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Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB


Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants1,2,3,4,5,6. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive1,7,8. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes1,2,3,4. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1–4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

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Figure 1: The Polycomb group proteins BAP1 and ASX form a conserved complex in vivo and in vitro.
Figure 2: PR-DUB is bound at Polycomb target genes in Drosophila.
Figure 3: Recombinant Drosophila and human PR-DUB deubiquitinate H2A in nucleosomes in vitro.
Figure 4: PR-DUB is required for H2A deubiquitination in Drosophila and its catalytic activity is essential for HOX gene repression.

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Data deposits

The microarray data have been deposited in the ArrayExpress database ( under the accession number E-TABM-908.


  1. Schwartz, Y. B. & Pirrotta, V. Polycomb silencing mechanisms and the management of genomic programmes. Nature Rev. Genet. 8, 9–22 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B. & Cavalli, G. Genome regulation by polycomb and trithorax proteins. Cell 128, 735–745 (2007)

    Article  CAS  PubMed  Google Scholar 

  3. Müller, J. & Verrijzer, C. P. Biochemical mechanisms of gene regulation by polycomb group protein complexes. Curr. Opin. Genet. Dev. 19, 150–158 (2009)

    Article  PubMed  Google Scholar 

  4. Simon, J. A. & Kingston, R. E. Mechanisms of polycomb gene silencing: knowns and unknowns. Nature Rev. Mol. Cell Biol. 10, 697–708 (2009)

    Article  CAS  Google Scholar 

  5. Pietersen, A. M. & van Lohuizen, M. Stem cell regulation by polycomb repressors: postponing commitment. Curr. Opin. Cell Biol. 20, 201–207 (2008)

    Article  CAS  PubMed  Google Scholar 

  6. Henderson, I. R. & Dean, C. Control of Arabidopsis flowering: the chill before the bloom. Development 131, 3829–3838 (2004)

    Article  CAS  PubMed  Google Scholar 

  7. Jürgens, G. A group of genes controlling the spatial expression of the bithorax complex in Drosophila . Nature 316, 153–155 (1985)

    Article  ADS  Google Scholar 

  8. Gaytán de Ayala Alonso, A. et al. A genetic screen identifies novel polycomb group genes in Drosophila . Genetics 176, 2099–2108 (2007)

    Article  PubMed  Google Scholar 

  9. Nijman, S. M. B. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005)

    Article  CAS  PubMed  Google Scholar 

  10. Larsen, C. N., Price, J. S. & Wilkinson, K. D. Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues. Biochemistry 35, 6735–6744 (1996)

    Article  CAS  PubMed  Google Scholar 

  11. Amerik, A. Y. & Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta 1695, 189–207 (2004)

    Article  CAS  PubMed  Google Scholar 

  12. Jensen, D. E. et al. BAP1: A novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene 16, 1097–1112 (1998)

    Article  CAS  PubMed  Google Scholar 

  13. Ventii, K. H. et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res. 68, 6953–6962 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sinclair, D. A. R. et al. The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes. Development 125, 1207–1216 (1998)

    CAS  PubMed  Google Scholar 

  15. Johnston, S. C., Larsen, C. N., Cook, W. J., Wilkinson, K. D. & Hill, C. P. Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 Å resolution. EMBO J. 16, 3787–3796 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ji, H. & Wong, W. H. TileMap: create chromosomal map of tiling array hybridizations. Bioinformatics 21, 3629–3636 (2005)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Gambetta, M. C., Oktaba, K. & Müller, J. Essential role of the glycosyltransferase Sxc/Ogt in Polycomb repression. Science 325, 93–96 (2009)

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Papp, B. & Müller, J. Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20, 2041–2054 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Lagarou, A. et al. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22, 2799–2810 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Weake, V. M. & Workman, J. L. Histone ubiquitination: triggering gene activity. Mol. Cell 29, 653–663 (2008)

    Article  CAS  PubMed  Google Scholar 

  23. Vassilev, A. P., Rasmussen, H. H., Christensen, E. I., Nielsen, S. & Celis, J. E. The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. J. Cell Sci. 108, 1205–1215 (1995)

    CAS  PubMed  Google Scholar 

  24. Buchwald, G. et al. Structure and E3-ligase activity of the Ring–Ring complex of Polycomb proteins Bmi1 and Ring1b. EMBO J. 25, 2465–2474 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Henry, K. W. et al. Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev. 17, 2648–2663 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Klymenko, T. et al. A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20, 1110–1122 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Beuchle, D., Struhl, G. & Müller, J. Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development 128, 993–1004 (2001)

    CAS  PubMed  Google Scholar 

  28. McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G. & Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. McGinty, R. K. et al. Structure-activity analysis of semisynthetic nucleosomes: mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958–968 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. White, R. A. H. & Wilcox, M. Protein products of the Bithorax Complex in Drosophila . Cell 39, 163–171 (1984)

    Article  CAS  PubMed  Google Scholar 

  31. Celniker, S. E., Sharma, S., Keelanm, D. J. & Lewis, E. B. The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 9, 4277–4286 (1990)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193 (2003)

    Article  CAS  PubMed  Google Scholar 

  33. Nekrasov, M. et al. Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes. EMBO J. 26, 4078–4088 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Fraterman, S., Zeiger, U., Khurana, T. S., Wilm, M. & Rubinstein, N. A. Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes. Mol. Cell. Proteomics 6, 728–737 (2007)

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  36. Francis, N. J., Saurin, A. J., Shao, Z. & Kingston, R. E. Reconstitution of a functional core polycomb repressive complex. Mol. Cell 8, 545–556 (2001)

    Article  CAS  PubMed  Google Scholar 

  37. Levine, S. S. et al. The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol. Cell. Biol. 22, 6070–6078 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mason, D. E., Ek, J., Peters, E. C. & Harris, J. L. Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry. Biochem. 43, 6535–6544 (2004)

    Article  CAS  Google Scholar 

  39. Thåström, A. et al. Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences. J. Mol. Biol. 288, 213–229 (1999)

    Article  PubMed  Google Scholar 

  40. Luger, K., Rechsteiner, T. J. & Richmond, T. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999)

    Article  CAS  PubMed  Google Scholar 

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We thank T. Sixma and G. Buchwald for the gift of proteins, H. W. Brock, R. E. Kingston, B. Korn and B. Turner for plasmids, baculoviruses and antibodies, V. Benes, J. de Graaf, S. Müller and A. Riddell for technical support, and W. Huber and J. Gagneur for discussions. T.W.M. is supported by NIH grant RC2CA148354. J.C.S., A.G.A.A., K.O., N.L.-H. and J.M. are supported by EMBL and by grants from the DFG.

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Author Contributions J.C.S., A.G.A.A., K.O., N.L.-H. and J.M. conceived the project, designed and carried out the experiments, discussed and interpreted the data and prepared the manuscript. R.K.M. synthesized H2Bub1 in the laboratory of T.W.M., S.F. performed the mass spectrometry analysis in the laboratory of M.W.

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Correspondence to Jürg Müller.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 with legends and Supplementary Table 1. (PDF 2989 kb)

Supplementary Table 2

This table shows the chromosomal coordinates of PR-DUB-bound regions and assigned target genes with gene ontology classifications. (XLS 667 kb)

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Scheuermann, J., de Ayala Alonso, A., Oktaba, K. et al. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465, 243–247 (2010).

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