Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis


Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants1,2,3,4,5. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

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Figure 1: Identification of Arabidopsis DNA fragments with PRE activity.
Figure 2: PRE-binding TFs physically interact with PRC2.
Figure 3: Cis motifs enriched in Arabidopsis PREs are required for PRE activity.
Figure 4: ChIP-seq analysis to test chromatin occupancy of FIE, H3K27me3, AZF1 and BPC1 in 30-h-old plants.
Figure 5: Class I BPC and C1-2iD ZnF TF families are required for Polycomb-mediated silencing and PRC2 recruitment in planta.
Figure 6: Tethering TFs from the class I BPC and C1-2iD ZnF subfamilies to the DNA to test PRC2 and FIE recruitment.

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We thank U.-S. Lee for help with manuscript preparation, students of the BIOL425 lab course at the University of Pennsylvania (Spring 2015 and Fall 2015) for the telobox interactome screen, J. He for help with the fluorometric assay of GUS activity, and W. Li (Chinese Academy of Sciences) for statistical analyses. Support for the study comes from NSF MCB-1243757 and MCB-1614355 to D.W., NSF IOS-1238142 to X.Z., AAFC GRDI-130 to R.S.A., BBSRC award G20266 to J.G., BBSRC award G20266 and DFG fellowship CL 393/1-1 to O.C., NIH NRSA 1F31GM112417-01 to M.F.G., NIH R01GM056006 to S.A.K. and J.L.P.-P. NIH R01GM067837 and RC2GM092412 to S.A.K., and NIH DP2MH107055, MoD 1-FY-15-344, and a Searle Scholar Program award to R.B.

Author information

D.W. and J.X. conceived of the study and J.X. conducted the majority of the experiments. O.C. and J.G. generated the CLF ChIP-chip data set. J.L.P.-P. and S.A.K. conducted the high-throughput DNA interactome screen. R.S.A. performed the motif analysis, C. Helliwell contributed to functional PRE analysis, and R.J. and M.S. contributed to TF–PRC2 interaction tests. A.P., C.S. and M.Z. identified telobox-binding TFs under the guidance of J.D.W. in the BIOL425 laboratory course held in the spring and fall semesters of 2015. C. He and R.B. performed library preps. R.B., X.Y., S.K., X.Z. and A.M.S. conducted bioinformatic analyses. M.F.G. developed the labeling protocol for EMSA. S.C. and X.L. raised anti-BPC1 antisera. D.W. wrote the paper with input from all authors.

Correspondence to Doris Wagner.

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Competing interests

The University of Pennsylvania has filed a provisional patent application, "Compositions and Methods for Epigenetic Modulation of Gene Expression in Higher Plants" (US provisional application no. 62/533,048, US Patent and Trademark Office), for development of synthetic PREs or destruction of endogenous PREs for possible epigenetic editing of plant traits.

Integrated supplementary information

Supplementary Figure 1 High-confidence PRC2 targets and candidate PREs.

(a) Flowchart for identification of candidate Arabidopsis PREs. We identified 1504 genomic regions marked by at least 3 of the following: H3K27me3, FIE, CLF or EMF115-17 (CLF ChIP-chip data: GSE7065) and linked these to 851 genes as previously described23. 132 of the 851 genes were significantly upregulated in prc2 mutants15,33 or strongly developmentally regulated48 and thus considered high confidence PRC2 regulated genes. 170 candidate PREs were associated with the 132 genes. From the 170 PREs we selected 5 associated with 3 genes for test of PRE activity. See methods for additional details. (b) Composition of the PRC2 complexes of the Arabidopsis sporophyte (diploid generation). Complex components: one of two SET domain methyltransferases (CURLY LEAF (CLF) or SWINGER (SWN)), one of two VEFS domain proteins (EMBRYONIC FLOWER2 (EMF2) or VERNALIZATION2 (VRN2)), a WD40 domain protein that can recognize H3K27me3 (FERTILIZATION INPENDPENT ENDOSPERM (FIE)) and a histone binding protein (MSI1)3.

