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Telobox motifs recruit CLF/SWN–PRC2 for H3K27me3 deposition via TRB factors in Arabidopsis

Nature Genetics volume 50, pages 638–644 (2018)Cite this article

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Abstract

Polycomb repressive complexes (PRCs) control organismic development in higher eukaryotes through epigenetic gene repression1,2,3,4. PRC proteins do not contain DNA-binding domains, thus prompting questions regarding how PRCs find their target loci5. Here we present genome-wide evidence of PRC2 recruitment by telomere-repeat-binding factors (TRBs) through telobox-related motifs in Arabidopsis. A triple trb1-2, trb2-1, and trb3-2 (trb1/2/3) mutant with a developmental phenotype and a transcriptome strikingly similar to those of strong PRC2 mutants showed redistribution of trimethyl histone H3 Lys27 (H3K27me3) marks and lower H3K27me3 levels, which were correlated with derepression of TRB1-target genes. TRB1–3 physically interacted with the PRC2 proteins CLF and SWN. A SEP3 reporter gene with a telobox mutation showed ectopic expression, which was correlated with H3K27me3 depletion, whereas tethering TRB1 to the mutated cis element partially restored repression. We propose that telobox-related motifs recruit PRC2 through the interaction between TRBs and CLF/SWN, a mechanism essential for H3K27me3 deposition at a subset of target genes.

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Fig. 1: The phenotype of trb1/2/3 mutants is similar to those of strong PRC mutants.
Fig. 2: Genome-wide changes of H3K27me3 in trb1/2/3 mutants.
Fig. 3: Lower H3K27me3 correlates with TRB1 binding and telobox enrichment.
Fig. 4: Teloboxes recruit CLF–PRC2 via TRBs.

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Acknowledgements

We thank P. Taenzler for excellent technical help and A. Brazel for critical reading of the manuscript. This work was supported by core funding from the Max Planck Society (Y. Zhou, K.K., J.A.D., and F.T.), the Chinese Scholarship Council (T.Y.) and the National Natural Science Foundation of China (grant no. 31570319; Y.W. and Y. Zhang). We thank K. Riha (Gregor Mendel Institute of Molecular Plant Biology) and J. Goodrich (University of Edinburgh) for providing materials.

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Authors and Affiliations

  1. Max Planck Institute for Plant Breeding Research, Department of Plant Developmental Biology, Köln, Germany

    Yue Zhou, Kristin Krause, Tingting Yang & Franziska Turck

  2. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Shanghai, China

    Yuejun Wang & Yijing Zhang

  3. University of Chinese Academy of Sciences, Shanghai, China

    Yuejun Wang & Yijing Zhang

  4. Max Planck Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Köln, Germany

    Joram A. Dongus

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Contributions

Y. Zhou performed and supervised all experimental work; K.K. performed the functional analysis of the SEP3 gene; J.A.D. assisted in Y2H assays and coimmunoprecipitation; K.K. and J.A.D. contributed to the development of theTRB2 CRISPR–CAS9 line; K.K. and T.Y. contributed the protoplast work; Y. Zhang and Y.W. carried out all bioinformatics analysis; and Y. Zhou, K.K., and F.T. planned the study and wrote the manuscript.

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Correspondence to Franziska Turck.

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Integrated supplementary information

Supplementary Figure 1 Phenotypes of trb, clf, and trb clf mutants.

