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RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ�2) counteracts Polycomb-mediated gene silencing by removing methyl groups from trimethylated histone H3 lysine 27 (H3K27me3) in hundreds of genes in Arabidopsis thaliana�. 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.

In animals and plants, H3K27me3 facilitates the maintenance of developmentally regulated genes in a transcriptionally repressed state. The establishment and removal of H3K27me3 at specific genes is therefore critically important for normal development2–4. In Arabidopsis, ~3,000–4,000 genes are marked by H3K27me3, many of which are important transcriptional regulators that respond to devel-opmental or environmental cues5–8. The REF6 protein (also known as JMJ12) specifically demethylates H3K27me3 at its target loci for transcriptional activation1. Loss of REF6 leads to the ectopic accu-mulation of H3K27me3 at hundreds of genes in seedlings1, as well as a number of developmental phenotypes such as late flowering and short petioles9–11. However, how REF6 is targeted to specific genes remains unknown.

REF6 contains a tandem array of four Cys2His2 zinc-finger (C2H2-ZnF) domains at its C terminus (Fig. 1a). To investigate whether REF6 function requires the C2H2-ZnF domains, we trans-formed the ref6 mutant (ref6-1 unless otherwise specified) with two different constructs, pREF6::REF6-HA (referred to as REF6-HA ref6 hereafter) encoding full-length REF6 and pREF6::REF6∆ZnF-HA (referred to as REF6∆ZnF-HA ref6 hereafter) encoding truncated REF6 without the C2H2-ZnF domains (amino acids 1,243–1,352). Transgenic lines that showed REF6 transcript levels comparable to those of wild-type Columbia (Col) plants were chosen for further analysis (Supplementary Fig. 1a,b). The pREF6::REF6-HA transgene largely rescued the late-flowering and short-petiole phenotypes of the ref6 mutant1, whereas the pREF6::REF6∆ZnF-HA transgene did not (Fig. 1b,c and Supplementary Fig. 1c). In addition to making phenotypic observations, we examined the expression of FLC and several brassinosteroid-inducible genes that are misregulated in the ref6 mutant1,12,13. Consistent with the morphological changes, these genes showed similar transcript levels in REF6∆ZnF-HA ref6 and ref6 plants (Fig. 1d,e). Taken together, these data indicate that REF6 function requires the C2H2-ZnF cluster.

The phenotypic and transcriptional defects seen in REF6∆ZnF-HA ref6 plants resembled those of the ref6 mutant, suggesting that the genome-wide H3K27me3 hypermethylation in ref6 is not rescued by REF6∆ZnF-HA. Therefore, we performed H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) in Col, ref6, REF6-HA ref6, and REF6∆ZnF-HA ref6 plants (Supplementary Table 1). A total of 1,471 regions covering 1,688 genes showed significantly higher levels of H3K27me3 in the ref6 mutant than in wild-type Col (Supplementary Fig. 2a and Supplementary Table 2). These H3K27me3-hypermethylated regions in ref6-1 significantly overlapped with those reported in the ref6-3 mutant

REF6 recognizes a specific DNA sequence to demethylate H3K27me3 and regulate organ boundary formation in ArabidopsisXia Cui1,8,9, Falong Lu1,2,8,9, Qi Qiu1,2,9, Bing Zhou1,8,9, Lianfeng Gu1,8, Shuaibin Zhang1,2, Yanyuan Kang1,2, Xiekui Cui1,2, Xuan Ma1,3, Qingqing Yao4,5, Jinbiao Ma4,5, Xiaoyu Zhang6 & Xiaofeng Cao1,5,7

1State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. 2University of Chinese Academy of Sciences, Beijing, China. 3Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, China. 4School of Life Sciences, Fudan University, Shanghai, China. 5Collaborative Innovation Center of Genetics and Development, Shanghai, China. 6Department of Plant Biology, University of Georgia, Athens, Georgia, USA. 7CAS Center for Excellence in Molecular Plant Sciences, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. 8Present addresses: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China (Xia Cui), Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, Massachusetts, USA (F.L.), Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA (B.Z.) and Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China (L.G.). 9These authors contributed equally to this work. Correspondence should be addressed to X. Cao (xfcao@genetics.ac.cn).

