SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level1,2,3,4,5, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase6, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM–REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Open Access 02 November 2022
aBIOTECH Open Access 14 March 2022
Stress Biology Open Access 09 December 2021
Subscribe to Journal
Get full journal access for 1 year
only $6.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Van der Meulen, J., Speleman, F. & Van Vlierberghe, P. The H3K27me3 demethylase UTX in normal development and disease. Epigenetics 9, 658–668 (2014).
Hargreaves, D.C. & Crabtree, G.R. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms. Cell Res. 21, 396–420 (2011).
Ho, L. & Crabtree, G.R. Chromatin remodelling during development. Nature 463, 474–484 (2010).
Clapier, C.R. & Cairns, B.R. The biology of chromatin remodeling complexes. Annu. Rev. Biochem. 78, 273–304 (2009).
Kooistra, S.M. & Helin, K. Molecular mechanisms and potential functions of histone demethylases. Nat. Rev. Mol. Cell Biol. 13, 297–311 (2012).
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).
Goldberg, A.D., Allis, C.D. & Bernstein, E. Epigenetics: a landscape takes shape. Cell 128, 635–638 (2007).
Li, B., Carey, M. & Workman, J.L. The role of chromatin during transcription. Cell 128, 707–719 (2007).
Bannister, A.J. & Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 21, 381–395 (2011).
Narlikar, G.J., Fan, H.-Y. & Kingston, R.E. Cooperation between complexes that regulate chromatin structure and transcription. Cell 108, 475–487 (2002).
Reyes, J.C. The many faces of plant SWI/SNF complex. Mol. Plant 7, 454–458 (2014).
Jerzmanowski, A. SWI/SNF chromatin remodeling and linker histones in plants. Biochim. Biophys. Acta 1769, 330–345 (2007).
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).
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).
Tang, X. et al. The Arabidopsis BRAHMA chromatin-remodeling ATPase is involved in repression of seed maturation genes in leaves. Plant Physiol. 147, 1143–1157 (2008).
Zhao, M. et al. Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture. PLoS Genet. 11, e1005125 (2015).
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).
Ho, L. et al. esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating Polycomb function. Nat. Cell Biol. 13, 903–913 (2011).
Li, C. et al. The Arabidopsis SWI2/SNF2 chromatin remodeler BRAHMA regulates Polycomb function during vegetative development and directly activates the flowering repressor gene SVP. PLoS Genet. 11, e1004944 (2015).
Yang, S. et al. The Arabidopsis SWI2/SNF2 chromatin remodeling ATPase BRAHMA targets directly to PINs and is required for root stem cell niche maintenance. Plant Cell 27, 1670–1680 (2015).
Smaczniak, C. et al. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc. Natl. Acad. Sci. USA 109, 1560–1565 (2012).
Vercruyssen, L. et al. ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development. Plant Cell 26, 210–229 (2014).
Han, S.K. et al. The SWI2/SNF2 chromatin remodeling ATPase BRAHMA represses abscisic acid responses in the absence of the stress stimulus in Arabidopsis. Plant Cell 24, 4892–4906 (2012).
Efroni, I. et al. Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev. Cell 24, 438–445 (2013).
Archacki, R. et al. BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in Arabidopsis. PLoS One 8, e58588 (2013).
Luo, C. et al. Integrative analysis of chromatin states in Arabidopsis identified potential regulatory mechanisms for natural antisense transcript production. Plant J. 73, 77–90 (2013).
Oh, E., Zhu, J.-Y. & Wang, Z.-Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat. Cell Biol. 14, 802–809 (2012).
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).
Klug, A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu. Rev. Biochem. 79, 213–231 (2010).
Brown, R.S. Zinc finger proteins: getting a grip on RNA. Curr. Opin. Struct. Biol. 15, 94–98 (2005).
Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).
Margueron, R. et al. Role of the Polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).
Hansen, K.H. et al. A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1291–1300 (2008).
Yuan, W. et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337, 971–975 (2012).
Hurtado, L., Farrona, S. & Reyes, J.C. The putative SWI/SNF complex subunit BRAHMA activates flower homeotic genes in Arabidopsis thaliana. Plant Mol. Biol. 62, 291–304 (2006).
Masiero, S. et al. INCOMPOSITA: a MADS-box gene controlling prophyll development and floral meristem identity in Antirrhinum. Development 131, 5981–5990 (2004).
Curtis, M.D. & Grossniklaus, U. A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003).
Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Gendrel, A.V., Lippman, Z., Martienssen, R. & Colot, V. Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2, 213–218 (2005).
Li, C. et al. Regulation of oleosin expression in developing peanut (Arachis hypogaea L.) embryos through nucleosome loss and histone modifications. J. Exp. Bot. 60, 4371–4382 (2009).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Lamesch, P. et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40, D1202–D1210 (2012).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Nicol, J.W., Helt, G.A., Blanchard, S.G. Jr., Raja, A. & Loraine, A.E. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25, 2730–2731 (2009).
Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).
Ye, T. et al. seqMINER: an integrated ChIP-seq data interpretation platform. Nucleic Acids Res. 39, e35 (2011).
Maere, S., Heymans, K. & Kuiper, M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005).
Lu, Q. et al. Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J. 61, 259–270 (2010).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
We thank the Arabidopsis Biological Resource Center (ABRC) for seeds for T-DNA insertion lines; A. Molnar for help with figure preparation; X. Shi of the Clinical Genomics Centre at Mount Sinai Hospital for overseeing the next-generation sequencing; and S. Rothstein for critical reading of the manuscript. C.C. is supported by a graduate fellowship from the Chinese Scholarship Council. This work was supported by funding from the Agriculture and Agri-Food Canada A-base and the National Science and Engineering Research Council of Canada (R4019A01) to Y.C., the Natural Science Foundation of China (31128001 to K.W. and Y.C. and 31210103901 to X. Cao and X. Chen), the State Key Laboratory of Plant Genomics (2015B0129-01 to X. Cao), and the US National Institutes of Health to X. Chen (GM061146) and Z.-Y.W. (R01GM066258).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 ChIP-seq genome browser views of BRM occupancy at previously identified BRM targets.