(a) Phenotype of 28-days-old Col-0, trb1-2, trb3-2, trb1-2 trb3-2, clf-28, trb1-2 clf-28, trb3-2 clf-28 and trb1-2 trb3-2 clf-2 plants grown at 22 °C in long-day (LD) conditions. Scale bar indicates 1 cm. (b) Flowering time scored for genotypes in (a) as number of leaves and average of rosette size at bolting. Error bars indicate mean ± s.e.m. (n = 9). Statistical significance was determined by one-way analysis of variance (ANOVA) and multiple comparison by the Holm–Sidak method (P < 0.05). Different letters indicate significance groups. (c) Phenotype of 28-day-old Ws-2, trb2-1, clf-50 and trb2-1 clf-50 plants grown at 22 °C in LD conditions. Scale bar indicates 1 cm. (d and e) Flowering time scored for genotypes in (c) as number of leaves and average rosette size at bolting. Significance tested as in (b). (f) Expression of TRB2 in Ws-2 and trb2-1 mutant backgrounds measured by qRT-PCR. PP2A was used as reference gene. The presence of full-length transcripts was analyzed.

Supplementary Figure 2 Phenotype of higher-order trb mutants and complementation of triple trb1/2/3.

(a) Phenotype of 10-day-old seedlings of Col-0, trb1/2, trb1/3 and trb2/3 mutants grown at 22 °C in LD conditions. Scale bar: 1 cm. (b) Phenotype of 10-day-old seedlings of Col-0, trb1/2/3, and gTRB2-Flag transgenic lines (L12 and L21) in trb1/2/3 background. Scale bar: 1 cm. (c) +1 insertion (2nd exon of TRB2) in trb2-2 line is identified in trb1/3 double mutant background. The predicted DNA cleavage site is marked with an arrow. (d) Phenotype of 10-day-old seedlings of Col-0, trb1/2/3 and trb2-2 trb1/3 mutants. Scale bar: 1 cm.

Supplementary Figure 3 Nuclear localization of TRB proteins and their effect on teleomere integrity.

(a) Co-localization of TRB2 and TRB1 in the nucleus via transient expression of TRB2-GFP and TRB1-RFP in Tobacco leaves. Scale bar indicates 10 μM. (b) Analysis of pixel density along the axis indicated by a white line in (a) was performed by ImageJ. GFP signal intensities are shown in green and RFP signals in red. (c,d) gTRB2-YFP (c) and gTRB3-YFP (d) fusion proteins are detected in the nuclei of almost all cells in transgenic Arabidopsis plants. Yellow fluorescence detected in the root tip (upper panel) and the first true leaves (lower panel). Scale bars: 50 µm. (e) Telomere length measured by TRF analysis of genomic DNA prepared from 10-day-old Col-0, Ws-2, ku70, fifth generation (G5) tert, second (G2), and third (G3) generation of trb1/2/3 or trb1/3 trb2-/+ seedlings with ku70 and tert being included as reference.

Supplementary Figure 4 Correlation between H3K27me3 and expression changes for genes misregulated in trb1/2/3.

(a) Boxplot shows H3K27me3 ChIP-seq read intensity across genes commonly down- or up-regulated in trb123 and clf swn compared to all annotated genes. Venn diagrams are a reproduced from Fig. 1d for better clarity. A Hypergeometric test was used for statistical analysis. (b) Scatter plot showing the negative correlation between H3K27me3 and expression changes for genes mis-regulated in trb1/2/3.

Supplementary Figure 5 Quantitative PCR validation of RNA-seq and H3K27me3 ChIP–seq data in Col-0 and trb1/2/3 seedlings.

(a) Genes up-regulated in trb1/2/3 mutants. (b) Genes down-regulated in trb1/2/3 mutants. RNA was extracted from 10 day-old seedlings grown at 22 °C in LDs. Values were normalized to the reference gene PP2A using the –ΔΔCT approach. Vertical central line and whiskers indicate mean ± s. d. of four independently grown and processed replicate experiments used for qRT-PCR, symbols indicate data points as averages of three technical qPCR replicates. Statistical significance was determined by a Mann–Whitney–Wilcoxon test (df = 6) (c) H3K27me3 level change in Col-0 biased genes. (d) H3K27me3 level change in trb1/2/3 biased genes. (e) H3K27me3 level in common genes. Vertical central line and whiskers indicate mean ± s. d. of three independently grown and processed replicate experiments used for ChIP-qPCR, symbols represent averages of three technical qPCR replicates. Statistical significance was determined by a Mann–Whitney–Wilcoxon test (df = 4).