Received 17 February 2015; accepted 1 April 2016; published online 25 April 2016; doi:10.1038/ng.3556

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(Fisher’s exact test, P < 2.2 × 10−16; Supplementary Fig. 2b)1. A total of 1,342 H3K27me3-hypermethylated regions covering 1,560 genes were found in REF6∆ZnF-HA ref6 (Fig. 2a, Supplementary Fig. 2a, and Supplementary Table 3), which significantly overlapped with those in ref6 (Fisher’s exact test, P < 2.2 × 10−16; Fig. 2a). In contrast, most of the H3K27me3 hypermethylation was eliminated in REF6-HA ref6 (Supplementary Fig. 2a). Notably, the distribution of H3K27me3 on REF6-targeted genes was highly similar in ref6 and REF6∆ZnF-HA ref6 plants, when compared with REF6-HA ref6 and wild-type plants

(Fig. 2b), whereas H3K27me3 signal at a set of randomly selected genes appeared similar in all genotypes (Supplementary Fig. 2c). Taken together, these results demonstrate that the C2H2-ZnF cluster is required for the function of REF6 in genome-wide H3K27me3 demethylation.

The C2H2-ZnF domains could be required for REF6 demethy-lase activity or for the recruitment of REF6 to its target genes. To distinguish between these possibilities, we first tested whether the REF6∆ZnF protein was enzymatically active. We found that

FLC

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Figure 1 The C2H2-ZnF domains of REF6 are essential for REF6 function. (a) Diagram of the proteins encoded by the pREF6::REF6-HA and pREF6: :REF6∆ZnF-HA constructs. (b,c) pREF6::REF6∆ZnF-HA cannot rescue the late-flowering (b) and short-petiole (c) phenotypes of the ref6 mutant. Flowering time for the different transgenic plants was assessed by counting leaf numbers in bolting plants. The petiole length of the fifth true leaf was measured at 40 d after germination. Data are shown as means ± s.d. (n = 20 for flowering time, n = 15 for petiole length). A two-sided Student’s t test comparing wild type (Col) with other genotypes was performed: *P < 0.05, **P < 0.01. (d) Expression of FLC in different transgenic plants detected by RNA blotting. FDH (AT5G43940) was used as a control. (e) Expression of REF6 target genes in different transgenic plants. Expression levels in 14-d-old transgenic plants were measured 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.m.

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Figure 2 The C2H2-ZnF cluster is essential for REF6 function in H3K27me3 demethylation by regulating REF6 genomic binding. (a) Venn diagram showing significant (Fisher’s exact test, P < 2.2 × 10−6) overlap of H3K27me3-hypermethylated genes in ref6 and REF6∆ZnF-HA ref6 plants. (b,c) Density profiles of H3K27me3 (b) and REF6 (c) signal at genes showing H3K27me3 hypermethylation in ref6 plants. The distribution of H3K27me3 and REF6 signal for each gene in fixed 1-kb regions around the transcription start site (TSS), transcription termination site (TTS), and center of the gene was summarized and plotted. Hierarchical clustering of these samples based on each gene profile is shown at the top. (d) Venn diagram showing significant (Fisher’s exact test, P < 2.2 × 10−6) overlap between the H3K27me3-hypermethylated genes in ref6 and REF6-binding genes. (e) Representative genome browser view of H3K27me3 and REF6-HA binding in different genotypes. (f) H3K27me3 and REF6-HA ChIP-seq data for the TCH4 locus (left) and qPCR validation using ChIP samples from another biological replicate (right). 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. ChIP–qPCR was performed with four technical replicates. Data are shown as means ± s.e.m. The locations of CTCTGYTY motifs are indicated by blue bars below the gene model. The regions validated by ChIP–qPCR are marked by black lines above the gene model. NA, not analyzed.

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REF6∆ZnF could demethylate H3K27me3 and dimethylated H3K27 (H3K27me2) in an in vivo histone demethylation assay1,14 (Supplementary Fig. 3a,b). In addition, overexpression of REF6∆ZnF in wild type (REF6∆ZnFox) led to a global reduction in H3K27me3 levels, as well as phenotypes similar to those seen in REF6ox and some Polycomb-deficient mutants1,15 (Supplementary Fig. 3c–e). Therefore, we conclude that the C2H2-ZnF domains are dispensable for the H3K27 demethylase activity of REF6.

