Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
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We thank F. Bantignies for his contribution on immuno-FISH experiments, S. Roux and C. Jacquier for producing and testing anti-SU(Z)12 rabbit antibodies. We thank the Montpellier RIO imaging facility for assistance with microscopy and the MGX facility for genomic data production and first-level processing. We are grateful to J. Kassis for anti-PHO antibodies, V. Pirrotta and R. Jones for anti-E(Z) antibodies and J. Müller for Sce-mutant fly lines. We thank the Bloomington Stock Center (Indiana University) and the Developmental Studies Hybridoma Bank (University of Iowa) for fly stocks and antibodies. We thank the laboratory of J. Wang for the phdel stock. We thank S. Perez-Lluch and Montserrat Corominas for providing the RNA-seq data on wild-type eye and wing imaginal discs before publication. We are grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees for providing computing and storage resources. Research in the laboratory of G.C. was supported by grants from the European Research Council (ERC-2008-AdG No 232947), CNRS, the European Network of Excellence EpiGeneSys, the European H2020 E-INFRASTRUCTURES Multiscale Genomics (MuG) grant, L'Agence Nationale de la Recherche, the Laboratory of Excellence EpiGenMed and the Fondation ARC pour la Recherche sur le Cancer. A.-M.M. was supported by a grant from the Canceropole Grand Sud-Ouest (2012-E10). B.B. is supported by the Sir Henry Wellcome Postdoctoral Fellowship (WT100136MA). V.L. is supported by a doctoral fellowship from the Laboratory of Excellence (Labex) EpiGenMed. B.S. is supported by L'Institut National de la Santé et de la Recherche Médicale. S.S. is supported by a postdoctoral fellowship from the Labex EpiGenMed.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Growth, proliferation and apical polarity are affected in PRC1 mutants independently of the PRC2 activity.
A- Dot plots showing the size of phdel, PcXT109 and Su(z)2-Psc1.b8 mutant eye discs. The central horizontal bar represents the mean and the whiskers the standard deviation. *** stands for pvalue < 0.001 using Wilcoxon signed-rank test. Mutations affecting any of the 3 PH, PC or PSC subunits of the PRC1 complex induce overgrowth compared to their genetic control.
B- Dot plots showing the size of E(z)731 and Su(z)121 mutant eye discs. The central horizontal bar represents the mean and the whiskers the standard deviation. *** stands for pvalue < 0.001 using Wilcoxon signed-rank test. Mutations affecting either E(Z) or SU(Z)12 subunit of the PRC2 complex induce small discs phenotype.
C- Comparative statistical analysis of immunostaining experiments done in phdel and E(z)731 mutants conditions, characterizing Hox gene derepression, ectopic Notch expression, apico-basal polarity, differentiation and proliferation defects.
D- Proliferation profiles are visualized by 2′-deoxy-5-ethynyluridine (EdU) incorporation during the replicative S-phase (in red). In a control disc, cells just posterior to the morphogenetic furrow (MF) undergo a synchronous S-phase and then stop to divide. In PRC1 phdel mutant discs, ectopic DNA replication is detected posterior to MF, showing that phdel mutant cells undergo unscheduled rounds of replication. Unlike PRC1 mutant discs, PRC2 E(z)731 mutant discs do not show ectopic DNA replication in the eye part of the disc, and even hypo-replicate their DNA in the second mitotic wave region. Scale bar, 50μm.
E- Apical polarity is analysed using a rhodamine-phalloidin staining detecting F-actin (in red). In control discs, a well-organized epithelium is apparent in a planar section. phdel mutant cells are detected by an anti-PH labelling and show a cellular-autonomous loss of F-actin organization while in H3K27me3 minus E(z)731 (PRC2) mutant cells detected by an anti-H3K27me3 labelling (in green), the apical polarity is maintained. Areas in dashed squares are shown at higher magnification. Scale bar, 50 μm.
Supplementary Figure 2 PcXT109 and Su(z)2-Psc1.b8 mutations show polarity defects in contrast to the Su(z)121 PRC2 mutant.