Supplementary Figure 2 Test of PRE activity.

(a) A schematic of the construct used to test the ability of candidate PREs or negative control DNA fragments (NC) to recruit PRC2 and H3K27me3. The thick black line indicates the regions (PRE or a distal site (Dist)) tested by chromatin immunoprecipitation followed by qPCR (ChIP-qPCR). (b) H3K27me3/H3 abundance (top) and occupancy of PRC2 components MSI1 (middle) and EMF2 (bottom) assessed by ChIP-qPCR. Mean ± s.e.m. from three ChIP experiments (red dots). Black asterisks, significantly different relative to NC_1. Grey asterisks – significantly different occupancy at the PREs relative to the distal site (Dist). * P < 0.05; ** P < 0.01, NS, not significant (P > 0.05); one-tailed unpaired t-test. (c) A schematic of the construct used to test the ability of PREs to silence active reporters. Candidate PREs and control fragments were placed between two constitutive promoters, pF3H-35S mini and pMAS, with pF3H-35S mini driving expression of GFP and the β-glucuronidase gene GUS, and pMAS driving expression of the herbicide-resistance gene BAR. (d) Fluorometric assay of beta-glucuronidase (GUS) activity of 15 independent transformants in the wild type (WT) (top) or a PRC2 mutant (clf-28) (bottom). Violin plot of GUS activity in presumptive PREs (left) or negative controls (right): red or green indicates range; white circle, median; white line, mean; vertical black line, lower to higher quartile. Black horizontal bars mark the median GUS fluorescence of the PRE populations in the wild-type background. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant (P > 0.25) relative to NC_1, one-tailed Mann-Whitney U-test. (e) Herbicide resistance (survival rate) conferred by the BAR gene product in n = 60 independent T1 plants in the wild type (top) or in the prc2 (clf-28) mutant (bottom) background. Box-and-whisker plots showing the median (red line), upper and lower quartile (box edges), and minima and maxima (whiskers). P - value (one-tailed Mann–Whitney U-test): * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant (P > 0.07) relative to NC_1.

Supplementary Figure 3 Physical interaction of PRE-binding TFs with PRC2.

(a) Domains of TFs important for interaction with PRC2. Yeast-two-hybrid interaction tests using EMF2 and CLF as bait and AZF1, BPC1 and TOE1 full-length proteins (FL), N-terminal domains (N), middle regions (M), or C-terminal domains (C) as prey. Known protein motifs20,21,78 are shown below. NA = Not applicable. No single known protein motif is responsible for the TF/PRC2 interactions. a.a.: amino acid. (b) Detection of TF abundance in plant cells used for the BiFC analyses in Fig. 2d, e. Above: mean ± s.e.m. of TF levels relative to that of histone H3 from three independent BiFC experiments. (c) Co-IP of Myc-tagged TFs and PRC2 in the presence or absence of the benzonase endonuclease79. TOE1, AZF1 and BPC1 co-immunoprecipitate with PRC2 (HA-tagged FIE) in the absence and presence of the nuclease. Above: Ratio of FIE co-immunoprecipitated in the presence relative to absence of benzonase, mean ± s.e.m. from two independent co-IP experiments. The TRB2 sequence-specific binding protein serves as negative control. For expression levels of all 4 Myc-tagged proteins see Fig. 2f.78. Wanke, D. et al. Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. PLoS One 6, e16070 (2011).79. Fiil, B.-K., Qiu, J.-L., Petersen, K., Petersen, M. & Mundy, J. Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis. CSH Protoc 2008, pdb prot5049 (2008).

Supplementary Figure 4 GA repeat and Telobox motifs are bound by class I BPC and C1-2iD Zn-finger TFs.

