Letter | Published:

Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development

Nature Genetics volume 48, pages 14361442 (2016) | Download Citation


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.


Polycomb group (PcG) proteins form two main classes of evolutionarily conserved complexes: PRC2 and PRC1. In Drosophila, PRC2 contains E(Z), the enzymatic subunit that deposits the H3K27me3 mark, as well as SU(Z)12, ESC and p55 (refs. 1,2,3,4). PRC1 contains either PH-P or the homologous PH-D (referred to collectively as PH), PC, PSC (or its homolog SU(Z)2) and SCE subunits5. These two complexes are recruited to their target sites by a set of DNA-binding proteins, notably PHO6. They colocalize almost perfectly during embryogenesis7, and their embryonic phenotypes are similar, with posterior homeotic transformations due to misexpression of Hox genes8. Later in development, alterations in PcG components induce cancer9,10, suggesting that PcG proteins may be dynamically recruited to new target genes. As previous work suggested that larval alterations in different PcG components can induce phenotypes of different severity11, we analyzed the effect of PRC1 and PRC2 alterations side by side using the FLP–cell-lethal system12 to generate eye discs composed predominantly of mutant cells.

There are different PRC1 complexes, but all of them contain a catalytic subunit, which in Drosophila is encoded by Sce. In addition, canonical PRC1 (cPRC1) complexes contain PC, PH and PSC proteins. Null mutations in Psc and Su(z)2 (Psc-Su(z)21.b8), ph-p and ph-d (ph505 or phdel)9,10 and Pc (PcXT109) induced tumors in a highly penetrant manner (Fig. 1a and Supplementary Figs. 1a and 2a,b), showing that cPRC1 is required for tumor suppression. In contrast, a null mutation in E(z) (E(z)731) and an antimorphic mutation in Su(z)12 (Su(z)121), which encode components of PRC2, generated eye discs with normal appearance and smaller size than that of wild-type controls13,14 (Fig. 1a and Supplementary Fig. 1a,b). ph-null cells were also negative for the ELAV neuronal differentiation marker, whereas E(z)-null mutant cells were ELAV positive even in the absence of the PRC2-dependent H3K27me3 mark (Fig. 1b and Supplementary Fig. 1c). ph-null mutant cells showed extensive overproliferation in the posterior part of the eye disc; in contrast, E(z)-null mutant cells hypoproliferated (Supplementary Fig. 1c,d). Furthermore, cell polarity was normal in E(z) and Su(z)12 PRC2 mutants, whereas it was dysregulated in ph-, Psc- and Pc-null mutant cells, with multilayered cell growth and disruption of the apical localization of F-actin (Fig. 1c and Supplementary Figs. 1e and 2a,b). The contrast between tumor induction by cPRC1 alteration and hypoproliferation in PRC2 mutants does not appear to depend on Hox genes, as they were strongly derepressed in both classes of mutants (Fig. 1d and Supplementary Figs. 1c and 2c). Whereas ph-null mutation induces expression of N (Notch) in a cell-autonomous manner10, no overexpression of Notch was observed in E(z)-null mutant, H3K27me3-negative cells (Fig. 1e and Supplementary Fig. 1c). Quantitative immunoprecipitation (qChIP) at the N locus showed that PC and PH bind to N in eye discs in the absence of H3K27me3 (Supplementary Fig. 3).

Figure 1: Differential effects of PRC1 and PRC2 alterations on tumorigenesis.
Figure 1

(a) DAPI staining of eye-antennal imaginal discs showing overgrowth of PRC1 mutants (bearing phdel, PcXT109 or Psc-Su(z)21.b8 null mutations) and small eye phenotype of PRC2 mutants E(z)731 and Su(z)121. Control eye discs (CTL) carry only an FRT sequence. (b) Double staining of PH or H3K27me3 and the ELAV neuronal differentiation marker in eye imaginal discs. Insets, higher magnification. MF, morphogenetic furrow (arrowheads). FRT2A and FRT19A, controls for the E(z) and ph mutations, respectively. (c) Staining of the F-actin polarity marker in eye imaginal discs. White dashed lines indicate the positions of xz and yz cross-sections (shown at bottom and right, respectively). (d) Staining of the Hox protein UBX in PRC1- and PRC2-mutant eye-antennal discs. (e) Staining of Notch and PH or H3K27me3 in PRC1- and PRC2-mutant eye discs. Scale bars, 50 μm (b,c,e) or 100 μm (a,d). Discs are oriented with the antenna to the left. When only eye discs are shown, the posterior of the disc is at the right.

To test whether other genes might acquire PRC1 binding in the absence of H3K27me3, we mapped the PRC1 subunits PH and PC as well as the H3K27me3 mark in embryos6 and in the larval eye and wing imaginal discs by ChIP-seq (Fig. 2a–e and Online Methods). PRC1-bound genes (co-bound by PC and PH) in embryos are usually marked by H3K27me3 (ref. 7). Most of the 176 canonical embryonic target genes marked by the two complexes maintained PRC1 and H3K27me3 in larval tissues (Fig. 2a,c, Supplementary Figs. 4a and 5a and Supplementary Table 1). H3K27me3 increased during development on these canonical targets (Supplementary Figs. 5a and 6). However, PcG-mediated silencing was dynamic, as shown by combined immunostaining and fluorescence in situ hybridization (immuno-FISH) experiments (Supplementary Fig. 7). The hh (hedgehog) gene is expressed in the latest phases of eye disc development posteriorly to the morphogenetic furrow15. hh was strongly localized within nuclear PH foci in anterior cells, before the passage of the furrow. However, it was released from PH in most of the cells after the passage of the furrow (Supplementary Fig. 7b), suggesting that, similarly to mammalian systems16, PcG-mediated cellular memory is dynamically regulated during Drosophila development17,18. Dynamic Polycomb binding also occurred on many PRC1–PRC2 targets (Supplementary Fig. 5a) where PcG binding strongly increases between embryonic and larval stages as illustrated for danr, dan and chinmo (Fig. 2c).