A- Apical polarity is analysed using a rhodamine-phalloidin staining detecting F-actin (in red). White dashed lines indicate the positions of the XZ and YZ cross-sections shown, respectively, below and to the right of each panel. In control discs, a well-organized epithelium is apparent. PcXT109 and Su(z)2-Psc1.b8 PRC1 mutants show defects of F-actin organization while Su(z)121 PRC2 mutant does not.
B- Table showing the number of discs observed for each F-actin immunostaining and the frequency of polarity defects in each mutant.
C- Staining of the Hox ABD-B protein in PRC1 and PRC2 mutants, as indicated. All mutants derepress the Abd-B HOX gene. Scale bar, 100µm.
Supplementary Figure 3 Binding of the PRC1 complex in the absence of the H3K27me3 mark to the N gene during larval stages.
A- qChIP assays performed in wild-type 3rd instar larval eye discs show PRC1-binding (PC in blue and PH in yellow) to the N promoter (N1 region) and first exon (N2). As a positive control, the Fab-7-PRE region from the homeotic gene Abd-B is strongly bound by both PC and PH proteins.
B- qChip assays performed in wild-type 3rd instar larval eye discs show the absence of the H3K27me3 mark (in green) at the Notch locus.
Error bars represent the standard deviation from three independent experiments. ChIP signals were normalized to a negative control, the PGRP-LE gene. The location of amplicons relative to each investigated region is shown on the bottom.
Supplementary Figure 4 Identification of novel PcG target genes during larval stages in eye and wing imaginal discs.
A- Venn diagram representing the number of PcG target genes that are both bound by PRC1 (PC and PH) and labelled by the H3K27me3 epigenetic mark (PRC2) in embryos (in orange), in eye imaginal discs (in green) and wing imaginal discs (in purple). As expected, a high proportion of canonical embryonic targets are maintained in eye and wing imaginal discs (respectively 80% and 74%). Among larval targets, 124 in eye discs and 246 in wing discs do not overlap with embryonic targets. When visually inspected however, these targets exhibit weak PcG binding at embryonic stage (see Supplementary Table 1). 82% of the PRC1-PRC2 targets that are detected in eye discs are also targeted in wing discs.
B- Venn diagram representing the number and depicting overlap of neo PRC1 target genes marked only by PC/PH in the absence of the H3K27me3 mark in eye and wing imaginal discs. 73% of the neo PRC1 targets in eye are also neo PRC1 targets in wing imaginal discs.
Supplementary Figure 5 Histograms of mean enrichments of PC, PH and H3K27me3 on the different categories of PcG targets in eye and wing imaginal discs.
A- Histogram representing the enrichments of PC/PH recruitment and of the H3K27me3 mark on PRC1-PRC2 target sites.
B- Histogram representing the enrichments of PC/PH and of the H3K27me3 mark on neo PRC1 target sites.
The Y axis represents the mean ratio of “ChIP reads/RPKM in the input”. The enrichment values have been computed in a 1kb sliding window along 1kb upstream of the first TSS and the end of the gene and have been normalized by the median enrichments of non-PcG target genes. RPKMs: Reads per kilo base per million of mapped reads.
Comparison of mean enrichment levels of the H3K27me3 mark calculated from ChIP-Seq data sets in wild type 4-12 hours embryos (in red), 16-18 hours embryos (in orange), in eye discs (in green) and in wing discs (in purple) on the 10 most highly enriched domains shared by these tissues. Positions of PcG domains (FlyBase annotation 5.46) are indicated below the respective bars.
Supplementary Figure 7 Dynamic PcG targeting visualized by immuno-FISH experiments during larval stages.
A - Immuno-DNA-FISH of robo3 and vestigial (vg) in combination with immuno-staining against PH in third-instar eye imaginal discs. Merged images of DAPI labelling (in blue), PH (PcG) foci (in green) and the FISH probe (in red). Robo3 is a non-PcG target and is used as a negative control. vg is a PcG target gene not expressed in eye discs and is used as a positive control for Immuno-FISH experiments done in eye discs. Orange arrows indicate cases of colocalization between vg loci and PcG foci. Colocalization between the robo3 and vg loci and PcG foci in eye imaginal discs are indicated in percentage. N indicates the total number of nuclei analyzed.