(a) Electrophoretic mobility shift assay to test association of BPC1 with the GA repeats or with mutated versions thereof. WT, M1 and M2 were also used as cold competitors. Class I BPC TFs are known to oligomerize22,28,78. This, combined with the presence of two GA repeats in the tested DNA fragment, may explain why multiple shifted bands are observed when the protein is complexed with the DNA. Below: fraction of shifted DNA (% complex). Mean ± s.e.m. of three EMSA experiments. (b) Top: Diagram for motif-based yeast one hybrid screen. Bottom: The screen preferentially identified C2H2 ZnF proteins, in particular those of the C1-2iD subfamily (AZF1 and ZAT6) as Telobox (AAACCCTA) binding transcription factors. (c) Confirmation of Telobox interactome screen. Ten-fold serial dilutions of the yeast strains containing the bait DNA (b) integrated into the genome and plasmids containing C1-2iD Zn-finger TFs were plated on growth media (left) or on selection media (containing 600ng/ml aureobasidin A fungicide; right). See Supplementary Fig. 7 for a phylogenetic tree of ZnF TFs. The thin white line indicates where the plate image was cut.78. Wanke, D. et al. Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus. PLoS One 6, e16070 (2011)

Supplementary Figure 5 FIE, AZF1, BPC1 and H3K27me3 ChIP-seq analysis.

(a,b) Principal component analysis (PCA) of RPM-normalized ChIP and input DNA reads for narrow (a) and broad (b) peak calling. (c) Peak size distribution. (d) Enrichment (P values; converted from the Z-scores of the motif enrichment calculations using a normal distribution) and frequency (%) of PRE cis motifs under FIE-bound peaks. NE: not enriched (P > 0.5). (e) Functional classification of PRC2 (FIE), C1-2iD ZnF (AZF1) and class I BPC (BPC1) peak associated genes. Enrichment of Gene Ontology terms (FDR < 10-5 in at least one of the datasets) for the genes associated with FIE, BPC1 and AZF1 peaks (Q < 10-10). The majority of the significant Gene Ontology (GO) terms of BPC1 and AZF1 targets are also significant GO terms of FIE targets. Enriched GO terms include regulation of transcription, postembryonic development (reproductive development, shoot development, gynoecium development) and hormone response.

Supplementary Figure 6 TF family knockdown in the wild type and control knockdown in clfR.

(a) Class I BPC knockdown in the wild type (WT) does not cause leaf curling. Top: Representative images of plants 4 weeks after germination. Scale bar, 1 cm. Bottom: quantification of phenotypes. Box and whisker plot with median (red line, n = 15 independent lines), upper and lower quartile (box edges), and minima and maxima (whiskers). Letters above boxes indicate significantly different groups (P < 0.05 based on Kruskal–Wallis test with Dunn's post hoc test). (b) Control knockdown (GFPKD) in the hypomorph clfR mutant does not enhance clfR leaf curling. Top: Representative images of plants 4 weeks after germination. Scale bar, 1 cm. Bottom: quantification of phenotypes. Box and whisker plot with median (red line), upper and lower quartile (box edges), and minima and maxima (whiskers). Letters above boxes indicate significantly different groups (P < 0.05 based on Kruskal–Wallis test with Dunn's post hoc test).

Supplementary Figure 7 RNAi-mediated knockdown of class I BPC and C1-2iD ZnF TF families.