To determine whether the C2H2-ZnF domains have a role in REF6 targeting, we profiled the genome-wide localization of REF6 (in REF6-HA ref6) and REF6∆ZnF (in REF6∆ZnF-HA ref6) using ChIP-seq with an antibody to HA. A wild-type sample was included as a negative control. The background signals were similar in all three genotypes (Supplementary Fig. 4a). A total of 3,094 peaks covering 2,836 genes were bound by REF6. In contrast, only 22 peaks cover-ing 20 genes were bound by REF6∆ZnF (Supplementary Fig. 4b and

Supplementary Table 4), and ChIP-seq signals at REF6 target genes were virtually identical in REF6∆ZnF-HA ref6 and the negative con-trol (Fig. 2c). Notably, REF6-bound genes significantly overlapped with those showing H3K27me3 hypermethylation in the ref6 mutant (Fisher’s exact test, P < 2.2 × 10−16), suggesting that REF6 binding provides the specificity for its H3K27 demethylase activity (Fig. 2d,e and Supplementary Fig. 5). Validation of H3K27me3 changes and REF6 binding by qPCR on an independent batch of ChIP samples (ChIP–qPCR) yielded highly consistent results (Fig. 2f and Supplementary Fig. 5). Taken together, these findings indicate that the C2H2-ZnF domains are essential for REF6 binding to its target genes.

Because C2H2-ZnF domains often possess sequence-specific DNA-binding activity16, it was of interest to determine whether REF6 can bind to its targets in a sequence-specific manner. A motif discov-ery analysis using MEME-ChIP17,18 identified the DNA sequence CTCTGYTY (where Y represents T or C) as the most significantly

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g h iFigure 3 The REF6 C2H2-ZnF cluster binds a specific DNA motif enriched in REF6-binding sites. (a) DNA sequence of the motif enriched in REF6-binding sites. The DNA sequences flanking REF6 binding summits (−400 bp to +400 bp) were used for de novo motif discovery with MEME-ChIP. (b) Distribution of the CTCTGYTY motif in REF6 binding peaks. (c) Sequences of the 50-bp DNA probe corresponding to a segment of the NAC004 locus with one CTCTGTTT motif (NAC004-WT) and mutant versions of this probe used in d and e. The motif is highlighted in red, and mutated bases are shown in lowercase. (d) EMSA showing that GST-REF6C containing the C2H2-ZnF cluster but not GST binds the NAC004 probe in vitro. ZnF mutants (cysteine to alanine) abolished (ZnF2mu–ZnF4mu) or severely attenuated (ZnF1mu) the protein–DNA interaction. (e) EMSA showing that single-base mutations in the CTCTGTTT motif of the NAC004 probe either abolish or weaken the interaction between GST-REF6C and the DNA probe. (f) EMSA showing that excess wild-type but not mutant unlabeled oligonucleotide can outcompete the wild-type labeled probe. (g) The distance between adjacent REF6-bound (REF6+) motifs is much shorter than that between unbound (REF6−) motifs. (h) The number of motifs in each 600-bp genomic window shows a positive correlation with the fraction of the windows bound by REF6. (i) The number of motifs in REF6 binding peaks shows a positive correlation with the level of REF6 binding (peak score). Box plots show the median (middle bar) and interquartile range (IQR; from the 25th to 75th percentile); whiskers extend to minimum and maximum values within 1.5 times the IQR. Further information about CTCTGYTY motifs and REF6 binding peaks can be found in supplementary tables 5–7.

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Figure 4 Distribution of CTCTGYTY motifs in the Arabidopsis genome. (a–c) The distribution (a), average profiles for histone variants and modifications19–21 (b), and overlap with DNase I–hypersensitive sites (DHSs)22 (c) are compared for bound and unbound motifs. The positions of the CTCTGYTY motifs in the genome and their relationship to REF6 peaks (REF6 bound/unbound) can be found in supplementary table 6. Promoters were defined as the 1-kb region immediately upstream of the TSS of each gene. IGR, intergenic region.

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enriched motif (P = 5.1 × 10−650), and this motif was supported by 76% of the detected peaks (Fig. 3a and Supplementary Table 5). Notably, this motif was centered on REF6 binding peaks (Fig. 3b), suggesting that it may have a role in REF6 recruitment.

To determine whether REF6 can directly bind the CTCTGYTY motif, we performed electrophoretic mobility shift assays (EMSAs) using a 50-bp DNA fragment from the NAC004 gene containing one CTCTGTTT motif (Fig. 3c) and recombinant protein correspond-ing to the C-terminal part of REF6 containing the four C2H2-ZnF domains with a GST tag at the N terminus (GST-REF6C; amino acids 1,239–1,360). We found that GST-REF6C but not GST bound the NAC004 probe (Fig. 3c,d). In addition, cysteine-to-alanine substitu-tion in the second, third, or fourth C2H2-ZnF domain abolished the protein–DNA interaction, while an analogous substitution in the first C2H2-ZnF domain severely reduced binding affinity (Fig. 3d).