Figure 2: Redeployment of PRC1 from canonical to neo-PRC1 target genes during development.
Figure 2

(a) Overlap of PcG PRC1–PRC2 target genes in embryos and eye imaginal discs. (b) Overlap of genes marked only by PC and PH in the absence of H3K27me3 (PRC1-only targets) in wild-type embryos and in third instar eye imaginal discs. (c) ChIP-seq for PC, PH and H3K27me3 in embryos and eye imaginal discs for PRC1–PRC2 targets illustrated at the Bithorax complex (Ubx, bxd, abd-A and Abd-B) and at chinmo, danr and dan. Significantly enriched regions are shown as horizontal bars under each ChIP-seq track. (d) Distribution of PC, PH and H3K27me3 in embryos and eye imaginal discs at neo-PRC1 target genes. (e) ChIP-seq signal levels of PC or PH versus H3K27me3 on their targets in embryos and larval eye discs. Regions that were already enriched in embryos are shown in orange; blue indicates targeting in the larval stage only. Random, control set of random H3K27me3-negative regions is represented in gray. (f) qChIP for PC, PH and PSC from the PRC1 complex; SU(Z)12 from the PRC2 complex; and H3K27me3. Gray, control regions; orange, PRC1–PRC2 target genes; blue, neo-PRC1 target genes. Results were normalized using the PGRP-LE gene (negative control). FC, fold change. Data are mean ± s.d. of 3 experiments. (g) Comparative Gene Ontology (GO) analysis of the maintained versus neo-PcG targets in eye imaginal discs. The number of PcG target genes in each GO category is indicated in brackets.

In addition, we found a large set of genes that are bound by cPRC1 proteins in larval tissues in the absence of H3K27me3 (Fig. 2b,d–f and Supplementary Table 1). This category, which we termed 'neo-PRC1', includes 894 genes in eye discs, 654 of which are also targeted in wing discs (Supplementary Fig. 4b). Neo-PRC1 targets were strongly enriched in genes regulating the cell cycle, cell polarity and cytoskeletal organization as well as genes involved in signaling and signal transduction pathways (Fig. 2g and Supplementary Tables 2 and 3). These data show that, after a first wave of deployment during embryogenesis, a second developmental wave recruits PRC1 components to a distinct set of genes during larval development.

We analyzed the neo-PRC1 category in more detail. First, we verified PRC1-specific targeting to six of these genes by qChIP in eye discs (Fig. 2f). H3K27me3 was absent from all of them, but it was clearly detected in PRC1–PRC2 targets. Notably, however, the SU(Z)12 and E(Z) subunits of the PRC2 were present on neo-PRC1 sites (Fig. 2f), and, in genome-wide mapping of SU(Z)12 (ref. 19), 66.5% of the neo-PRC1 genes were bound by SU(Z)12 (Supplementary Fig. 8). We therefore compared PC binding and H3K27me3 in ph- and in E(z)-null eye disc tissue. H3K27me3 at PRC1–PRC2 target genes is lower in E(z)-null mutants. As expected, PC binding also decreased in both ph- and in E(z)-null mutants (Supplementary Fig. 9a). H3K27me3 also decreased slightly upon null mutation of ph at PRC1–PRC2 target genes (Supplementary Fig. 9a), suggesting that PRC1 may have an effect in stabilizing PRC2 function. Although PC and PH binding at neo-PRC1 was generally weaker than at PRC1–PRC2 targets (Fig. 2e,f and Supplementary Fig. 5), we observed decreased PC levels at neo-PRC1 target genes in ph-null mutant tissue. However, PC levels were not affected in E(z)-null mutant tissue, showing that these peaks reflect specific binding and that PC is recruited on neo-PRC1 targets independently from PRC2 (Supplementary Fig. 9b). We next analyzed histone H2A ubiquitination at Lys118 (H2AK118ub) of PRC1. Mutant discs for the Drosophila PRC1 catalytic subunit Sce showed no defects in growth20 (Supplementary Fig. 10a) or polarity (Supplementary Fig. 10b,c), suggesting that PRC1 acts on neo-PRC1 targets independently of its associated histone mark. qChIP experiments showed that H2AK118ub was not substantially enriched on neo-PRC1 targets (Supplementary Fig. 10d). We showed earlier7 that the DNA-binding protein PHO, a known recruiter of PcG components, also binds at low levels to many other target loci in fly embryos. Genome-wide comparisons and qChIP show that many neo-PRC1 peaks colocalize with these PHO binding sites, suggesting that neo-PRC1 genes correspond to progressive assembly of PRC1 to weaker PHO sites during development7 (Supplementary Fig. 11). In contrast to PRC1–PRC2 targets, neo-PRC1 genes were robustly transcribed (Supplementary Figs. 12 and 13). Binding is not simply the consequence of the presence of open chromatin, however, as genes that were similarly or more highly transcribed than the neo-PRC1 group are mostly devoid of PC or PH proteins (Supplementary Fig. 12). Because acetylation of histone H3 at Lys27 (H3K27ac) is frequently associated with active genes, and neo-PRC1 targets lack the counteracting H3K27me3 mark, we mapped H3K27ac in eye discs (Fig. 3a–e). This mark was mostly absent from canonical PcG target genes (Fig. 3a,e), but we observed strong H3K27ac at neo-PRC1 target genes (Fig. 3b,c,e), slightly downstream of the transcription start site (TSS) (Supplementary Fig. 14). The presence of H3K27me3 and H3K27ac were anticorrelated, with genes bound by both PRC1 and PRC2 carrying the former and the neo-PRC1 targets carrying the latter (Fig. 3d). Therefore, the level of expression and the mark at H3K27 define PcG target gene categories (Fig. 3e and Supplementary Fig. 12).

Figure 3: PRC1 represses transcriptionally active neo-PRC1 targets.
Figure 3

(a) ChIP-seq profiles for PC, PH, H3K27me3 and H3K27ac in the Bithorax complex (Ubx, bxd, abd-A and Abd-B) in eye imaginal discs. Enriched regions are shown under each ChIP-seq track. (b) ChIP-seq profiles as above in a set of neo-PRC1 target loci. (c) qChIP against H3K27ac on a set of canonical and neo-PRC1 target genes. Results are normalized using the Antp gene (negative control). Data represent mean ± s.d. of 3 experiments. (d) ChIP-seq distribution of H3K27me3 and H3K27ac. PRC1–PRC2 target sites (black) carry mainly H3K27me3, whereas neo-PRC1 target sites (red) carry H3K27ac. H3K27ac and H3K27me3 enrichment are anticorrelated (Spearman's rank correlation coefficient ρ = −0.79). (e) Overlap of the different classes of PcG target genes with the H3K27ac mark, based on genome-wide ChIP-seq data analysis in eye imaginal discs. (f) Percentage of upregulated genes in RNA-seq of PRC1-mutant (ph505 and Psc1.b8) or PRC2-mutant (E(z)731 and Su(z)121) eye discs. (g) Comparative GO analysis of genes upregulated in PRC1 (ph505 and Psc1.b8) or PRC2 (E(z)731 and Su(z)121) mutants, stratified by categories of PcG ChIP-seq targets. The total number of upregulated genes in each GO category is indicated in brackets.