(a,b) Class I BPC TF family knockdown by RNAi (BPCKD, a) or C1-2iD C2H2 Zn-finger TF knockdown (ZnFKD, b) was assayed in independent transgenic lines in the hypomorph clfR mutant background. Expression of all genes tested is relative to that in clfR. Mean ± s.e.m. shown from one experiment and 3 independent lines. In each case, strong knockdown of the targeted TF family is observed (class I BPC and AZF1, AZF3, ZAT6), with more minor effects on distantly related BPC or ZnF TFs (BPC6, At2g26940, At3g46080). The translation initiation factor EIF4 served as qRT-PCR control. (c) Simultaneous knockdown of Class I and C1-2iD C2H2 Zn TFs by RNAi (BPC + ZnFKD) in the wild type. Shown are 2 independent double knockdown lines analyzed as described in (a, b). (d,e) Phylogenetic tree of the BBR-BPC family of TFs (see also22) (d) and of select C2H2 Zinc finger TFs (see also20) plus a subset of additional C1-2i C2H2 ZnF proteins in Arabidopsis (e). Branch length is indicated. Light green shading highlights the class I BPC TFs and light purple shading the C1-2iD Zn-finger proteins targeted for knockdown. Triangles point to the genes tested by qRT-PCR in (a-c) or used as controls in BiFC (Fig. 2d,e; BPC6 and ZAT5). Triangle color indicates RNAi targets (green) or distantly related genes (red). Note that the primer pair employed for class I BPC qRT-PCR amplifies all 3 genes in that clade.

Supplementary Figure 8 The effect of knockdown of class I BPC or C1-2iD Zn-finger TF family in the hypomorph clfR mutant.

(a) Leaf curling (inset) and flowering time in wild type (WT), the weak clfR mutant, class I BPC knockdown in clfR, C1-2iD ZnF knockdown in clfR and in clf-50 RNA null mutant. Photographs show plants 6 weeks after germination. Scale bar, 1cm. (b) Leaf curling phenotype of independent BPCKD clfR (top) and ZnFKD clfR (bottom) transgenic lines. Photographs show plants 4 weeks after germination. Scale bar, 1cm. (c) Quantification of the phenotypes in (b). Box and whisker plot with median (red line, n = 15 plants), upper and lower quartile (box edges), and minima and maxima (whiskers). Letters above boxes indicate significantly different groups (P < 0.05 based on Kruskal–Wallis test with Dunn's post hoc test). (d) De-repression of Polycomb target gene expression (AG, SEP3, LEC2) in the genotypes in (b). Expression is shown relative to the parental line (clfR). The housekeeping gene EIF4 served as control. (e) The abundance of the CLF mRNA relative to the wild type as measured by qRT-PCR. CLF levels are unchanged in the BPCKDclfR and the ZnFKDclfR plants shown in (b) compared to the parental line (WT). Shown are mean ± s.e.m. of one experiment for three independent transgenic lines (d,e).

Supplementary Figure 9 Phenotype of simultaneous knockdown of C1-2iD ZnF and class I BPC transcription factors.

(a) Leaf curling (inset) and early flowering in the wild type (WT), two independent BPCKD ZnFKD TF family knockdown lines, the hypomorph clfR mutant and the null clf-50 mutant. Photographs show plants 6 weeks after germination. Scale bar, 1cm. (b) Quantification of the phenotypes in (a). Box and whisker plot with median (red line, n = 15 plants), upper and lower quartile (box edges), and minima and maxima (whiskers). Letters above boxes indicate significantly different groups (P < 0.05 based on Kruskal–Wallis test with Dunn's post hoc test). (c) Misexpression of Polycomb target genes in the genotypes listed in (a). Expression in the mutant lines is shown relative to that of the wild type. Mean ± s.e.m. from three experiments (red dots). * P < 0.05; ** P < 0.01, NS, not significant (P > 0.05); relative to the wild type, one-tailed unpaired t-test. The two independent BPCKD ZnFKD TF family knockdown lines are not significantly different from each other, NS, not significant (P > 0.05); one-tailed unpaired t-test.

Supplementary Figure 10 FIE ChIP in wild type or BPC+ZnF KD plants.