To further characterize the sequence specificity of the C2H2-ZnF domains, we mutated each base within the motif as well as one flanking base of the NAC004 probe to adenosine (Fig. 3c and Supplementary Fig. 6a). In EMSA results with the mutant probes, CTCTGY seemed to be the core sequence recognized by REF6, whereas TY contrib-utes additional REF6 binding affinity (Fig. 3e,f and Supplementary Fig. 6b). Furthermore, GST-REF6C bound three other probes that contained the CTCTGYTY motif, NAC004_GTTC, NAC004_GCTT, and NAC004_GCTC (Supplementary Fig. 6a,c). Taken together, these results indicate that the C2H2-ZnF domains target REF6 by directly recognizing the CTCTGYTY motif.

Of the 37,812 CTCTGYTY motifs in the Arabidopsis genome, ~15% were within REF6-bound regions, indicating that other factors may affect recognition of the motifs by REF6. We found that REF6-bound

CTCTGYTY motifs tended to be enriched in genic regions, where they cluster together (Figs. 3g–i and 4a, and Supplementary Table 6). Indeed, genomic regions with higher densities of these motifs were preferentially bound by REF6 (Fig. 3h and Supplementary Table 7), and the number of motifs was positively correlated with peak score (Fig. 3i and Supplementary Table 5).

In addition to motif density, we also compared chromatin states in the regions with and without REF6 binding. Motifs bound by REF6 tended to be more enriched for histone modifications or variants associated with active transcription (H3K4me3 (ref. 19), H3K4me2 (ref. 20), and H2A.Z21), more accessible to DNase I (ref. 22), and depleted for a heterochromatic modification (H3K9me2) (Fig. 4b,c). Taken together, these results suggest that the recognition of a particu-lar CTCTGYTY motif by REF6 may be modulated by motif density as well as associated chromatin states.

Because H3K27me3 is often found throughout the entire length of target genes8, it was of interest to determine whether REF6 demethylase activity extends beyond the immediate boundaries of REF6-binding sites. We therefore compared REF6-demethyl-ated regions (those with H3K27me3 hypermethylation in ref6) to REF6-binding sites. Interestingly, although the lengths of REF6-bound regions and REF6-demethylated regions were both positively correlated with the number of CTCTGYTY motifs (Supplementary Fig. 7), the full width at half maximum (FWHM) of REF6 peaks was around 400 bp, whereas the FWHM of H3K27me3 demethylation peaks was around 1 kb, indicating that REF6-mediated H3K27me3 demethylation is confined to REF6-binding sites, different from the activity of the H3K9me2 demethylase AtIBM1 (also known as JMJ25)23.

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Figure 5 CUC1 is a direct target of REF6. (a) Normally shaped (top) and heart-shaped (bottom) cotyledons seen in ref6. Scale bars, 2 mm. (b) Genome browser view of REF6 binding and H3K27me3 signal for different genotypes at the CUC1, CUC2, and CUC3 loci. Gene models from TAIR10 are shown at the bottom. The region validated by ChIP–qPCR in c and d is marked by a red bar above the CUC1 gene model. The locations of CTCTGYTY motifs are indicated by blue bars below the gene models. (c,d) ChIP–qPCR validation of REF6 binding (c) and H3K27me3 levels (d) at CUC1 using ChIP samples from another biological replicate. Data are shown as assay-site fold enrichment of the signal from immunoprecipitation over the background. Col was used as the negative control. ChIP–qPCR was performed with four technical replicates. Data are shown as means ± s.e.m. (e) Lower expression of CUC1 in the ref6 mutant. Transcript levels of CUC1 in 14-d-old seedlings were measured by RT–qPCR and normalized to those of ACTIN7 (AT5G09810). Data are shown as means ± s.e.m. from four technical replicates. NA, not analyzed.