Although the finding of PcG protein binding to active genes is not unprecedented21,22,23,24, the coordinate recruitment of PRC1 components to genes involved in cell proliferation, signaling and polarity was notable. We thus performed RNA-seq of eye discs mutated in PRC1 (ph505 and Psc–Su(z)21.b8 null mutations) or PRC2 (E(z)731 null mutation and Su(z)121 antimorphic mutation) components (Supplementary Table 4). We found that alterations in different components of the same PcG complex regulate similar sets of genes (72% of the genes upregulated in ph mutants were also induced upon Psc–Su(z)2 mutation, and 68% of the genes upregulated in E(z) mutants were also induced in Su(z)12-null tissues) (Supplementary Table 4 and Supplementary Fig. 15a). Hox and other canonical PcG targets were derepressed in PRC1 and PRC2 mutants (Fig. 3f, Supplementary Fig. 15b and Supplementary Table 4). Some PRC1–PRC2 target genes were more strongly derepressed in PRC1 mutants (Supplementary Fig. 15b). This includes genes of the JAK–STAT pathway that were previously shown to be linked to Polycomb-dependent tumorigenesis9,25. A substantial fraction of neo-PRC1 genes were upregulated in PRC1 mutants but unaffected in PRC2 mutants (Fig. 3f and Supplementary Fig. 15c). Upregulated neo-PRC1 genes, representing putative direct targets of PRC1, were strongly enriched in cancer-related Gene Ontology (GO) categories such as cell cycle, cytoskeleton organization and tissue polarity (Fig. 3g, Supplementary Fig. 16 and Supplementary Table 5). Many of these genes have key roles in signaling pathways that promote cell proliferation and are involved in tumorigenesis, such as the Notch pathway (including N, sgg, Nedd4 and Apc)10,26,27, the JAK–STAT pathway (including dome, stat92E)9,28 and the Rho–JNK pathway (including Ran, Rho1 and puc)29. Two of these, N and stat92E, were previously shown to mediate tumorigenesis in null ph or Psc mutants (refs. 9,10). Therefore, Drosophila PRC1 components might suppress tumor formation by dampening the expression of a set of cell proliferation, cell signaling and polarity genes independently of PRC2.

Although PcG proteins and many of their target genes are strongly conserved in evolution30,31, the prevalent view is that mammalian PcG components act as oncoproteins, both through INK4A–ARF-dependent and independent mechanisms32,33. However, recent evidence suggests that several PcG members can act as tumor suppressors32 and that PRC1 may be present at a large subset of sites devoid of H3K27me3 (ref. 34). Mammalian cells contained a variety of PRC1-like complexes, all of which contain RING1B34. We therefore analyzed genome-wide maps of H3K27me3 and the PRC1 RING1B subunit in human embryonic stem (ES) cells, myelogenous leukemia cell (K562) and normal fibroblasts (Hs68) (Fig. 4a–d). As expected, a large fraction (96%) of genes bound by RING1B in ES cells showed colocalization of RING1B with H3K27me3. Notably, this fraction decreased in the two differentiated cell types, with only 33% and 36% of RING1B target genes marked by H3K27me3 in K562 and Hs68 fibroblast cells, respectively (Supplementary Table 6). Whereas canonical targets were found to encode transcriptional regulators involved in developmental pathways (Fig. 4e), noncanonical targets (bound by RING1B without H3K27me3) showed a difference in gene ontologies, with functions predominantly in cell cycle regulation, DNA repair, cytoskeleton organization and signaling pathways (Fig. 4e–g). Similarly to fly larval targets, they are generally strongly expressed in both differentiated cell types (Supplementary Fig. 17a,b). As RING1B is involved in the formation of non-PRC1 complexes35, we analyzed ChIP-seq profiles of BMI1, another PRC1 subunit, as well as EZH2 and SUZ12 (for PCR2), H3K27ac and DNA methylation in K562 cells. We found that the majority of the targets containing both RING1B and BMI1 did not colocalize with H3K27me3 (Supplementary Fig. 18a,b). Although EZH2 and SUZ12 were bound to them, these PRC1 targets were strongly marked by H3K27ac. Most of them correspond to DNA–unmethylated CpG islands and are located close to TSSs of highly expressed genes (Fig. 4h and Supplementary Figs. 17c, 18c and 19), highly reminiscent of the situation in Drosophila.

Figure 4: PcG proteins localize to a large set of DNA–unmethylated CpG islands in the absence of H3K27me3 in differentiated human cells.
Figure 4

(a) Overlap of RING1B target genes (red) and genes marked by H3K27me3 (white) in human ES cells, K562 myelogenous leukemia cells and Hs68 foreskin fibroblasts. (b) ChIP-seq profiles for RING1B and H3K27me3 in ES at the HOXD complex. Significantly enriched regions are shown as horizontal bars under each ChIP-seq track (q value cutoff = 0.01 for RING1B and 0.1 for H3K27me3). Tracks are normalized for sequencing depth. (c) ChIP-seq profiles for RING1B and H3K27me3 at a set of PRC1 loci in K562 cells. (d) ChIP-seq profiles for RING1B and H3K27me3 at a set of PRC1 loci in Hs68 cells. (e) Comparative GO analysis of canonical versus noncanonical PcG targets in ES, K562 and Hs68 cells. The total number of PcG target genes in each GO category is indicated in brackets. (f) KEGG pathways enriched in noncanonical targets in K562 cells. (g) KEGG pathways enriched in noncanonical targets in Hs68 cells. (h) Heat maps and profiles of percentage of DNA methylation, occupancies of RING1B, BMI1, EZH2, SUZ12, H3K27me3 and H3K27ac within ±5 kb from RING1B peaks in K562 cells. Clusters were defined by k-means clustering based on ChIP-seq signals around RING1B peaks (±5 kb). CpGi, CpG islands (UCSC definition); RPM, reads per million.

