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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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).
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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.
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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 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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DOI: https://doi.org/10.1038/ng.3555
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