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In addition to the previously characterized phenotypes, we observed some heart-shaped cotyledons in ref6 mutants at a low fre-quency and, in some rare cases, cup-shaped cotyledons, indicating that the ref6 mutant may be defective in organ boundary formation (Fig. 5a). These phenotypes are characteristic of cuc mutants. In Arabidopsis, three homologous transcription factors (CUC1, CUC2, and CUC3) act as key regulators of boundary formation in cotyledons and other organs24–27. cuc1, cuc2, and cuc3 single mutants develop heart-shaped or cup-shaped cotyledons at low frequencies because of functional redundancy, whereas double mutants involving any two CUC genes show more severe phenotypes24,25. Consistent with the phenotypic similarities between ref6 and the cuc mutants, we found that REF6 bound strongly to the first intron of CUC1 and weakly to the third exon of CUC3. No REF6 binding signal was found in CUC2 (Fig. 5b). Consistent with the sequence specificity of the REF6 C2H2-ZnF domains, the REF6 binding peak was centered on six CTCTGYTY motifs in CUC1 and two CTCTGYTY motifs in CUC3, whereas CUC2 had no detectable CTCTGYTY motif (Fig. 5b and Supplementary Fig. 8a,b). REF6 binding and H3K27me3 hypermethylation in ref6 were further validated at CUC1 by ChIP–qPCR with an independent batch of seedlings (Fig. 5c,d). Additionally, ChIP–qPCR data showed strong REF6 binding at CUC1 and weak binding at CUC3 in samples enriched for shoot apical meristem (Supplementary Fig. 8a–c), where CUC1 and CUC3 are mainly expressed. Consistent with the ChIP data, GST-REF6C bound two CTCTGYTY motifs in CUC1 in EMSA analysis (Supplementary Fig. 6d,e).

Notably, REF6 binding at CUC1 and CUC3 had functional conse-quences, as H3K27me3 levels were dramatically higher at CUC1 and moderately higher at CUC3 in the ref6 mutant in comparison to wild type, but there was no difference at CUC2 (Fig. 5b). Consistent with the changes in H3K27me3, the expression of CUC1 in ref6 was ~90% lower in seedling and shoot apical samples than it was in wild-type counter-parts (Fig. 5e and Supplementary Fig. 8d). No significant difference in expression was observed for CUC2 or CUC3 (Supplementary Fig. 8e,f). Moreover, the expression level of CUC1 was rescued in REF6-HA ref6 but not REF6∆ZnF-HA ref6 (Fig. 5e). Taken together, these results indicate that REF6 is recruited to CUC1 through sequence-specific binding by the C2H2-ZnF domains to demethylate H3K27me3 and maintain CUC1 in a transcriptionally active state.

To further characterize the role of REF6 in organ boundary forma-tion, we generated double mutants by crossing the ref6 mutant with the cuc mutants. Of these double mutants, the cuc2 ref6 mutant showed

the most severe phenotype, with ~16% of individuals having heart-shaped cotyledons and ~1% of individuals having cup-shaped cotyledons (Table 1 and Supplementary Fig. 9). The phenotype of cuc3 ref6 was stronger than that of cuc1 ref6. Furthermore, cuc1 cuc3 and cuc1 cuc3 ref6-3 plants showed similar phenotypes. These genetic interac-tions indicate that REF6 regulates organ boundary formation predominantly through CUC1 and to a lesser extent through CUC3 but does not act through CUC2. Although we did not see a difference in CUC3 expression between seedlings with and without REF6, our genetic data suggest that REF6 might regulate CUC3 expression at other stages important for cotyledon separation.

Taken together, the findings described here support a model in which REF6 is recruited to

its target genes through the sequence-specific DNA-binding activity of the C2H2-ZnF domains (Supplementary Fig. 10). This new target-ing mechanism for chromatin-modifying enzymes may be conserved in plants: in addition to REF6 and its close homolog in Arabidopsis, EARLY FLOWERING 6 (ELF6, also known as JMJ11)10,28,29, there are many Jmjc domain–containing proteins with tandem arrays of C2H2-ZnF domains in other plant species, including angiosperms (basal angiosperms30, monocots, and eudicots), lycophytes (Selaginella moel-lendorffii), and bryophytes (Physcomitrella patens) (Supplementary Fig. 11). C2H2-type ZnF domains are capable of specific recognition of an enormous assortment of DNA motifs31. It will be of interest to determine the sequence specificities of the C2H2-ZnF domains in other Jmjc domain–containing proteins and explore how these proteins may function at specific genes to regulate local histone methylation levels.

URLs. Plant Regulome, http://www.plantregulome.org/.

METHOdsMethods and any associated references are available in the online version of the paper.