The present work uncovers a substantial redeployment of PRC1 during development. PRC1 components prevent tumorigenesis both by silencing signaling pathway genes in conjunction with PRC2 and by limiting the expression of neo-PRC1 genes coordinately regulating cell cycle, signaling and cell polarity. Therefore, PRC1's role in control of cell proliferation and tissue polarity is as important as its canonical developmental-patterning role that involves silencing of transcription factor genes. Furthermore, the analysis of PcG targeting in human cells suggests a parallel with Drosophila. In human ES cells, whereas non-canonical PRC1 complexes can bind to active genes in the absence of H3K27me3 (refs. 36,37), cPRC1 and PRC2 components colocalize with H3K27me3 similarly to their behavior in fly embryogenesis. In contrast, other normal differentiated cells or cancer cells23,34,38 may feature widespread association of PRC1 components to a large set of target genes in the absence of H3K27me3. Of note, the Mel18 subunit of cPRC1 was suggested to be also capable of activating a subset of its target genes. A similar function was reported for part of the PRC1 targets in Drosophila24, suggesting possible conserved activation function for Polycomb components. Finally, it will be important to analyze whether the tumor suppressor function that was recently identified for several PRC1 components32,39 involves regulation of neo-PRC1-target genes, similarly to the Drosophila case.



Flies were raised in standard cornmeal yeast extract medium at 25 °C. The Oregon-R w1118 line (referred to as wild type) was obtained from R. Paro (ZMBH, University of Heidelberg, Germany). Mosaic imaginal discs were generated through the eye-FLP-cell lethal clonal method as described12 using UAS-flp under the control of ey-GAL4 to induce recombination. Discs are composed predominantly of mutant cells (referred to in the text as mutant discs). The strong and null mutants used in this study are: ph-p505 and phdel (both null), Psc-Su(z)21.b8 (null), E(z)731 (null), Su(z)121 (antimorphic) and SceKO (null). Genotypes of control and PcG-mutant flies are as follows: FRT19A CTL (control line for ph mutations): FRT19A/ FRT19A GMR-hid; ey-GAL4 UAS-FLP1/+; FRT2A CTL (control line for Pc, E(z) and Su(z)12 mutations): UAS-GFP/ ey-GAL4 UAS-FLP1; FRT2A/ FRT2A GMR-hid; FRT42D CTL (control line for Psc mutations): FRT42D/ FRT42D GMR-hid; UAS-GFP/ ey-GAL4 UAS-FLP1; FRT82B CTL (control line for Sce mutations): Ey-GAL4 UAS-FLP/+; FRT82B/ FRT82B GMR-hid; ph505 mutants (neoplastic tumors): ph505, FRT19A/ FRT19A GMR-hid; ey-GAL4 UAS-FLP1/+; phdel mutants (neoplastic tumors): phdel, FRT19A/ FRT19A GMR-hid; ey-GAL4 UAS-FLP1/+; Psc-Su(z)21.b8 mutants (neoplastic tumors): Su(z)21.b8, FRT42D/ FRT42D GMR-hid; UAS-GFP/ ey-GAL4 UAS-FLP1; E(z)731 mutants (small discs phenotype): UAS-GFP/ ey-GAL4 UAS-FLP1; E(z)731, FRT2A/ FRT2A GMR-hid; Su(z)121 mutants (small discs phenotype): UAS-GFP/ ey-GAL4 UAS-FLP1; Su(z)121, FRT2A/ FRT2A GMR-hid; SceKO mutant (no obvious phenotype with the FLP–cell-lethal system in eye discs): ey-GAL4 UAS-FLP/+; SceKO FRT82B/ FRT82B GMR-hid.

Staining procedures.

Eye-antennal imaginal discs were dissected in PBS from L3 wandering larvae and fixed in 4% PFA in PBS for 20 min at room temperature. Discs were permeabilized for 1 h at room temperature in PBS + 0.5% Triton X-100 (0.5% PBTr) and blocked for 1 h at room temperature with 3% BSA in 0.025% PBTr. Discs were then incubated overnight at 4 °C on a rotating wheel with the primary antibodies in 0.025% PBTr and 1% BSA. The following antibodies were used: goat anti-PH (1:500)8, rabbit anti-H3K27me3 (Diagenode, C15410195, 1:1,000), mouse anti-ELAV (DSHB, 9F8A9-c, 1:500), mouse anti-UBX (DSHB, FP3.38-s, 1:20), mouse anti-ABD-B (DSHB, 1A2E9-s, 1:10), chicken anti-GFP (Life Technologies, A10262, 1:200), rabbit anti-H2AK119ub (Cell Signaling Technology, D27C4, 1:500), mouse anti-NOTCH (DSHB, C17.9C6, 1:1,000). The second day, discs were washed three times with 0.025% PBTr and incubated for 2 h at room temperature on a rotating wheel with secondary antibodies diluted in 0.025% PBTr + 1% BSA. The following secondary antibodies were used: donkey anti-rabbit Alexa Fluor 488 (Life Technologies, A-21206, 1:200), donkey anti-rabbit Alexa Fluor 555 (Life Technologies, A-31572, 1:200), donkey anti-rabbit Alexa Fluor 647 (Life Technologies, A-31573, 1:200), donkey anti-goat Alexa Fluor 555 (Life Technologies, A-21432, 1:200), donkey anti-mouse Alexa Fluor 488 (Life Technologies, A-21202, 1:200), donkey anti-mouse Alexa Fluor 555 (Life Technologies, A-31570, 1:200), donkey anti-goat Alexa Fluor 647 (Life Technologies, A-31571, 1:200) and goat anti-chicken Alexa Fluor 488 (Life Technologies, A-11039, 1:200). F-actin was stained by adding rhodamine phalloidin Alexa Fluor 555 (Life Technologies, R415, 1:200) to secondary antibodies. Eye-antennal discs were then washed and stained with DAPI diluted in 0.025% PBTr (1 μg/mL final) for 20 min at room temperature. Discs were rinsed in 0.025% PBTr and put in PBS. Discs were mounted in Vectashield medium (Vector Laboratories) and visualized on Leica SP8-UV confocal microscope. EdU-incorporation experiments were done with EDU Click-iT kit according to the manufacturer's recommendations (Alexa Fluor 488, Invitrogen, C10337). Eye disc sizes were measured by manually defining the limits of the eye discs using ImageJ.