(a) Principal component analysis (PCA) of RPM-normalized ChIP and input DNA. (b) Comparison of FIE ChIPseq reads from wild-type (WT) and BPC+ZnF KD plants. Shown are ChIPseq reads mapping to the WT-FIE peak regions. Regions with significantly (P-value < 0.01 and 2-fold change) increased and decreased read density in BPC+ ZnF KD are indicated by blue and red color, respectively. (c) Heatmap of RPM normalized read counts of the FIE ChIPseq at the targets tested by qRT-PCR in Figure 5. (d) Percent overlap table (by row) of 170 candidate PREs with significant (Q < 10-10) ChIPseq peaks for FIE in the wild type background (from Figure 5) as well as FIE and H3K27me3 (from Figure 4). Shading highlights strength of overlap. (e) Piechart of the overlap between the 170 computationally defined candidate PREs we identified from the vegetative phase of development (supplementary Fig. 1) and significant (Q < 10-10) FIE, TF (BPC1/AZF1) and H3K27me3 peaks identified by ChIPseq in 30-h-old plants.

Supplementary Figure 11 BPC1 TF chromatin occupancy in prc2 mutant.

ChIP using antiserum against class I BPC proteins to test BPC1 occupancy in the wild type (WT), the bpc123 triple mutant and the clf-28 (prc2) mutant. Binding was assayed at endogenous PREs (AG_2, SEP3_1, PC_LEC2), a control locus (NC_1) or a PRE reporter (Arti-AG_2). Shown are mean ± s.e.m. of three experiments (red dots). ** P < 0.01, relative to bpc123 mutant, one-tailed unpaired t-test. NS, no significant difference (P > 0.08) between BPC1 binding in Col and clf-28, one-tailed unpaired t-test.

Supplementary Figure 12 Gain-of BPC1 or AZF1 function rescues a hypomorph prc2 mutant.

(a) ChIP using antiserum against class I BPC proteins (see Supplementary Fig. 11) to test BPC1 occupancy in the wild type (WT), the hypomorph clfR mutant and 35S:BPC1 clfR. Binding was assayed at endogenous PREs (AG_2, PC_LEC2) or an endogenous control region (NC_ACT2). Shown is the mean ± s.e.m. of one experiment for two independent TF overexpression lines. (b) ChIP using antiserum against FIE (PRC2; left) or H3K27me3/H3 (right) in the wild type (WT), clfR, 35S:BPC1 clfR and 35S:AZF1 clfR. Shown is the mean ± s.e.m. of one experiment for two independent TF overexpression lines. (c,d) Overexpression of PRC2-recruiting TFs largely rescues leaf curling defects of clfR. Leaf curling in wild type (WT), two independent lines (L1 and L2) each of 35S:BPC1 clfR and of 35S:AZF1 clfR and the hypomorph clfR mutant. (c) Representative images of plants 4 weeks after germination, scale bar, 1 cm. (d) Quantification of leaf curling in the genotypes shown in (c). Box and whisker plot with median (red line, n = 15 plants), upper and lower quartile (box edges), and minima and maxima (whiskers). Letters above boxes indicate significantly different groups (P < 0.05 based on Kruskal–Wallis test with Dunn's post hoc test).

Supplementary Figure 13 Full-length gel images for all figures.

Left: Images for Co-IP. Two images are provided for the lower panel, the left image has non-specific signal at the top. Center (top): Image for telobox EMSA; (below): Co-IP in the absence and presence of Benzonase. Right (top): Image for GA repeat EMSA; (below): Quantification of proteins in protoplasts used for BiFC.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13. (PDF 2313 kb)

Supplementary Table 1

CLF binding site (significant region), ChIP-chip in seedlings. (XLSX 152 kb)

Supplementary Table 2

170 computationally defined candidate PRE regions associated with the 132 high-confidence PcG targets. (XLS 302 kb)

Supplementary Table 3

Transcription factors binding to functional PREs. (XLS 1210 kb)

Supplementary Table 4

FIE-bound regions (Q < 10–10) in BPC and ZnF double-knockdown plants (BPC+ZnF KD), ChIP-seq, germinating embryos. (XLSX 620 kb)

Supplementary Table 5

Oligos used in this study. (XLS 54 kb)

Supplementary Table 6

Exact P values for all statistical tests used in this study. (XLSX 53 kb)

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Xiao, J., Jin, R., Yu, X. et al. Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nat Genet 49, 1546–1552 (2017).

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