Accession codes. The ChIP-seq data sets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession GSE65329.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKnowLedGMentSWe 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 ContRIBUtIonSXia 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,

table 1 Penetrance of the fused-cotyledon phenotype Cotyledon phenotype

Genotype Number Normal (%) Heart-shaped (%) Cup-shaped (%) stm-like (%)

Col 100.00 0.00 0.00 0.00

cuc1 1,294 99.85 0.15 0.00 0.00

cuc2 1,503 99.87 0.13 0.00 0.00

cuc3 903 99.34 0.66 0.00 0.00

ref6-1 4,091 99.95 0.05 0.00 0.00

cuc1 ref6-1 488 99.39 0.61 0.00 0.00

cuc2 ref6-1 759 84.06 15.68 0.26 0.00

cuc3 ref6-1 714 94.54 5.32 0.14 0.00

ref6-3 1,787 99.94 0.06 0.00 0.00

cuc1 ref6-3 535 96.07 3.93 0.00 0.00

cuc2 ref6-3 606 83.00 16.01 0.99 0.00

cuc3 ref6-3 517 89.17 10.06 0.77 0.00

cuc1 cuc3 670 0.60 21.19 71.64 6.57

cuc1 cuc3 ref6-3 464 1.72 29.31 60.13 8.84

stm-like, mild fusion at the base of cotyledons25.

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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.

CoMPetInG FInAnCIAL InteReStSThe authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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ONLINE METHOdsPlant materials. All Arabidopsis materials were grown in soil or on plates at 23 °C under long-day conditions (16-h light/8-h dark). The ref6-1 (SALK_001018), ref6-3 (SAIL_747_A07), cuc1-13 (SALK_006496), cuc2-3 (SAIL_605_C09), and cuc3-105 (GABI_302G09) mutants were provided by the Arabidopsis Biological Resource Center. Nicotiana benthamiana was grown at 25 °C under long-day conditions.

Transgenes. Sequences for the primers used in this study can be found in Supplementary Table 8. The REF6∆ZnF constructs were made by site-directed deletion from the pREF6::REF6-HA templates1 using the QuikChange kit (Stratagene) with primers CX5117 and CX5118. The constructs were transformed into Agrobacterium tumefaciens cells (strain EHA105). The pREF6::REF6∆ZnF-HA and p35S::REF6∆ZnF-YFP-HA transgenes were introduced into ref6-1 mutants and wild type (Col), respectively, by the floral dip method32. T1 Arabidopsis seeds were planted in soil and selected by spray-ing with 100 mg/L BASTA at 3–5 d after germination. For transient expression in tobacco, Agrobacteria carrying p35S::REF6∆ZnF-YFP-HA were infiltrated into N. benthamiana leaves14. Forty-eight hours after infiltration, tobacco leaves were collected for nuclei isolation and immunostaining.

Flowering time and petiole length assessment. Mutants and control plants were grown in soil side by side in a greenhouse at 23 °C under long-day con-ditions. Flowering time was assessed by counting the number of rosette and cauline leaves when the plants flowered. The petiole length of the fifth true leaf was measured for each plant at 40 d after germination. More than 20 plants were assessed for each line.

Chromatin immunoprecipitation. H3K9me2, H3K27me3, and HA ChIP were performed as previously described33 with minor modifications. Briefly, 2 g of 12-d-old seedlings, grown on half-strength Murashige and Skoog (MS) medium plus 1% sucrose, was vacuum infiltrated with cold PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM NaHPO4, and 1.5 mM KH2PO4, pH 7.4) containing 1% formaldehyde for 12 min (3 min × 4 times) to cross-link pro-tein–DNA complexes. The nuclear pellet was isolated according to Bowler et al.33. The pellet was resuspended with ChIP lysis buffer (50 mM Tris-HCl, 10 mM EDTA, and 1% SDS, pH 8.0) and kept on ice for 30 min. One volume of ChIP dilution buffer (16.7 mM Tris-HCl, 167 mM NaCl, and 1.1% Triton X-100, pH 8.0) was added to the samples before sonication for 12 min (30 s on, 30 s off, high level) in a Bioruptor (Diagenode, UCD-200) to yield DNA fragments of 0.2–1.0 kb in length. The lysates were diluted fivefold in ChIP dilution buffer to decrease the concentration of SDS to 0.1% and cleared by centrifugation (16,000g for 15 min at 4 °C). After keeping 5% of the sam-ple as input, the rest of the supernatant was incubated with antibody-bound Dynabeads Protein A or G (Life Technologies, 10001D or 10003D, bound to antibodies according to the user’s manual) overnight at 4 °C. The washing, elution, reverse cross-linking, and DNA purification steps were performed according to Bowler et al.33.