Chromatin immunoprecipitation experiments on whole Drosophila embryos or larval imaginal discs.

ChIP of whole embryos (16–18 h) was essentially performed as previously described40. Briefly, cross-linking was performed for 15 min in the presence of 1.8% formaldehyde during tissue homogenization. Chromatin extracts of embryos were sonicated using a Bioruptor (Diagenode) for 15 min (settings 30 s on, 30 s off, high power).

ChIP in Drosophila imaginal discs was carried out on third instar larval eye-antennal and wing imaginal discs as previously described7 with the following modifications: after dissection and fixation of the imaginal discs the chromatin was sonicated using a Bioruptor (Diagenode) for 15 min (settings 30 s on, 60 s off, high power) in A2 buffer at 1% SDS. The size of the sheared chromatin fragments ranged from 500 to 1,000 bp. After sonication, SDS concentration was brought back to 0.1%. For ChIP-seq experiments, immunoprecipitation (IP) was carried out in a total volume of 250 μl using the following antibodies (diluted 1:100): previously described PC- and PH-specific antibodies7, anti-H3K27ac (Abcam #4729), anti-H3K27me3 (Upstate Biotechnology #07–449) and anti-PHO, provided by J.A. Kassis and described previously41.

Sequencing of the ChIP samples was performed by the sequencing platform MGX. To obtain the recommended quantity of DNA, several IPs were prepared (using 50 discs per IP), pooled and resuspended in a volume of 20 μl (12–14 IPs for PC or PH, 3–4 IPs for H3K27me3 and 3 IPs for H3K27ac). Sample preparation was done with Illumina kit (ref. IP-102-1001) following the manufacturer's instructions.

ChIP followed by qPCR (qChIP) was carried out with the same protocol, using 500 eye-antennal discs per IP. The following antibodies were used: rabbit anti-PC, anti-PH (1:100)7, rabbit anti-PSC (1:200)42, rabbit anti-H3K27ac and anti-H3K27me3 (Active Motif, #39134 and #39155, 1:100), rabbit anti-H2AK119Ub (Cell Signaling Technology, D27C4, 1:100), rabbit anti-SU(Z)12 (Supplementary Fig. 8b) (1:100), rabbit anti-E(Z) (Santa Cruz Biotechnology, #98265, lot A0109, 1:50). Primers for qPCR reactions are listed in Supplementary Table 7. For ChIP experiments performed in PcG-mutant eye-antennal discs, the antenna was removed to work with as many mutant cells as possible.

Drosophila ChIP-seq analysis.

ChIP experiments were performed in duplicates, and DNA samples were sequenced on HiSeq2000 and filtered and aligned with CASAVA (Illumina). The number of reads and the correlations between replicates are provided in Supplementary Tables 8 and 9, respectively.

PC, PH and H3K27ac ChIP-seq data were analyzed using MACS version 1.3.7 (ref. 43) with standard parameters, except genome size had a value of 120 Mb and tag size had a value of 36 nt. Only the peaks from MACS with a minimum enrichment of twofold and a maximum FDR of 10% were considered as enriched.

The H3K27me3 ChIP-seq data were analyzed using SICER44 with input as control library, a redundancy threshold of 4, a window size of 500 bp, an effective genome fraction of 0.7, a gap size of 2 kb and a threshold of 10%. To define highly confident targets, the 500-bp windows with twofold enrichment were considered. However, for assignment of neo-PRC1 target genes, H3K27me3-enriched regions detected using default settings were used.

For each condition, the final list of peaks was obtained as the intersection between the peaks of both replicates.

SU(Z)12 ChIP-seq data (GEO GSE36039) were reanalyzed with the same settings used for the other ChIP-seq analyses in the present study. PHO-enriched regions were obtained from ChIP-on-chip data (ArrayExpress E-MEXP-1708).

To visualize and present ChIP-seq data, we used Integrative Genomics Viewer (IGV)45,46.

For the scatter plots showing PC (or PH) versus H3K27me3 enrichments at PC (or PH) sites and a random set of H3K27me3-negative sites in embryos and in eye discs, ChIP-seq signals were quantified using EaSeq version 1.01 (ref. 47). Enrichment values were then plotted using GraphPadPrism 6. The scatter plot showing H3K27ac and H3K27me3 enrichments were calculated through 500-bp-enriched windows (enrichment ≥ 2) sliding along the genome. Density plots showing the average enrichment of ChIP-seq tracks around the TSS of target genes were obtained using the Bioconductor package seqplots with default parameters.

Assignment of genes to enriched regions.

The method is based on the one used by ModEncode to map regulatory elements in Drosophila48. A gene is considered marked if an enriched region is located between 1 kb upstream of one of the TSS of the gene and a distance equal to the length of the longest transcript downstream of the TSS, with a maximum of 2 kb.

The annotations used were computed with dedicated scripts using the API EnsEMBL Core with the EnsEMBL v73 database, which corresponds to release 5.46 in FlyBase.

Assignment of genes to the different categories of Polycomb targets.

Larval PcG targets were analyzed using the following scheme: PRC1–PRC2 target genes were enriched for PRC1 (PC and PH) and for H3K27me3 using a stringent twofold enrichment cut-off. Neo-PRC1 target genes were strictly defined as enriched for PRC1 (PC and PH) but not enriched for H3K27me3 when using default settings of SICER. Genes already bound in embryos by PC and/or PH and/or H3K27me3 using SICER default settings were not considered neo-PRC1 targeted during larval stages.

Immuno-FISH in Drosophila imaginal discs.

The FISH protocol was as described on EpiGeneSys. Briefly, wild-type eye-antennal third instar imaginal discs were dissected and fixed in PBT (PBS + 0.1% Tween) 4% PFA. Hybridization of 10 ng probe was done overnight in FISH hybridization buffer (FHB). For immunostaining, after post-hybridization washes, discs were blocked in PBSTr + 10% normal goat serum (NGS) for 2 h at room temperature and incubated overnight at 4 °C with an anti-PH7 at a dilution of 1:700 in PBSTr + 10% NGS. Discs were washed several times in PBSTr, blocked again in PBSTr + 10% NGS for 1 h at room temperature and incubated for 1 h at room temperature with an anti-rabbit-Cy5 (Jackson Laboratories) at a dilution of 1:200 in PBSTr + 10% NGS. Discs were then stained with DAPI (0.1 μg/mL in PBT for 10 min) and mounted in ProLong antifade (Molecular Probes).

