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Nature杂志2010


Vol 465 | 13 May 2010 | doi:10.1038/nature08966

LETTERS
Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB
? ? Johanna C. Scheuermann1*, Andres

Gaytan de Ayala Alonso1*, Katarzyna Oktaba1, Nga Ly-Hartig1, ¨ ¨ Robert K. McGinty2, Sven Fraterman1, Matthias Wilm1, Tom W. Muir2 & Jurg Muller1

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 plants1–6. 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 genes1–4. 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. A genetic screen for Drosophila mutants with PcG phenotypes recently identified calypso as a complementation group with two lethal alleles that complemented mutations in any other PcG gene8. We mapped the calypso mutations (see Methods), and the following findings established that calypso corresponds to the uncharacterized gene CG8445. First, calypso1 and calypso2, two independently isolated lethal calypso alleles8, both contained a cytosine to thymine mutation in CG8445 that creates a premature termination codon (Fig. 1a), whereas the parental chromosome on which these mutations had been induced contained a wild-type cytosine. Second, a transgene expressing a tandem affinity purification (TAP)-tagged form of the CG8445 protein under control of the a-tubulin1 promoter rescued calypso mutant animals into viable and fertile adults (see Methods). Third, calypso1 and calypso2 mutants did not express detectable levels of CG8445 protein (see later and Supplementary Fig. 5). We therefore named the CG8445 gene calypso. calypso encodes a polypeptide of 471 amino acids that is a member of the ubiquitin C-terminal hydrolase (UCH) subclass of deubiquitinating enzymes (Fig. 1a)9. UCH domains are cysteine proteases that hydrolyse the isopeptide bond between the C-terminal glycine of ubiquitin and the lysine side chain in the conjugated protein9–11.
1

The closest human homologue of Calypso is BAP1 (Supplementary Fig. 1), a nuclear protein that possesses tumour suppressor activity9,12,13. The Calypso protein thus represents Drosophila BAP1. Western blot analyses of Drosophila nuclear extracts and staining of imaginal discs with anti-Calypso antibodies showed that the Calypso protein is localized in nuclei (Supplementary Fig. 1c and see later). To identify interaction partners of Calypso, we purified proteins associated with a TAP–Calypso fusion protein from nuclear extracts of embryos that carried the a-tubulin1-TAP-calypso transgene. The purified material was separated on SDS-polyacrylamide gels and four major protein bands were identified (Fig. 1b). Sequencing of peptides from these bands by nanoelectrospray tandem mass spectrometry identified the 55-kDa band as the Calypso bait protein, whereas the other three bands all represented fragments of the PcG protein Additional sex combs (ASX) (Fig. 1b, Supplementary Fig. 2 and Supplementary Table 1). Analysis of other gel regions and liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis of total purified material confirmed that ASX was the main protein co-purifying with TAP–Calypso (Supplementary Table 1). ASX is a PcG protein required for long-term repression of HOX genes during Drosophila development7,8,14, but it had not been identified in previously characterized PcG protein complexes and its molecular function has remained largely elusive. Calypso and ASX are thus components of a new, bona fide PcG protein complex that we named Polycomb repressive deubiquitinase (PR-DUB). We tested whether Drosophila PR-DUB complexes could be reconstituted from recombinant Calypso and ASX proteins. Using baculovirus vectors, we expressed Flag–Calypso and haemagglutinin (HA)–ASX(1–1668) or HA–ASX(2–337) as individual proteins in Sf21 cells, mixed the cell lysates and performed Flag affinity purification. This strategy resulted in the isolation of stable Calypso–ASX complexes and showed that Calypso interacts with the aminoterminal 337 amino acids of ASX (Fig. 1c, left, lane 3 and Supplementary Fig. 3). ASX also formed stable complexes with Calypso(C131S), a mutant Calypso protein in which the predicted catalytic cysteine in the UCH domain10,15 had been substituted by serine (Fig. 1c, left, lane 4 and Supplementary Fig. 3). The interaction between Calypso and ASX(2–337) was specific because ASX(2–337) did not bind to the PcG proteins Flag–ESC or Flag–Sce under the same assay conditions (Fig. 1c, left, lanes 5 and 6). Using the same strategy, we found that human BAP1 also forms a stable complex with the N-terminal domain of human ASXL1 (ASXL1(2–365)) but not with the human PcG proteins BMI1 or RING1A (Fig. 1c, right, lanes 8–10). Like Drosophila Calypso and ASX, human BAP1 and ASXL1 proteins could thus also be assembled into a stable PR-DUB complex (Fig. 1c, right, lane 8), demonstrating the evolutionary conservation of this interaction.