Supplementary Figure 6 List of cis elements enriched in different H3K27me3 gene categories detected in trb1/2/3 mutants.

Sequence logos of the 15 most enriched elements detected by MEME analysis.

Supplementary Figure 7 ChIP–qPCR analysis of H3K27me3 coverage in transiently FIE-GFP- and TRB1-GFP-expressing protoplasts.

(a) Western blot of GFP tagged FIE or TRB1 proteins expressed in protoplasts prepared from Col-0 and trb1/2/3-2 or Col-0 and clf-28. Anti-H3 was used as a loading control. Displayed is a representative blot of three replicated experiments. See Supplementary Fig. 14 for full scan of cropped image. (b) H3K27me3 level at SEP3, EMF1, AG and ACT2 in protoplast prepared from Col-0 and clf-28 seedlings. ACT2 was used as negative control for H3K27me3 distribution. Vertical central line and whiskers indicate mean ± s. d. of ChIP-qPCR of three independently transfected and processed batches of protoplasts. Statistical significance was determined by Mann–Whitney–Wilcoxon test (df = 4).

Supplementary Figure 8 Interaction of TRBs with CLF and SWN.

(a) TRB1-nYFP co-infiltrated with cYFP-CLF (1) or ATJ3 (2) and ATJ3 co-infiltrated with cYFP-CLF (3) in Nicotiana tabacum leaves. LHP1-RFP was used as reference for co-infiltration and ATJ3 was used as control for interactions. Scale bar = 10 μM. (b) Percentage of RFP expressing nuclei as in (a) expressing YFP and RFP signal. Total cells = 100, counted in 5 independently infiltrated leaves; Statistical analysis was a one-tailed Mann–Whitney U-test (df = 8). Vertical central line and whiskers indicate mean ± s. d. (c) Co-immunoprecipitation of CLF and (b) SWN with TRB1-3 expressed in Arabidopsis mesophyll protoplasts. TRB1-GFP, TRB2-GFP or TRB3-GFP were immunoprecipitated with anti-GFP trap beads from protoplasts co-transfected with either HA-CLF or HA-SWN. The precipitates were analyzed by western blotting with anti-GFP and anti-HA antibodies. See Supplementary Fig. 14 for full scan of cropped images.

Supplementary Figure 9 Interaction of TRBs with CLF and SWN in yeast cells.

(a) GAL4 DNA-binding domain (BD) or BD-CLF and BD-SWN fusion proteins were tested for their ability to bind to an activation domain (AD) fused to different TRB proteins. (b) Mutual interactions between TRB1-3 were tested as in (a). (c) Schematic presentation of the TRB protein domain structure. (d-e) Interaction between the MYB, H1/5, or Coiled-coil domain of TRB3 and CLF (d) and SWN (e). (f) Interaction between the MYB, H1/5, or Coiled-coil domain of TRB1 with SWN. Growth shown for three dilutions (10−1, 10−2 and 10−3) of the yeast culture on SD medium lacking LW (-LW) and LWH (-LWH) in all panels.

Supplementary Figure 10 Histochemical detection of GUS activity in transgenic SEP3pro-GUS lines with or without an intact telobox.

(a) 10-day-old seedlings carrying wild type pSEP3pro-WT-GUS. (b) 10-day-old seedlings carrying mutated SEP3pro-M-GUS. Picture in (a-b) show representative pattern observed in four single copy insertion lines transformed in the Col-0 background per construct and (c) an untransformed Col-0 control. (d) GUS activity at the inflorescence of representative SEP3pro-WT-GUS and (e) SEP3pro-M-GUS line. Scale bars in all panels indicate 1 mm.

Supplementary Figure 11 ChIP–qPCR analysis of TRB2 or TRB3 binding to the SEP3 promoter.