Fluorescence high-resolution wide-field image acquisition was performed on a Leica DMRXA equipped with a micromax YHS1300 CCD camera (Roper Scientific), a 100×/NA 1.40 oil immersion objective (Leica Microsystems).

Generation of fluorescent probes.

For each gene, the FISH probes cover a 12-kb region significantly enriched for PRC1 proteins (PC and PH) together with H3K27me3. FISH probes were generated using 4–6 genomic PCR fragments of approximately 1.5 kb. Primer sequences to generate these fragments are listed in Supplementary Table 10. Probes were labeled using FISH Tag kits (Invitrogen Life Technologies) following the manufacturer's instructions. The specificity of each probe has been systematically tested by FISH on polytene chromosomes.

To calculate the percentage of colocalization, genes and PH foci were considered colocalized when FISH signal overlapped or was juxtaposed to the immunostaining signal in a nucleus. For each condition, 3D stacks were collected from 3–4 different tissues (optical sections were collected at 0.5-μm intervals along the z axis), and 50–100 nuclei were observed in 3D stack using Metamorph software (Universal Imaging Corp.) to obtain the percentage of colocalization.

Drosophila RNA-seq data.

RNA-seq data from mutant conditions were compared to their respective control genotypes, i.e., neutral clones generated in the same genetic background. Expression levels were calculated on the basis of 2 biological replicates for each condition.

RNA-seq libraries were constructed with the TruSeq RNA sample preparation (low-throughput protocol) kit from Illumina (performed by Montpellier MGX; part number 15008136). 1 μg total RNA was used for the construction of the libraries. The RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase and random hexamer primers. The second-strand cDNA was synthesized. These cDNA fragments were then subject to an end-repair process, the addition of a single A base and the subsequent ligation of the adaptor. The products were then purified and enriched with 15 cycles of PCR, as per the manufacturer's instructions (TruSeq RNA sample preparation kit). The final cDNA libraries were validated with a DNA 1000 Labchip on a Bioanalyzer (Agilent) and quantified with a KAPA qPCR kit.

For one sequencing lane of a flowcell V3, three libraries were pooled in equal proportions, denatured with NaOH and diluted to 7 pM in hybridization buffer. Cluster formation, primer hybridization and 50 single-read cycles of sequencing were performed on cBot and HiSeq2000 (Illumina) respectively.

The RNA-seq data were aligned only on the transcripts using TopHat49 version 2.0.8b and Bowtie with standard parameters. For each gene, a reads per kilobase per million mapped reads (RPKM) score was computed.

Image analysis and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. The quality of the data was assessed using fastqc (Babraham Institute) and Sequence Analysis Viewer (SAV (Illumina)). Demultiplexing, alignment and RNA counting was performed using CASAVA 1.8.2 (Illumina). Alignment was made with eland_rna on the dm3 version of Drosophila melanogaster genome (BDGP Release 5) and on several contaminants (the ribosomal RNA sequences, the mitochondrial chromosome, the PhiX genome and the Illumina adaptors). The transcript annotation was retrieved from UCSC Genome Browser (assembly BDGP R5/dm3, Table refFlat), version dated 14 August 2012. Gene IDs are from NCBI (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2refseq.gz; 14 August 2012). Before statistical analysis, genes with fewer than 10 reads (cumulating all the analyzed samples) were filtered and thus removed.

Differentially expressed genes were identified using the Bioconductor50 package edgeR 2.6.2 (ref. 51). Data were normalized in parallel using the Trimmed Mean of M values (TMM)52. Genes with adjusted P value < 0.05 (according to the Benjamini–Hochberg FDR method) were considered differentially expressed.

Wild-type RNA-seq from eye discs and wing discs were obtained from Gene Expression Omnibus (GEO GSE43341).

RNA interference in S2 cells.

Double-stranded RNA (dsRNA) against Su(z)12 and GFP were synthesized by in vitro transcription (Ambion Megascript T7 kit) of PCR products amplified from w1118 genomic DNA or from GFP coding sequence from pAWG (the Drosophila Gateway Vector Collection) using gene-specific primers that included T7 promoter sequences at their 5′ ends (Supplementary Table 11). S2 cells were cultured at a concentration of 106 cells/ml and incubated with 6 μg/ml of dsRNA for 4 d before harvesting.

Production of rabbit anti-Drosophila SU(Z)12 antibody.

A rabbit anti-SU(Z)12 antiserum was raised against a peptide containing the residues 624–900 from Drosophila SU(Z)12 (Covalab, Inc.).


To assess the specificity of the SU(Z)12 rabbit antibody, equal amounts of proteins from nuclear extracts were boiled for 5 min in Cracking Buffer (125 mM Tris-HCl, pH 7, 5% 2-mercaptoethanol, 2% SDS, 4 M urea) and separated on 4–20% gradient SDS–PAGE (TGX stain-free precast gels, Bio-Rad). After activation of stain-free gel by UV light and transfer onto nitrocellulose membrane using Trans-Blot Turbo (Bio-Rad), the membrane was imaged under UV light for loading control. The membrane was blocked in 5% milk, 1× PBS and 0.1% Tween, and incubated overnight with anti-SU(Z)12 (1:1,000) followed by incubation for 2 h at room temperature with anti-rabbit secondary antibody (Sigma-Aldrich A0545; 1:5,000) coupled to HRP. Detection was performed using Clarity Western ECL substrate (Bio-Rad) using a ChemiDoc XRS+ Imager (Bio-Rad).

Gene Ontology (GO) analysis.

The ontologies were obtained using the DAVID53,54 database through the DAVID web interface. We applied the 'functional annotation' function considering only 'biological process' (BP) ontologies. Gene Ontologies were visualized using the R package clusterProfiler55 with 'row.percentage' settings.

Human ChIP-seq analysis.