About 0.5 g of shoot-apex-enriched tissue from 12-d-old seedlings was collected by manual dissection (removing hypocotyls, leaves, and roots) and stored at –80 °C until use. Plant tissues were ground to a fine powder, and 10 ml of ChIP extraction buffer 1 (0.4 M sucrose, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, and protease inhibitor cocktail, pH 8.0) was added. 270 µl of 37% formaldehyde solution (to a final concentration of 1%) was added, and samples were incubated at 4 °C for 10 min to cross-link DNA to protein. Cross-linking was quenched by adding 0.63 ml of 2 M glycine, followed by incubation at 4 °C for 5 min. The nuclear pellet was isolated, and ChIP assays were performed as described above.

The antibodies used for ChIP were to H3K27me3 (Millipore, 07-449), H3K9me2 (Abcam, ab1220), H3 (Abcam, ab1791), and HA (Sigma, H6908). The DNA isolated by ChIP was used for qPCR analysis or Illumina single-end sequencing. ChIP-seq libraries were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina (New England BioLabs, E6040S) according to the manufacturer’s protocol. Primers for qPCR are listed in Supplementary Table 8. One intergenic region without H3K27me3 modification and REF6 binding was used as a negative control (NC3)1.

ChIP-seq analysis. ChIP-seq reads were aligned to Arabidopsis genome build TAIR10 by Bowtie 2 (ref. 34) using default parameters with a local alignment model. Duplicated reads and reads with low mapping quality were identified and removed with SAMtools35. Enriched intervals were identified by MACS version 2.1.0 (ref. 36) with default parameters. The parameter ‘--broad’ was used in the calling of H3K27me3-enriched regions, whereas ‘--call-summits’ was used in peak calling for the REF6 binding signal. Hypermethylation of H3K27me3 in ref6, REF-HA ref6, and REF6∆ZnF-HA ref6 plants was called by requiring more than twofold change in RPKM in comparison with wild type. The differentially enriched peaks of REF6 binding in REF6-HA ref6 and REF6∆ZnF-HA ref6 plants were also determined by >2-fold change in RPKM in comparison with Col. Density maps of reads for visualization were based on reads with a 200-bp extension in the 3′ direction as described previously37 after normalization.

DNA sequence motif analysis. To discover the consensus DNA sequence motifs underlying the REF6 binding peaks, all the REF6 binding peaks with summit score >10 in REF-HA ref6 were first trimmed to 800-bp genomic regions centered on their peak summits. Then, DNA motifs within these 800-bp DNA sequences were identified by MEME-ChIP17,18, an online tool for de novo motif discovery, with default parameters. On the basis of the observation that the majority of the CTCTGYTY motifs were within the 600 bp surrounding peak summits, we further narrowed down the REF6 binding peak to a 600-bp genomic region for further analysis of motif distribution and annotation.

To evaluate the role of the CTCTGYTY motif in the recruitment of REF6 and H3K27me3 demethylation, we integrated histone variants and modifications (H3K9me2, H3K4me3 (ref. 19), H3K4me2 (ref. 20), and H2A.Z21) and DNase I– hypersensitive site (DHS) information22. For the DHS information, we used the processed DNase I–sensitive loci in 7-d-old wild-type seedlings (HotSpots data at the website, DS21094) for further analyses. We used FIMO38 to scan all possible loci containing the CTCTGYTY motif in the genome, with default parameters. Then, we manually filtered out loci located in the mitochondria and chloroplast genomes. BEDTools39 was employed in identifying the overlap and difference between loci of motifs and regions bound by REF6.

In vivo histone demethylation assays. The demethylation assay was carried out as previously described1. Half of each tobacco leaf was infiltrated with A. tumefaciens EHA105 containing REF6∆ZnF-YFP-HA, and the other half without infiltration was used as a control. Immunostaining assays were performed using histone-methylation-specific antibodies (H3K27me3: Millipore, 07-449, 1:200 dilution; H3K27me2: Millipore, 07-452, 1:100 dilu-tion). The modified histones were identified by Alexa Fluor 555–conjugated goat anti-rabbit secondary antibody (1:200 dilution; Thermo Scientific, A31629). After staining, the slides were mounted in Vectashield Antifade Mounting Medium with DAPI (Vector Laboratory, H-1200) and photo-graphed with an Olympus BX51 fluorescence microscope. More than 25 pairs of transfected or non-transfected nuclei were imaged in the same field of view. Quantification was performed using ImageJ software.