B - Immuno-DNA-FISH of eyeless (ey) and hh (hedgehog) in combination with immuno-staining against PH compared between anterior and posterior compartments of the eye imaginal discs. ey is exclusively expressed in the anterior compartment whereas hh is expressed exclusively in the posterior compartment. Histograms show the percentage of co-localization with PH for each locus either in the anterior or in the posterior compartment.
C- Percentage of co-localization of three genes implicated in the eye specification, ey, eyes absent (eya) and optix, with PH in wing discs. robo3 is used as a negative control.
Figure images are representative examples of deconvolved single slices from 3D stacks. Asterisks indicate that the differences in co-localization rates are significant (p<0.001). The scale bars represent 1 μm.
A- qChIP experiments for E(Z) in Drosophila eye imaginal discs. Peptidoglycan recognition protein LE (PGRP-LE), Msta, Chorion protein 36 (Cp36) are used as negative controls. Antennapedia (Antp), Bxd, distal antenna (dan) and distal-antenna related (danr) are canonical target genes. Notch (N), myospheroid (mys), Suppressor of cytokine signaling at 36E (Socs36E), Sec61 β subunit (Sec61 β), Lk6, TRAM and Signal-tranducer and activator of transcription protein at 92E (Stat92E) are examples of the neo PRC1 category. Results are normalized using the PGRP-LE locus (negative control).
B- Top panel shows Western blot analysis of anti-SU(Z)12 rabbit antibody specificity. The specific band corresponding to SU(Z)12 at 100kDa is strongly reduced upon Su(z)12 knockdown by RNAi in S2 cells. Histone H3 is used as a loading control.
C- ChIP-Seq profiles for PC, PH and H3K27me3 in eye imaginal discs and for SU(Z)12 in L3 larvae for PRC1-PRC2 targets (top) and at neo PRC1 target genes (bottom). Significantly enriched regions are shown under each ChIP-Seq track.
D- Venn diagram showing the overlap between genes targeted by SU(Z)12 in the L3 larvae and neo PRC1 target genes in the eye disc. 67% of neo PRC1 genes are also targeted by SU(Z)12.
A- Comparative qChIP performed either in control discs (in grey) or in phdel-PRC1 mutant eye discs (in dark red) using anti-PC (top) or anti-H3K27me3 antibodies (bottom). As expected, in PRC1-mutant discs, PC recruitment is lost on PRC1-PRC2 and neo PRC1 targets. The H3K27me3 mark is slightly reduced at PRC1-PRC2 targets.
B- Comparative qChIP performed either in control discs (in grey) or in E(z)731-PRC2 mutant eye discs (in blue) using anti-PC (top) or anti-H3K27me3 antibodies (bottom). As expected, both PC recruitment and the H3K27me3 mark are significantly lost on PRC1-PRC2 targets. Remarkably, PC levels are maintained at neo PRC1 targets.
Antennapedia (Antp), engrailed (en), chronologically inappropriate morphogenesis (chinmo), distal antenna (dan) and danr (distal antenna-related) illustrate PRC1-PRC2 targets. Notch (N), myospheroid (mys), Suppressor of cytokine signaling at 36E (Socs36E), Sec61 β subunit (Sec61 β), Lk6 and TRAM illustrate the neo PRC1 class of PcG targets. PGRP-LE, a non-PcG target gene, is used as a negative control. Results are expressed as percentage of Input. For each value, mean enrichments of at least two independent qChIP experiments are shown. P-values are calculated using a T-test to evaluate the statistical significance of differences between the control and mutant eye discs. Note that enrichment levels for the same protein at the same sites differ between panels A, B and Fig. 2F, because for each of these panels a different set of experiments was performed. Nevertheless, binding trends are maintained in each case.
A- DAPI staining showing that SceKO mutant discs do not overgrow compared to their genetic controls. Dot plots show the corresponding measurement of eye discs sizes (no significant difference).
B- Double staining of H2AK118Ub and F-actin in control eye discs (FRT82B CTL) and SceKO mutants. While the H2AK118Ub mark deposited by SCE shows a stark decrease in SceKO mutant eye discs generated using the HID-lethal system, F-actin organization is not affected.
C- Statistics showing the percentage of discs with polarity defects (%) and the number of observed eye discs (n).
D- qChIP experiments performed against H2AK118Ub in WT eye-antennal imaginal discs show no enrichment of H2AK118Ub at neo PRC1 target genes while some H3K27me3 enriched PcG target genes like dan and danR are enriched for this PRC1 deposited mark.