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 2The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA. *These authors contributed equally to this work.

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NATURE | Vol 465 | 13 May 2010

a
1 CAA Gln 41

Calypso
UCH 471 TAA Stop

c
+ + + + +
kDa 63

Proteins expressed in Sf21 cells Flag–Calypso Flag–Calypso(C131S) Flag–ESC Flag–BAP1 BMI1–Flag Flag–RING1A HA–ASXL1(2–365)

+

+ + + + + +
kDa 63

b
M kDa

TAPWT – calypso

+ * * *
#

+ *
#

+ *

+ Flag–Sce + HA–ASX

45 126 63 45 35 25 17 12 7 ASX fragments CBP–Calypso ASX fragment 35 1 2

*
4 5

Purified protein complexes (Coomassie) 6

*

*
#

*
8 9

*
10

45

3

Anti-HA Anti-HA Anti-Flag

HA–ASX/HA–ASXL1 in purified complexes (western) Sf21-cell extract (input) (western)

7

Anti–HA Anti–HA Anti–Flag

Drosophila PR-DUB Human PR-DUB

Figure 1 | The Polycomb group proteins BAP1 and ASX form a conserved complex in vivo and in vitro. a, Domain architecture of the Drosophila Calypso protein and molecular lesions in calypso mutant alleles. UCH, ubiquitin C-terminal hydrolase domain. b, Calypso complexes isolated by TAP26 from wild-type (WT) or TAP-calypso transgenic embryos. Input material for purification was normalized by protein concentration, and equivalent amounts of eluate from calmodulin-affinity resin were separated on a 4–12% polyacrylamide gel and visualized by silver staining together with a molecular mass marker (M). Calypso bait protein containing the calmodulin-binding tag (CBP–Calypso), and bands representing ASX fragments were identified by mass spectrometry (Supplementary Table 1 and Supplementary Fig. 2). No band corresponding to full-length ASX (180 kDa) was detected in several independent purifications, even though

ASX is present as a single polypeptide of 180 kDa in total embryo extracts (Supplementary Fig. 5). This suggests that ASX is degraded during nuclear extract preparation or TAP purification. c, Reconstitution of recombinant Calypso–ASX and BAP1–ASXL1 complexes. Proteins were extracted by Flag-affinity purification from cell lysates containing the indicated Flagtagged proteins and HA–ASX(2–337) (left) or HA–ASXL1(2–365) (right). Experiments with full-length ASX are shown in Supplementary Fig. 3. Proteins were visualized by Coomassie staining or western blotting analysis, as indicated. Input material for experiments in lanes 3–6 (left) and 7–10 (right) were probed by western blotting to ensure that comparable amounts of proteins were present in cell lysates. On the Coomassie-stained gel, Flagtagged proteins are marked with an asterisk, HA–ASX(2–337) and HA–ASXL1(2–365) are marked with a hash symbol.

