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Concerted genomic targeting of H3K27 demethylase REF6 and chromatin-remodeling ATPase BRM in Arabidopsis

Abstract

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.

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Figure 1: Genome-wide occupancy of BRM and REF6.
Figure 2: BRM and REF6 co-occupy a large number of genomic regions.
Figure 3: REF6-dependent recruitment of BRM to genomic loci.
Figure 4: A DNA motif required for REF6 genomic targeting.
Figure 5: The REF6 zinc-finger domains are essential for the binding of REF6 to chromatin.
Figure 6: Expression of BRM–REF6 co-target genes in the brm-1, ref6-1, and brm-1 ref6-1 backgrounds.

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References

  1. Van der Meulen, J., Speleman, F. & Van Vlierberghe, P. The H3K27me3 demethylase UTX in normal development and disease. Epigenetics 9, 658–668 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Hargreaves, D.C. & Crabtree, G.R. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms. Cell Res. 21, 396–420 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ho, L. & Crabtree, G.R. Chromatin remodelling during development. Nature 463, 474–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Clapier, C.R. & Cairns, B.R. The biology of chromatin remodeling complexes. Annu. Rev. Biochem. 78, 273–304 (2009).

    CAS  PubMed  Google Scholar 

  5. Kooistra, S.M. & Helin, K. Molecular mechanisms and potential functions of histone demethylases. Nat. Rev. Mol. Cell Biol. 13, 297–311 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. 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 

  7. Goldberg, A.D., Allis, C.D. & Bernstein, E. Epigenetics: a landscape takes shape. Cell 128, 635–638 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Li, B., Carey, M. & Workman, J.L. The role of chromatin during transcription. Cell 128, 707–719 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Bannister, A.J. & Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 21, 381–395 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Narlikar, G.J., Fan, H.-Y. & Kingston, R.E. Cooperation between complexes that regulate chromatin structure and transcription. Cell 108, 475–487 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Reyes, J.C. The many faces of plant SWI/SNF complex. Mol. Plant 7, 454–458 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Jerzmanowski, A. SWI/SNF chromatin remodeling and linker histones in plants. Biochim. Biophys. Acta 1769, 330–345 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. 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 

  14. 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 

  15. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  17. 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 

  18. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Smaczniak, C. et al. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc. Natl. Acad. Sci. USA 109, 1560–1565 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Efroni, I. et al. Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev. Cell 24, 438–445 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    Article  CAS  PubMed  Google Scholar 

  27. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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 

  29. 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 

  30. Brown, R.S. Zinc finger proteins: getting a grip on RNA. Curr. Opin. Struct. Biol. 15, 94–98 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. 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 

  32. Margueron, R. et al. Role of the Polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hansen, K.H. et al. A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1291–1300 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Yuan, W. et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337, 971–975 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. 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).

    Article  CAS  PubMed  Google Scholar 

  36. Masiero, S. et al. INCOMPOSITA: a MADS-box gene controlling prophyll development and floral meristem identity in Antirrhinum. Development 131, 5981–5990 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 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 

  39. 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).

    Article  CAS  PubMed  Google Scholar 

  40. 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).

    Article  CAS  PubMed  Google Scholar 

  41. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Lamesch, P. et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40, D1202–D1210 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ye, T. et al. seqMINER: an integrated ChIP-seq data interpretation platform. Nucleic Acids Res. 39, e35 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. 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).

    Article  CAS  PubMed  Google Scholar 

  48. Lu, Q. et al. Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J. 61, 259–270 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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).

Author information

Authors and Affiliations

Authors

Contributions

C.L. and Y.C. conceived the project. C.L. performed most of the experiments. C.-Q.W., L.-F.A., C.-W.C., M.P.S., L.J., A.L.B., and Z.-Y.W. performed BRM-GFP IP–MS assays. L. Gu, L. Gao, C.L., and C.C. conducted bioinformatics analyses. C.L., Q.Q., S.W., Y.Q., S.Y., C.-Y.C., V.N., S.E.K., S.H., X. Cao., and K.W. analyzed data. C.L., Y.C., and X. Chen wrote the manuscript.

Corresponding author

Correspondence to Yuhai Cui.

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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.

Supplementary Figure 2 pREF6::REF6-GFP complements ref6-1 phenotypes.

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.

Supplementary Figure 5 Gene Ontology (GO) analysis of the BRM and REF6 target genes.

Supplementary Figure 6 REF6 expression is not reduced in brm-1.

(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.

Supplementary Figure 7 BRM expression is not reduced in ref6-1.

(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.

Supplementary Figure 8 REF6 physically interacts with BRM.

(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.

Supplementary Figure 9 DNA sequence from YUC3 (701–977 bp downstream of the ATG start codon).

The CTCTGYTY motifs are highlighted in red. Nucleotides in exons and the intron are shown in upper and lowercase letters, respectively.

Supplementary Figure 10 The CTCTGYTY motif contributes to the recruitment of BRM.

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.

Supplementary Figure 12 Phenotypes of Col, ref6-1, brm-1, and brm-1 ref6-1 plants.

Top, 4-week-old plants. Bottom, rosette leaves from each genetic background as indicated. Scale bar, 1 cm.

Supplementary Figure 13 BRM and REF6 directly co-activate a set of genes.

(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.

Supplementary Figure 15 Correlation analyses of biological replicates of ChIP-seq data.

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 information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1 and 2. (PDF 2241 kb)

Supplementary Data 1

List of genes occupied by BRM in 14-d-old Col seedlings. (XLSX 649 kb)

Supplementary Data 2

List of genes occupied by REF6 in 14-d-old Col seedlings. (XLSX 419 kb)

Supplementary Data 3

List of genes co-occupied by BRM and REF6. (XLSX 177 kb)

Supplementary Data 4

List of genes showing REF6-dependent BRM occupancy. (XLSX 68 kb)

Supplementary Data 5

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)

Supplementary Data 6

Uncropped immunoblot images. (JPEG 347 kb)

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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

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