The ChIP-seq data sets for RING1B in human ES cells were obtained from Ku et al.56 (GEO: GSE13084); H3K27me3 data sets in ES cells were from Gifford et al.45 (GEO GSM772750). The ChIP-seq data sets for RING2 (RING1B) and H3K27me3 in K562 cells were from the ENCODE consortium (ENCSR138FUZ and ENCSR000AKQ); data in fibroblasts (Hs68) were from Pemberton et al.23 (GEO GSE40740). All of the data sets were analyzed according to the following methodology: sequences were aligned to the human genome (UCSC build hg38) with Bowtie2 using default parameters. PCR duplicates and reads with low mapping quality (MAPQ < 30) were removed, and only sequences that mapped uniquely to the genome were used for further analysis. Individual replicates were pooled, and peaks were called using MACS2.1 with default parameters for RING1B and with the -broad option on for H3K27me3 to identify domains, using whole-cell extract input from the corresponding cell line as control. RING1B peaks were associated with a gene using the same criteria as for Drosophila.

The data for BMI1, EZH2, SUZ12, H3K27ac and DNA methylation in K562 cells are from ENCODE (ENCSR782WRO, ENCSR000AQE, ENCSR000AUC, ENCSR000AKP and ENCSR765JPC). The definition for CpG islands was obtained from the UCSC Genome Browser (assembly hg38, table:cpgIslandExt).

For the clustering analysis, the normalized enrichment values were extracted from the ChIP-seq tracks in a window of 10 kb centered either on the RING1B peaks or on CpG islands in K562 cells. These values were then used for k-means-based clustering with 3 clusters. The data were visualized using Java TreeView. The average enrichment per cluster was plotted using R.

Human RNA-seq analysis.

Stranded RNA-seq data from K562 cells (ENCODE ENCSR000AEM) and Hs68 cells23 was mapped to the human genome (UCSC version hg38) using STAR (version 2.4) with the following parameters: –outFilterMultimapNmax 20–alignSJoverhangMin 8–alignSJDBoverhangMin 1–outFilterMismatchNmax 999–outFilterMismatchNoverLmax 0.04–alignIntronMin 20–alignIntronMax 1000000–alignMatesGapMax 1000000–outFilterIntronMotifs RemoveNoncanonical. Gene expression was quantified using cufflinks (version 2.2.1) against the GENCODE v22 reference transcriptome and normalized across all data sets using cuffnorm (version 2.2.1).


Montpellier GenomiX (MGX), http://www.mgx.cnrs.fr/; Integrative Genomics Viewer (IGV), https://www.broadinstitute.org/igv/; EaSeq, http://easeq.net; Bioconductor seqplots package, http://github.com/przemol/seqplots; FlyBase, http://flybase.org; EpiGeneSys two-color FISH protocol, http://www.epigenesys.eu/en/protocols/fluorescence-microscopy/182-two-colour-fluorescent-in-situ-dna-hybridization-on-whole-mount-drosophila-embryos-and-larval-imaginal-discs; UCSC Table Browser, https://genome.ucsc.edu/cgi-bin/hgTables; GENCODE v22, http://www.gencodegenes.org/releases/22.html.

Accession codes.

Gene Expression Omnibus: Drosophila ChIP-seq and RNA-seq data have been deposited under accession number GSE74080.


Primary accessions

Gene Expression Omnibus

Referenced accessions


  1. 1.

    et al. Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, 185–196 (2002).

  2. 2.

    et al. Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111, 197–208 (2002).

  3. 3.

    et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).

  4. 4.

    , , , & Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16, 2893–2905 (2002).

  5. 5.

    et al. Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98, 37–46 (1999).

  6. 6.

    et al. Cooperativity, specificity, and evolutionary stability of Polycomb targeting in Drosophila. Cell Rep. 9, 219–233 (2014).

  7. 7.

    et al. Functional anatomy of Polycomb and trithorax chromatin landscapes in Drosophila embryos. PLoS Biol. 7, e13 (2009).

  8. 8.

    , & From genetics to epigenetics: the tale of Polycomb group and trithorax group genes. Chromosome Res. 14, 363–375 (2006).

  9. 9.

    , , , & A tumor suppressor activity of Drosophila Polycomb genes mediated by JAK-STAT signaling. Nat. Genet. 41, 1150–1155 (2009).

  10. 10.

    et al. Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling. Nat. Genet. 41, 1076–1082 (2009).

  11. 11.

    , & Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development 128, 993–1004 (2001).

  12. 12.

    & A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152, 1631–1639 (1999).

  13. 13.

    , , & Drosophila ESC-like can substitute for ESC and becomes required for Polycomb silencing if ESC is absent. Dev. Biol. 313, 293–306 (2008).

  14. 14.

    & Mutations in polycombeotic, a Drosophila polycomb-group gene, cause a wide range of maternal and zygotic phenotypes. Genetics 125, 91–101 (1990).

  15. 15.

    , , & Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell 71, 33–50 (1992).

  16. 16.

    et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).

  17. 17.

    et al. Dynamic regulation by Polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila. Dev. Cell 15, 877–889 (2008).

  18. 18.

    et al. Stability and dynamics of Polycomb target sites in Drosophila development. PLoS Genet. 4, e1000178 (2008).

  19. 19.

    et al. Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila. Mol. Cell. Biol. 32, 1683–1693 (2012).

  20. 20.

    et al. The role of the histone H2A ubiquitinase Sce in Polycomb repression. Development 139, 117–127 (2012).

  21. 21.

    , , & Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol. Cell 45, 255–262 (2012).

  22. 22.

    et al. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science 338, 1465–1469 (2012).

  23. 23.

    et al. Genome-wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts. Genome Biol. 15, R23 (2014).

  24. 24.

    et al. Cohesin and polycomb proteins functionally interact to control transcription at silenced and active genes. PLoS Genet. 9, e1003560 (2013).

  25. 25.

    , , , & The transcriptional response to tumorigenic polarity loss in Drosophila. eLife 4 2015).

  26. 26.

    et al. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature 458, 987–992 (2009).

  27. 27.

    , , & Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila. Development 138, 1501–1506 (2011).

  28. 28.

    , , , & Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway. BMC Genomics 13, 506 (2012).

  29. 29.

    et al. A Drosophila overexpression screen for modifiers of Rho signalling in cytokinesis. Fly (Austin) 1, 13–22 (2007).

  30. 30.

    & Transcriptional silencing by Polycomb-group proteins. Cold Spring Harb. Perspect. Biol. 6, a019331 (2014).

  31. 31.

    & Recruitment of Polycomb group complexes and their role in the dynamic regulation of cell fate choice. Development 136, 3531–3542 (2009).

  32. 32.