On polytene chromosomes, ASX protein binds at chromosome intervals encompassing the HOX genes and at many other chromosomal sites that co-map with binding sites for other PcG proteins14. We determined the genome-wide PR-DUB binding profile in the chromatin of Drosophila larvae by performing chromatin immunoprecipitation (ChIP) assays with antibodies against Calypso and ASX proteins. The precipitated material was hybridized to high-density whole-genome tiling arrays and analysed with TileMap16, using a stringent cutoff. We only considered genomic regions that were significantly enriched by both anti-Calypso and anti-ASX antibodies, and thus obtained a high-confidence set of 879 genomic sites bound by PR-DUB (Fig. 2a, b and Supplementary Table 2). We compared the PR-DUB binding profile with the profiles of the PRC1 subunit Ph and the PhoRC subunit Pho in imaginal disc cells17,18. PR-DUB is cobound together with Ph and Pho at Polycomb response elements (PREs) of a large set of PcG target genes, such as the HOX genes (Fig. 2a, b and Supplementary Table 2). PR-DUB is thus a core PREbinding complex, like PhoRC, PRC1 and PRC2. To extend these analyses, we compared the binding of Calypso and ASX in wing imaginal disc cells in which the HOX gene Ultrabithorax (Ubx) is inactive, and in haltere/third leg imaginal disc cells in which Ubx is expressed, at the same 16 locations across the Ubx transcription unit where we had previously analysed binding of the PcG protein complexes PhoRC, PRC1 and PRC2 (ref. 19). Like these other PcG protein complexes19, PR-DUB was bound at Ubx PREs both in cells where Ubx is repressed and in cells where it is active (Fig. 2c). To characterize the deubiquitinase activity of PR-DUB, we tested whether the Drosophila complex could cleave the fluorogenic substrate ubiquitin-amidomethylcoumarin (Ub-AMC). Calypso alone hydrolysed the Ub-AMC bond, but the Calypso–ASX(2–337) complex was substantially more active in catalysing this reaction (Fig. 3a). In contrast, Calypso(C131S)–ASX(2–337) was virtually inactive (Fig. 3a). PR-DUB thus functions as a deubiquitinase in vitro and the catalytic activity of Calypso is strongly enhanced by association
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with the N-terminal domain of ASX. Because Drosophila PR-DUB associated with the chromatin of target genes, we then asked whether PR-DUB deubiquitinates histone H2A or H2B. Monoubiquitination of H2A (H2Aub1) at Lys 119 in vertebrates and Lys 118 in Drosophila by PRC1-like and dRAF, respectively, is thought to be critical for PcG repression20–22. Monoubiquitination of H2B (H2Bub1) at Lys 120 in vertebrates (corresponding to Lys 117 in Drosophila) is catalysed by a different E3 ligase, RNF20 (also known as BRE1), and has been implicated in transcriptional elongation22. We reconstituted recombinant mononucleosomes that contained either H2Aub1 or H2Bub1 (Fig. 3b) and used them as substrates in deubiquitination assays. Notably, the Drosophila Calypso–ASX(2–337) and Calypso–ASX(1–1668) complexes and the human BAP1– ASXL1(2–365) complex all deubiquitinated H2Aub1 but not H2Bub1 in nucleosomes (Fig. 3c, lanes 3–5, 14–16 and Supplementary Fig. 4c). Deubiquitination of H2Aub1 required both the presence of the catalytic cysteine in Calypso (Fig. 3c, lanes 6–8) and the association of ASX with Calypso (Supplementary Fig. 4) or of ASXL1 with BAP1, respectively (Fig. 3c, compare lanes 14–16 with 11–13). Moreover, PR-DUB showed only very poor activity for cleaving polyubiquitin chains that were linked through either Lys 63 or Lys 48 (Fig. 3d). PR-DUB thus specifically deubiquitinated H2Aub1 in nucleosomes in these assays. We next investigated how the lack of PR-DUB affects H2Aub1 levels in developing Drosophila. In embryos that are homozygous for Asx22P4, ASX protein is undetectable and Calypso protein levels are very drastically diminished (Supplementary Fig. 5). Asx22P4 mutant embryos thus have severely reduced levels of PR-DUB. We isolated bulk histones from wild-type and Asx22P4 homozygous embryos by acid extraction, and compared the levels of H2Aub1, H2Bub1, H3K27me3 and H4K4me3 in the two genotypes. Bulk H2Aub levels were almost tenfold increased in Asx22P4 mutant embryos (Fig. 4a). In contrast, the level of the PcG-specific histone tri-methylation mark H3K27me3 was comparable in Asx22P4 mutant

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NATURE | Vol 465 | 13 May 2010

LETTERS
Figure 2 | PR-DUB is bound at Polycomb target genes in Drosophila. a, PR-DUB is bound at PREs of PcG target genes in Drosophila. ChIP profiles of PR-DUB subunits ASX (dark blue) and Calypso (light blue), and of Ph18 (grey) and Pho17 (grey) at the Antennapedia HOX gene cluster in imaginal disc and CNS tissues from third instar Drosophila larvae. Hybridization intensities for oligonucleotide probes are plotted as coloured bars above the genomic map (release 5, kilobase coordinates) of Drosophila melanogaster; significantly enriched regions are marked below plots. HOX genes labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp) and other genes on the plus (above) or minus (below) strand are represented with exons (black boxes) and introns (thin black lines). b, Venn diagrams showing the overlap of 879 PR-DUB-bound regions with 1,681 Ph-bound and 670 Pho-bound regions in larval cells. c, PR-DUB is bound at the inactive and at the active Ubx gene. ChIP analyses monitoring ASX and Calypso binding in wing and haltere/third leg imaginal discs from wildtype third instar Drosophila larvae. Graphs show results from independent ChIP reactions (n 5 3 ChIP reactions) with ASX or Calypso antibodies. ChIP signals, measured by qPCR, are presented as the mean percentage of input chromatin precipitated at each region; error bars indicate 6s.d. (see Methods). Locations of Ubx PREs (boxes) and other regions relative to the Ubx transcription start site are indicated in kilobases. As control, binding was monitored at two euchromatic (eu) and one heterochromatic (het) region elsewhere in the genome, and at the PREs of the HOX genes Abd-B and Scr that are both inactive in wing and haltere/third leg imaginal discs. Calypso and ASX are bound at Ubx PREs both in wing and in haltere/third leg disc cells; at the 230-kb PRE, Calypso and ASX ChIP signals were comparable in wing and haltere/third leg chromatin, at the 132-kb PRE, the signal in haltere/third leg chromatin is about 2–4-fold lower than in wing chromatin, paralleling PRC1 and PRC2 binding at both these PREs19.

