) and CUC3 (b) loci. The numbers indicate base positions from the TSS. The motifs in the sense strand are labeled in red, and the motifs in the anti-sense strand are labeled in blue. Nucleotides in exons and introns are shown in uppercase and lowercase letters, respectively. Nucleotides in qPCR-detected regions are shown in bold letters. (c,d,f) In shoot apical tissues (Online Methods), HA ChIP–qPCR (c) and RT–qPCR (d,f) showed that REF6 binds CUC3 but does not affect the transcript level of CUC3. (e) Transcript levels of CUC2 and CUC3 are not changed in ref6 mutants (14-d-old seedlings). Data from ChIP–qPCR analysis are shown as assay-site fold enrichment of the signal from immunoprecipitation over the background. Col was used as the negative control. Gene expression is normalized to that of the control gene ACTIN7 (AT5G09810). ChIP–qPCR and RT-qPCR were performed with four technical replicates. Data are shown as means ± s.e. (n = 4). NA, not analyzed.

Supplementary Figure 9 Phenotype of ref6 cuc double and triple mutants.

Representative 10-d-old seedlings with different phenotypes are shown. Scale bars, 2 mm.

Supplementary Figure 10 Role of the CTCTGYTY motif and chromatin states in REF6 targeting to activate gene expression through H3K27me3 demethylation.

REF6 binds the CTCTGYTY motif through the C2H2-ZnF cluster and removes local H3K27me3 methylation. Genes containing clusters of CTCTGYTY motifs recruit REF6 more efficiently (top), whereas motifs in heterochromatic regions cannot recruit REF6 (bottom).

Supplementary Figure 11 Phylogenetic tree of REF6 homologs in plants.

The phylogenetic tree was constructed with MEGA (ver.5.05)44 using the neighbor-joining method. The red branches mark the monocot species, and the blue branches mark the eudicots. Amborella trichopoda is the most basal lineage in the clade of angiosperms (‘basal angiosperms’)30. Selaginella moellendorffii and Physcomitrella patens are outgroups.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Table 1. (PDF 2056 kb)

Supplementary Table 2

Chromosomal regions in which H3K27me3 sites were hypermethylated by more than 2-fold in ref6. (XLS 345 kb)

Supplementary Table 3

Chromosomal regions in which H3K27me3 sites were hypermethylated by more than 2-fold in REF6ΔZnF-HA ref6. (XLS 326 kb)

Supplementary Table 4

Chromosomal regions of REF6 binding peaks. (XLS 611 kb)

Supplementary Table 5

REF6 binding peaks for MEME-ChIP analysis. (XLSX 198 kb)

Supplementary Table 6

CTCTGYTY motifs in the Arabidopsis genome. (XLSX 1556 kb)

Supplementary Table 7

List of CTCTGYTY motif clusters. (XLSX 99 kb)

Supplementary Table 8

Primers used in this study. (XLSX 21 kb)

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Cui, X., Lu, F., Qiu, Q. et al. REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in Arabidopsis. Nat Genet 48, 694–699 (2016). https://doi.org/10.1038/ng.3556

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