Distal promoter region of FT was used to as negative region for TRB2 or TRB3 binding. Bars display the mean of three biological replicates used for ChIP-qPCR, Vertical central line and whiskers indicate mean ± s. d. Statistical significance was determined by Mann–Whitney–Wilcoxon test (df = 4).

Supplementary Figure 12 TRB binding is decreased at a SEP3 promoter carrying a mutated telobox element.

(a) ChIP-PCR of TRB1-GFP binding to pSEP3pro-WT-GUS or pSEP3pro-M-GUS transgenic lines; empty plasmids were used as a negative transfection control. Three independent biological experiments are shown. Note that PCR fragment necessary to distinguish the endogenous and transgenic SEP3 promoter is 800 bp long and therefore not compatible with qPCR. (b) Coomassie brilliant blue staining of recombinant His-TRB3 protein purified from E.coli. (c) Illustration depicting the SEP3 promoter region. Blue line indicates telobox; yellow lines indicate four telobox-related motifs; black line indicates fragment used for EMSA. (d) TRB3 binding to Cy5 labelled PCR fragments of SEP3 promoter with WT or mutated telobox.

Supplementary Figure 13 AZF1- and TRB1-target genes do not show significant overlap.

Numbers of significant AZF1 and TRB1 target genes overlapping with Col-0 biased genes. The p-value of the only significant overlap is indicated (Hypergeometric test).

Supplementary Figure 14

Original uncropped scans of representative western blots displayed in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Table 1

Reporting Summary

Supplementary Data Set 1: Summary table of gene expression and H3K27me3 changes in trb1/2/3 for all Arabidopsis genes annotated in TAIR10

Sheet 1: Columns indicate 1: Presence or absence of teloboxes, 2: differential expression in trb1/2/3, 3: differential expression in clf/swn, 4: TRB1 binding according to Zhou et al. (2016)5, 5: TRB1 binding according to the present analysis, 6: H3K27me3 change in trb1/2/3 seedlings. Sheet 2: Gene expression fold-changes in trb1/2/3, clf-29swn-21 and siFIE mutants.

Supplementary Data Set 2: Summary table for GO enrichment analysis of genes differentially expressed in trb1/2/3 seedlings

Columns indicate 1: Cluster type, 2: enriched GO-term, 3: GO-term description, 4: GO-term count, 5: %, 6: p-value, 7: AGI codes, 8: length of list, 9: hits in list, 10: total gene list, 11: enrichment, 12: Bonferroni corrected p-value, 13: Benjamini corrected p-value, 14: FDR corrected p-value.

Supplementary Data Set 3: Summary table of MAnorm analysis for regions differentially enriched in H3K27me3

Columns indicate 1: Chromosome, 2: start of peak, 3: end of peak, 4: position of peak summit, 5: M-value, 6: A-value, 7: pvalue, 8: peak category, 9: normalized reads, 10: normalized read density in trb1/2/3.

Supplementary Data Set 4: Summary table for GO enrichment analysis of Col-0 biased and trb1/2/3 biased genes

Columns indicate 1: Cluster type, 2: enriched GO-term, 3: GO-term description, 4: GO-term count, 5: %, 6: p-value, 7: AGI codes, 8: length of list, 9: hits in list, 10: total gene list, 11: enrichment, 12: Bonferroni corrected p-value, 13: Benjamini corrected p-value, 14: FDR corrected p-value.

Supplementary Data Set 5: Genomic coordinates of FIE and TRB1 enriched peaks

Columns indicate 1: Chromosome, 2: start of peak, 3: end of peak, 4: length, 5: position of peak summit, 6: read tags, 7: corrected p-Value, 8: enrichment.

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Zhou, Y., Wang, Y., Krause, K. et al. Telobox motifs recruit CLF/SWN–PRC2 for H3K27me3 deposition via TRB factors in Arabidopsis. Nat Genet 50, 638–644 (2018). https://doi.org/10.1038/s41588-018-0109-9

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