RNA blotting and transcript level analysis. Total RNA was extracted using TRIzol reagent (Invitrogen) from whole seedlings 10 d after germination grown under the indicated long-day conditions on half-strength MS plus 1% sucrose (1/2 MS) plates. RNA (20 µg per lane) was separated in an agarose gel containing 1% formaldehyde, blotted onto HyBond N+ membrane (GE Healthcare), and probed with PCR-amplified DNA fragments using specific primer pairs CX6403 and CX7244 for FDH and CX461 and CX462 for FLC. FDH (AT5G43940) was used as control40. Reverse transcription reactions were performed using oligo(dT) primer. qPCR was performed using a CFX96 Real-Time PCR Instrument (Bio-Rad) with the SYBR Green (Invitrogen, S-7567) method (the final concentration of SYBR Green used in reaction mix was 0.15×). The sequences of the primers used for qPCR can be found in Supplementary Table 8. The qPCR primers for AP1, AP3, PI, AG, SEP3, and TCH4 were described previously1.

Total histone immunoblotting. Whole 3-week-old plants grown in soil were ground to a fine powder in liquid nitrogen and resuspended in 3× SDS

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loading buffer followed by boiling at 99 °C for 5 to 10 min. The total protein was used for immunoblotting with the antibodies listed below. Immunoblotting for H3 was used as a loading control. Antibodies were to H3 (Abcam, ab1791; 1:30,000 dilution), H3K27me3 (Millipore, 07-449; 1:2,000 dilution), H3K27me2 (Millipore, 07-452; 1:2,500 dilution), H3K27me1 (Millipore, 07-448; 1:3,000 dilution), H3K4me3 (Millipore, 07-473; 1:3,000 dilution), H3K9me2 (Millipore, 07-441; 1:2,000 dilution), and H3K36me3 (Millipore, 05-801; 1:2,000 dilution).

Extraction of REF6-HA protein. The nuclear pellet without cross-linking was prepared with the ChIP protocol33 from 2 g of 12-d-old seedlings and then resuspended in ChIP lysis buffer (50 mM Tris-HCl, 10 mM EDTA, and 1% SDS, pH 8.0) on ice for 3 h. The extract was diluted with 1 volume of ChIP dilution buffer (16.7 mM Tris-HCl, 167 mM NaCl, and 1.1% Triton X-100, pH 8.0) and centrifuged at 4 °C (16,000g for 10 min) to remove debris. The samples were boiled at 99 °C for 5 min after adding SDS loading buffer and separated in a 10% polyacrylamide gel. REF6-HA was detected by antibody to HA (Sigma, H6908; 1:1,000 dilution). LHP1 was used as a loading control. The result of immunoblotting was visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).

Electrophoretic mobility shift assays. The REF6C fragment (encoding amino acids 1239–1360 with the stop codon) was first cloned into pGEX-6p-1 (GE Healthcare) using primers HX7329 and HX7330 (Supplementary Table 8). Then, each of the ZnF domains was mutated using the QuikChange kit (Stratagene) with the primers listed in Supplementary Table 8. GST and GST-REF6C recombinant fusion proteins were expressed in Escherichia coli (BL21 codon plus, Stratagene) and purified using Glutathione Sepharose 4B beads (GE Healthcare). EMSAs were performed as described41 with some minor modifications. The complementary oligonucleotides (Supplementary Table 8) were annealed and labeled with [α-32P]dATP using T4 polynucleotide kinase (New England BioLabs, M0201). About 100 ng of GST or GST-REF6C protein and 0.3 pM 32P-labeled probes were incubated in a 10-µl reaction mixture (con-taining 25 mM Tris-HCl, 100 mM NaCl, 2.5 mM MgCl2, 0.1% CA-630, 10% glycerol, 1 µM ZnSO4, and 1 mM DTT, pH 8.0) for 1 h on ice and then sepa-rated by 6% polyacrylamide gel in Tris-glycine buffer (50 mM Tris, 380 mM

glycine, and 2 mM EDTA, pH 8.5) at 80 V for about 70 min. For competition assays, 50- or 100-fold more non-labeled competitor DNA was added in the reaction 10 min before addition of the labeled probe.

Phylogenetic analysis. The homologs of REF6 and ELF6 were identified by comparing the RefSeq protein sequences with full-length REF6 or ELF6 using BLASTP42. Multiple-sequence alignment of all these proteins was performed using ClustalW43 with default parameters, and the phylogenetic tree was then constructed with MEGA (ver.5.05)44 using the neighbor-joining method.

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

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

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

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

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

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

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).

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