    & Context-dependent actions of Polycomb repressors in cancer. Oncogene 35, 1341–1352 (2016).

  33. 33.

    et al. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication. Nat. Commun. 5, 3649 (2014).

  34. 34.

    et al. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol. Cell 45, 344–356 (2012).

  35. 35.

    Role of Polycomb proteins Ring1A and Ring1B in the epigenetic regulation of gene expression. Int. J. Dev. Biol. 53, 355–370 (2009).

  36. 36.

    , , , & RYBP and Cbx7 define specific biological functions of Polycomb complexes in mouse embryonic stem cells. Cell Rep. 3, 60–69 (2013).

  37. 37.

    et al. KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. eLife 1, e00205 (2012).

  38. 38.

    et al. The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation. Nat. Struct. Mol. Biol. 23, 682–690 (2016).

  39. 39.

    et al. Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the Polycomb group genes. J. Exp. Med. 209, 445–454 (2012).

  40. 40.

    et al. Chromosomal distribution of PcG proteins during Drosophila development. PLoS Biol. 4, e170 (2006).

  41. 41.

    , , & The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130, 285–294 (2003).

  42. 42.

    & The Polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol. 17, 6773–6783 (1997).

  43. 43.

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

  44. 44.

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

  45. 45.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  46. 46.

    , & Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).

  47. 47.

    , , & An interactive environment for agile analysis and visualization of ChIP-sequencing data. Nat. Struct. Mol. Biol. 23, 349–357 (2016).

  48. 48.

    et al. A cis-regulatory map of the Drosophila genome. Nature 471, 527–531 (2011).

  49. 49.

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

  50. 50.

    et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

  51. 51.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  52. 52.

    & A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).

  53. 53.

    , & Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

  54. 54.

    , & Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

  55. 55.

    , , & clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).

  56. 56.

    et al. Genome-wide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).

Download references


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.

Author information

Author notes

    • Vincent Loubiere
    •  & Anna Delest

    These authors contributed equally to this work.


  1. Institute of Human Genetics, UPR1142 Centre National de la Recherche Scientifique (CNRS), Montpellier, France.

    • Vincent Loubiere
    • , Anna Delest
    • , Aubin Thomas
    • , Boyan Bonev
    • , Bernd Schuettengruber
    • , Satish Sati
    • , Anne-Marie Martinez
    •  & Giacomo Cavalli
  2. Department of Biology and Health, University of Montpellier, Montpellier, France.

    • Vincent Loubiere
    • , Anna Delest
    • , Aubin Thomas
    • , Boyan Bonev
    • , Bernd Schuettengruber
    • , Satish Sati
    • , Anne-Marie Martinez
    •  & Giacomo Cavalli


  1. Search for Vincent Loubiere in:

  2. Search for Anna Delest in:

  3. Search for Aubin Thomas in:

  4. Search for Boyan Bonev in:

  5. Search for Bernd Schuettengruber in:

  6. Search for Satish Sati in:

  7. Search for Anne-Marie Martinez in:

  8. Search for Giacomo Cavalli in:


V.L. and A.D. performed developmental genetics, molecular biology, microscopy and genomics experiments. A.T. and B.B. performed bioinformatic analysis. B.S. and S.S. performed molecular biology experiments. A.-M.M. performed developmental genetics and molecular biology experiments. A.-M.M., V.L., A.D. and G.C. wrote the manuscript. A.-M.M. and G.C. have jointly supervised the work. All authors commented on and corrected the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Anne-Marie Martinez or Giacomo Cavalli.

Integrated supplementary information

Supplementary figures

  1. 1.

    Growth, proliferation and apical polarity are affected in PRC1 mutants independently of the PRC2 activity.

  2. 2.

    PcXT109 and Su(z)2-Psc1.b8 mutations show polarity defects in contrast to the Su(z)121 PRC2 mutant.

  3. 3.

    Binding of the PRC1 complex in the absence of the H3K27me3 mark to the N gene during larval stages.

  4. 4.

    Identification of novel PcG target genes during larval stages in eye and wing imaginal discs.

  5. 5.

    Histograms of mean enrichments of PC, PH and H3K27me3 on the different categories of PcG targets in eye and wing imaginal discs.

  6. 6.

    Increase of H3K27me3 levels during development.

  7. 7.

    Dynamic PcG targeting visualized by immuno-FISH experiments during larval stages.

  8. 8.

    PRC2 components E(Z) and SU(Z)12 are bound at neo-PRC1 target genes.

  9. 9.

    PRC1 can be recruited independently of PRC2 and vice versa.

  10. 10.

    Sce is not a tumor suppressor gene.

  11. 11.

    Embryonic PHO peaks mark future binding sites for PC and PH in larvae.

  12. 12.

    Neo-PRC1 target genes are highly expressed.

  13. 13.

    Neo-PRC1 PcG binding sites are frequently found at TSS regions in eye and wing imaginal discs.

  14. 14.

    Comparative H3K27ac profiles between the different categories of PcG targets.

  15. 15.

    Characterization of transcriptional changes in PRC1 and PRC2 mutants at the different categories of PRC1 and PRC2 targets.

  16. 16.

    Comparative Gene Ontology (GO) of upregulated and downregulated genes in PRC1 and PRC2 mutants.

  17. 17.

    RNA-seq expression levels of the different categories of PcG targets.

  18. 18.

    BMI1 colocalizes with RING1B at noncanonical target genes in K562 cells that are enriched in DNA–unmethylated CpG islands.

  19. 19.

    Density plots of sequencing data in K562 cells.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–19 and Supplementary Tables 7–11

Excel files

  1. 1.

    Supplementary Table 1

    List of PcG target genes in eye and wing imaginal discs.

  2. 2.

    Supplementary Table 2

    Gene Ontology analysis of the PRC1–PRC2 and neo-PRC1 targets in third instar larval eye imaginal discs.

  3. 3.

    Supplementary Table 3

    Gene Ontology analysis of the PRC1–PRC2 and neo-PRC1 targets in third instar larval wing imaginal discs.

  4. 4.

    Supplementary Table 4

    RNA-seq data for PRC1 and PRC2 mutants in eye discs.

  5. 5.

    Supplementary Table 5

    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.

  6. 6.

    Supplementary Table 6

    List of PcG target genes in human ES, K562 and Hs68 cells.

About this article

Publication history






Further reading