Gene structures are shown underneath each panel.
Rosette leaf number was counted at bolting. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey’s HSD test (n = 17).
Supplementary Figure 3 ChIP-seq genome browser views of REF6 occupancy at previously published REF6 targets.
The positions of the CTCTGYTY motifs underlying the REF6 peaks (Fig. 4) are indicated by orange vertical bars. Gene structures are shown underneath each panel. The FLC locus is not targeted by REF6.
Supplementary Figure 4 Overlap of BRM- and REF6-occupied genes and BRM–REF6 co-target genes with various epigenetic marks.
(a) qRT–PCR analysis of REF6 transcript levels in brm-1 compared to wild type. (b) Immunoblot analyses showing the protein levels of REF6-GFP in wild type and brm-1. Histone 4 was used as the loading control. Numbers at the top represent relative levels of the proteins after normalization to the loading control. (c) Confocal images of root tips showing nuclear localization of GFP-tagged REF6 in brm-1 and wild-type plants. Scale bar, 20 μm.
(a) BRM transcript levels in ref6-1 compared to wild type. (b) Immunoblot analyses showing the protein levels of BRM-GFP in wild type and brm-1. Histone 4 was used as the loading control. Numbers at the top represent relative levels of the proteins after normalization to the loading control. (c) Confocal images of root tips showing nuclear localization of GFP-tagged BRM in ref6-1 and wild-type plants. Scale bar, 20 μm.
(a) A list of peptides from SWI/SNF subunits and REF6 identified in the BRM-GFP IP–MS experiments. All of these proteins were identified only in the pBRM::BRM-GFP sample but not in the p35S::YFP control sample. (b) BiFC assays showing the interaction between BRM and REF6 in vivo. An unrelated nuclear protein encoded by At3G60390 was used as a negative control. (c) Immunoblot analysis showing the protein levels of the YN- or YC-tagged proteins used in the BiFC assays. YN- and YC-tagged proteins were probed with antibodies to HA and FLAG, respectively. Histone 4 was used as the loading control.
The CTCTGYTY motifs are highlighted in red. Nucleotides in exons and the intron are shown in upper and lowercase letters, respectively.
For the full sequence of the transgene, see Supplementary Figure 9. ChIP–qPCR results show that binding of BRM at the transgene without the motifs (YUC3∆) is significantly less than that at the transgene containing the motifs (YUC3wt). Three independent transgenic lines were analyzed for each construct. ChIP signals are shown as percentage input. The endogenous YUC3 locus and the TA3 locus were used as the positive and negative control, respectively. Error bars, s.d. from three biological replicates. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey’s HSD test.
Supplementary Figure 11 Flowering time of wild-type, ref6-1, ref6-1 pREF6::REF6-GFP, and ref6-1 pREF6::REF6ΔZnFs-GFP plants as determined by rosette leaf number.
Error bars, s.d. from 17 plants. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey’s HSD test.
Top, 4-week-old plants. Bottom, rosette leaves from each genetic background as indicated. Scale bar, 1 cm.
(a) Venn diagrams showing a statistically significant overlap between genes upregulated in brm-1 and ref6-1. (b) Venn diagrams showing statistically significant overlaps between BRM–REF6 co-target genes and genes reduced in brm-1 or ref6-1. (c) Venn diagrams showing the lack of statistically significant overlaps between BRM–REF6 co-bound genes and genes induced in brm-1, ref6-1, or brm-1 ref6-1.
Supplementary Figure 14 BRM is not required for the ability of REF6 to remove trimethyl groups from H3K27me3.
Top, mean density of H3K27me3 at REF6-occupied genes in wild type (WT), brm-1, ref6-1, and brm-1 ref6-1. The average H3K27me3 signal within 2-kb genomic regions flanking the center of REF6 peaks is shown. Bottom, ChIP-seq genome browser views of REF6 occupancy at selected loci in wild type and brm-1. Gene structures are shown underneath each panel.
The x axis represents normalized signal intensity from the first replicate in log2 scale. The y axis represents the log2 value of normalized signal intensity from the second replicate. The correlation analyses show highly positive correlations between the biological repeats, suggesting that the binding profiles are generally similar for the two ChIP-seq replicates.
Supplementary Figures 1–15 and Supplementary Tables 1 and 2. (PDF 2241 kb)
List of genes occupied by BRM in 14-d-old Col seedlings. (XLSX 649 kb)
List of genes occupied by REF6 in 14-d-old Col seedlings. (XLSX 419 kb)
List of genes co-occupied by BRM and REF6. (XLSX 177 kb)
List of genes showing REF6-dependent BRM occupancy. (XLSX 68 kb)
List of genes differentially expressed in brm-1, ref6-1, and brm-1 ref6-1 compared to wild-type seedlings by RNA-seq. (XLSX 47 kb)
Uncropped immunoblot images. (JPEG 347 kb)
About this article
Cite this article
Li, C., Gu, L., Gao, L. et al. Concerted genomic targeting of H3K27 demethylase REF6 and chromatin-remodeling ATPase BRM in Arabidopsis. Nat Genet 48, 687–693 (2016). https://doi.org/10.1038/ng.3555
This article is cited by
Comprehensive characterization of three classes of Arabidopsis SWI/SNF chromatin remodelling complexes
Nature Plants (2022)
Bulletin of Mathematical Biology (2022)
Molecular Horticulture (2021)