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The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation

Abstract

Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.

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Figure 1: PRC2 interactors and architecture during stem-cell differentiation.
Figure 2: ChIP–seq of core PRC2 subunits during stem-cell differentiation.
Figure 3: PR-DUB interactors during stem-cell differentiation.
Figure 4: PRC1 interactors and architecture during stem-cell differentiation.
Figure 5: ChIP–seq of core PRC1 subunits during stem-cell differentiation.
Figure 6: Overexpression of NPC-enriched PRC1 subunits affects genomic localization of Ring1b.

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Acknowledgements

We are grateful to I. Poser (MPI-CBG Dresden) for contributing the BAC lines and to D. Reinberg (NYU), J. Gil (MRC Clinical Sciences), and M. van Lohuizen (NKI) for contributing antibodies and plasmids. We thank the Sequencing and Bioinformatics teams of the RIMLS Mol(Dev)Bio departments for ChIP–seq support. We thank N. Hubner and H. Stunnenberg (RIMLS) for providing access to the QExactive mass spectrometer. The Vermeulen and Di Croce groups are supported by the EU FP7 framework programme 277899, 4DCellFate. The Vermeulen group is supported by the EU FP7 ITN 607142, DevCom, and ERC Starting Grant 309384.

Author information

Authors and Affiliations

Authors

Contributions

S.L.K. performed most of the experiments and analyzed the data. M.M.M. performed and analyzed the cross-linking MS experiments. H.I.B. purified PR-DUB from ESCs and NPCs. L.v.V. purified PRC1 from ESCs. I.D.K. purified PRC1 and PRC2 from the NP fraction. P.W.T.C.J. assisted with the MS experiments. A.S. performed the coimmunoprecipitation for PRC2. L.D.C. supervised A.S. S.L.K. and M.V. wrote the paper. M.V. supervised the project.

Corresponding author

Correspondence to Michiel Vermeulen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Extended analysis of PRC2 pulldown data.

(a) qRT-PCR of core PRC2 subunits in ESCs and NPCs (n = 2 RNA isolations). Error bars represent the s.d. of technical triplicates. (b) On the left: co-immunoprecipitation (co-IP) and Western blotting of GFP-Eed and substoichiometric PRC2 interactors in mouse ESCs. On the right: endogenous co-IP of Suz12 and Jarid2 in mESCs. (c) Volcano plot of interactors from triplicate pulldowns on benzonase-treated nuclear pellets from Eed-GFP ESCs. (d) Comparison of stoichiometries from Eed-GFP nuclear extract (NE) and nuclear pellet (NP) pulldowns in ESCs. Error bars represent the s.d. of technical triplicates.

Supplementary Figure 2 Extended analysis of PRC2 ChIP–seq.

(a) GO biological process of PRC2-bound genes with ESC enriched, shared, or NPC enriched binding. (b,c) ChIP-qPCR of Suz12 (b) or Ezh2 (c) bound loci from each set of PRC2-bound peaks (n = 2 ChIPs). Error bars represent the s.d. of technical triplicates. (d) RPKM values of genes <5kb from PRC2-bound peaks in ESCs or NPCs from each set of PRC2-bound peaks. Center values represent the median. (e) Fold change in expression during differentiation at genes <5kb from PRC2 ESC-bound peaks (ESC over NPC, RPKM > 1) compared to a size-matched random transcript set. Center values represent the median. (f-h) qRT-PCR to determine expression level changes at each set of PRC2-bound peaks in ESCs and NPCs (n = 2 RNA isolations). Error bars represent the s.d. of technical triplicates.

Supplementary Figure 3 Asxl2-GFP pulldowns and stoichiometry.

(a) Volcano plots from label-free GFP pulldowns on Asxl2-GFP ESC nuclear extracts graphed as in Fig. 1b. (b) Stoichiometry of Asxl2-GFP interactors in mESCs and NPCs. The iBAQ value of each protein group is divided by the iBAQ value of Asxl2, then graphed with Asxl2 set to 1. Error bars represent the s.d. of technical triplicates.

Supplementary Figure 4 Extended analysis of PRC1 pulldown data.

(a) Stoichiometry values of all known and detected Ring1b interactors from ESCs and NPCs. The Ring1b iBAQ value was set to 1 for pulldowns from both cell types. Interactors are grouped according to Pcgf protein + interactors. These values were used to generate Fig. 4d. Error bars represent the s.d. of technical triplicates. (b) Co-immunoprecipitation of endogenous Ring1b from ESCs and NPCs with Western blotting for dynamically-bound subunits (Cbx7, Pcgf4). (c) Volcano plot of interactors from triplicate pulldowns on benzonase-treated nuclear pellets from Ring1b-GFP mESCs. (d) Comparison of stoichiometries from Ring1b-GFP nuclear extract (NE) and nuclear pellet (NP) pulldowns in mESCs. Error bars represent the s.d. of technical triplicates. (e) Volcano plot from label-free Flag M2 pulldowns on Pcgf2-Flag ESC nuclear extracts graphed as in Fig. 1b. (f) Stoichiometry of Pcgf2-Flag interactors in mESCs and NPCs. The iBAQ value of each protein group is divided by the iBAQ value of Pcgf2, then graphed with Pcgf2 set to 1. Error bars represent the s.d. of technical triplicates.

Supplementary Figure 5 Extended analysis of PRC1 ChIP–seq.

(a) GO biological process of PRC1-bound genes with ESC enriched, shared, or NPC enriched binding. (b,c) ChIP-qPCR of Ring1b (b) or Pcgf2 (c) bound loci from each set of PRC1-bound peaks. (d) RPKM values of PRC1-bound peaks in ESCs or NPCs from each set of PRC1-bound peaks. Center values represent the median. (e-g) qRT-PCR of expression levels changes at each set of PRC1-bound peaks in ESCs and NPCs (n = 2 RNA isolations). Error bars represent the s.d. of technical triplicates. (h-j) GO biological process of genes <5kb from the Ring1b peaks clustered as shown in Fig. 5e-f.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 2105 kb)

Supplementary Table 1

List of genes within 5 kb of Suz12 and Ring1B peaks (XLSX 159 kb)

Supplementary Table 2

Filtered MaxQuant protein group files used to generate volcano plots (XLSX 1580 kb)

Supplementary Table 3

Filtered list of cross-linked peptides (XLSX 13 kb)

Supplementary Table 4

List of antibodies, primers and ChIP–seq tracks used in this study (XLSX 19 kb)

Supplementary Data Set 1

Uncropped western blots (PDF 1160 kb)

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Kloet, S., Makowski, M., Baymaz, H. et al. The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation. Nat Struct Mol Biol 23, 682–690 (2016). https://doi.org/10.1038/nsmb.3248

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