A- ChIP-Seq profiles for PRC1 (PC and PH) and the H3K27me3-PRC2 mark in embryos (orange) and eye imaginal discs (green) aligned with embryonic PHO binding sites. At the embryonic stage, PHO colocalizes with 86% of the embryonic PRC1-PRC2 PcG targets, such as at the BXC complex, vestigial (vg), Antennapedia (Antp) and engrailed (en) genes. Remarkably, 72% of neo PRC1 target genes, as exemplified by Notch (N), myospheroid (mys), Suppressor of cytokine signaling at 36E (Socs36E) and Lk6 are already occupied by PHO peaks in embryos at the positions that will recruit PcG complexes later during development. Enriched regions are shown under each ChIP-Seq track.
B- qChIP quantifying PHO-binding levels at the PRC1-PRC2 and neo PRC1 PcG targets. The PRC1-PRC2 class is illustrated by chronologically inappropriate morphogenesis (chinmo), Ets at 21C (Ets21C), distal antenna (dan), distal antenna-related (danr). Notch (N), myospheroid (mys), Suppressor of cytokine signaling at 36E (Socs36E), Sec61 β subunit (Sec61β), Lk6 and TRAM are examples of the neo PRC1 category. Asterisks distinguish genes in each of the categories that are not bound by PHO in embryos. PHO recruitment is maintained during larval stages at genes that were already bound in embryos. PRC1-PRC2 and neo PRC1 genes that were not bound by PHO in embryos recruit PHO during larval stages. qChIP enrichment are represented as percentage of Input. Each histogram corresponds to the mean value for three independent experiments. The horizontal red band illustrates the background level observed at the non-PcG target gene PGRP-LE.
A- RNA-seq expression levels of the different categories of larval PcG targets in eye imaginal discs. Box-plots show the distribution of expression levels based on RPKM values. Boxes depict data between the 25th and 75th percentiles with central horizontal lines representing the median. As a comparison, expression levels of active genes marked by H3K27ac, either in the top 5th or in the top 15-20th expression percentiles are also shown. *** stands for pvalue<0.001, calculated with a Wilcoxon signed-rank test.
B- RNA-seq expression levels of the different categories of larval PcG targets in wing imaginal discs. Box-plots show the distribution of expression levels based on RPKM values. Boxes depict data between the 25th and 75th percentiles with central horizontal lines representing the median. As a comparison, expression for genes in the top 5th and the top 15-20th percentiles are also shown. *** stands for pvalue<0.001, calculated with a Wilcoxon signed-rank test.
Supplementary Figure 13 Neo-PRC1 PcG binding sites are frequently found at TSS regions in eye and wing imaginal discs.
A- Histogram shows cumulative percentage of PcG-binding sites located within 1kb, 2kb and 5kb from the closest transcription start site (TSS) in eye imaginal discs.
B- Histogram shows cumulative percentage of PcG-binding sites located within 1kb, 2kb and 5kb from the closest transcription start site (TSS) in wing imaginal discs.
C- Density plots show the average enrichment of PC and PH at PRC1-PRC2 genes, in a window of 4kb centered on the TSS. Dashed lines correspond to the mean value, the dark area the Standard error and the light area is the 95% Confidence Interval.
D- Density plots show the average enrichment of PC and PH at neo PRC1 genes, in a window of 4kb centered on the TSS. Dashed lines correspond to the mean value, the dark area the Standard error and the light area is the 95% Confidence Interval.
Supplementary Figure 14 Comparative H3K27ac profiles between the different categories of PcG targets.
Density plots show the average enrichment of H3K27Ac at genes in the top 5th expression percentile (5% highest RPKMs of the genome), at neo PRC1 genes, at genes in the top 15-20th expression percentile (a median expression level close to that of neo PRC1 genes) and at PRC1-PRC2 target genes. Average enrichments are shown in a 4kb window centered at the TSS. Dashed lines correspond to the mean value, the dark area the Standard error and the light area is the 95% Confidence Interval.
Supplementary Figure 15 Characterization of transcriptional changes in PRC1 and PRC2 mutants at the different categories of PRC1 and PRC2 targets.
A- Venn Diagrams showing the overlap between the genes significantly upregulated in both PRC2 mutants (left, E(z)731, Su(z)121) or in both PRC1 mutants (right, ph505 and Psc-Su(z)21.b8) in eye imaginal discs.
B- List of selected relevant genes from PcG targets with the associated Fold Change in the 4 different mutants. N.S. = not significant.
C- Histogram showing the p values obtained using an hypergeometrical statistical test taking in account the total number of genes in the genome (15682, Flybase genome release 5.46), the total number of upregulated genes in each mutant, the total number of neo PRC1 genes in eye discs (894), and the number of neo PRC1 genes that are upregulated in each mutant. Overall, the overlap between upregulated genes and neo PRC1 genes is higher than expected by chance for PRC1 mutants (ph505 and Su(z)2-Psc1.b8), but not for PRC2 mutants (E(z)731 and Su(z)121). The ratios between number of upregulated neo PRC1 genes and the total number of upregulated genes are shown at the right of each bar.
Supplementary Figure 16 Comparative Gene Ontology (GO) of upregulated and downregulated genes in PRC1 and PRC2 mutants.
GO terms (Biological Process) enriched in genes that are down-regulated or upregulated (pvalue=0.05) in both PRC2 mutants (E(z)731 and Su(z)121) or in both PRC1 mutants (phdel and Psc-Su(z)21.b8). The GO terms with the strongest enrichment are shown, and p-values are represented using a color code ranging from blue (highest p-values) to red (smaller p-values). For each GO term, the amount of genes deregulated in the different categories of PcG targets is written in brackets. Size of the circles represent the percentage of the total number of genes in each GO term category.
A- Notched box-plots are used to show the distribution of expression level (in FPKM) of genes belonging to different categories in K562 human cells. The boxes depict data between the 25th and 75th percentiles with central horizontal lines representing the median values. Hyphens indicate the 95% confidence intervals. RING1B targets show highest expression levels and H3K27me3 domains are used as a negative control.
B- Same as above for Hs68 human cells.
C- Average enrichment of RING1B ChIP-Seq signal density on RING1B+/H3K27me3- genes in K562 cells (green), or Hs68 cells (orange).
Supplementary Figure 18 BMI1 colocalizes with RING1B at noncanonical target genes in K562 cells that are enriched in DNA–unmethylated CpG islands.
A- Venn diagram showing the overlap of BMI1 target genes (in red) and genes carrying H3K27me3 (in white) in K562 cells.
B- Venn diagram showing the overlap of BMI1+/RING1B+ target genes (in orange) and genes carrying H3K27me3 (in white) in K562 cells.
C- Heat map of whole-genome bisulfite sequencing signals (DNA methylation) and ChIP-Seq signals (RING1B, BMI1,EZH2, SUZ12, H3K27me3 and H3K27Ac) at CpG islands in K562 cells.
Density plots showing the average enrichment of RING1B, SUZ12, EZH2, DNA methylation (CpGi), H3K27me3 and H3K27ac at RING1B peaks in K562 cells, in a window of 10kb centered on RING1B peaks. Box plots show the distribution of enrichment values for the three clusters.
Supplementary Figures 1–19 and Supplementary Tables 7–11 (PDF 5249 kb)
List of PcG target genes in eye and wing imaginal discs. (XLSX 81 kb)
Gene Ontology analysis of the PRC1–PRC2 and neo-PRC1 targets in third instar larval eye imaginal discs. (XLSX 178 kb)
Gene Ontology analysis of the PRC1–PRC2 and neo-PRC1 targets in third instar larval wing imaginal discs. (XLSX 130 kb)
RNA-seq data for PRC1 and PRC2 mutants in eye discs. (XLSX 870 kb)
Ontologies of genes repressed and derepressed in PRC1 (ph505 and Psc-Su(z)121.b8) and PRC2 mutants (E(z)731 and Su(z)121) per category of PcG target genes. (XLSX 318 kb)
List of PcG target genes in human ES, K562 and Hs68 cells. (XLSX 88 kb)
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Loubiere, V., Delest, A., Thomas, A. et al. Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development. Nat Genet 48, 1436–1442 (2016). https://doi.org/10.1038/ng.3671
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