a
15 Pho 30 Ph 40 Calypso 30 ASX

Taf1 2,450 2,500 lab 2,550 pb 2,600 bcd

Dfd 2,650 Scr

ftz 2,700

2,750 Antp Antp

2,800

2,850 kb

b

c

4 ASX 3

Wing discs Haltere/3rd leg discs

Pho 257 141 979 Ph 193 79 193 368 239 Calypso ASX

Percentage of input

2 1

0.8 0.6 0.4 0.2
–3 8 –3 3 –3 1 –3 0

Calypso

Wing discs Haltere/3rd leg discs

–2 9 –1 6 –2

Ultrabithorax

and wild-type embryos (Fig. 4a). Unexpectedly, we also found a weak increase in H2Bub levels and a very slight concomitant increase in H3K4me3 levels (Fig. 4a). The higher H2Bub levels could be an indirect consequence of widespread global H2A ubiquitination, but it is also possible that, in vivo, PR-DUB deubiquitinates both H2A and H2B. Previous studies reported that the monoclonal antibody E6C5 specifically recognizes H2Aub1 in mammalian cells23, but we have not been able to specifically monitor H2Aub1 levels by ChIP in Drosophila using the commercially available E6C5 antibody (Supplementary Fig. 6 and Methods). Finally, we tested whether the deubiquitinase activity of PR-DUB is required for PcG repression. To this end we used a transgene rescue assay and asked whether the catalytically inactive Calypso(C131S) protein can repress the PcG target gene Ubx in Drosophila larvae, as follows. Clones of calypso2 mutant cells in larval imaginal discs lack detectable Calypso protein and fail to repress Ubx (Fig. 4b)8. However, a regular supply of wild-type Calypso protein from a heat-inducible hsp70-calypso transgene fully rescues repression of Ubx in such clones (Fig. 4b). In contrast, the catalytically inactive Calypso(C131S) protein expressed from a hsp70-calypso(C131S) transgene failed to rescue repression, and Ubx was misexpressed as in control animals lacking any hsp70-calypso transgene (Fig. 4b). PRDUB deubiquitinase activity is thus critically required for repression of PcG target genes in Drosophila.

0 2 8 27 32 34 71 77

kb:

Abd-B Scr eu het

The following conclusions can be drawn from the work reported here: PR-DUB is a new PcG protein complex that comprises the Calypso and ASX proteins; PR-DUB is bound at the PREs of PcG target genes in Drosophila; reconstituted recombinant Drosophila or human PR-DUB deubiquitinate H2A in nucleosomes in vitro; Drosophila mutants lacking PR-DUB show an increase in global H2Aub1 levels; and a mutation in Calypso that disrupts H2A deubiquitinase activity in vitro impairs repression of HOX genes in Drosophila. Our analyses identified nucleosomal H2Aub1 as the preferred PR-DUB substrate; the complex failed to deubiquitinate nucleosomal H2Bub1 and showed only very poor activity for cleaving polyubiquitin chains. It is possible that PR-DUB also deubiquitinates other proteins but here we shall discuss its possible role in H2A deubiquitination. The observation that repression of PcG target genes in Drosophila requires not only the H2A ubiquitinase activity of PRC1 and dRAF but also PR-DUB may seem surprising. However, simultaneous depletion of Sce (that is, the H2A ubiquitinase subunit of PRC1 and dRAF20,21,24) and PR-DUB in embryos results in a more rapid loss of HOX gene repression and consequently more severe transformation of body segments than the depletion of Sce or PRDUB alone (Supplementary Fig. 7). This suggests that appropriately balanced H2Aub1 levels in target gene chromatin may be critical for maintaining a Polycomb-repressed state. One possibility would be that PRC1/dRAF and PR-DUB act locally within target gene chro245

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NATURE | Vol 465 | 13 May 2010

Fluorescence (a.u.)

160 Calypso–ASX(2–337) Calypso 120 Calypso(C131S)– ASX(2–337) 80 40 0 0 200 400 Time (s) 600 800

kDa 25 17 12

U nm od . H 2A ub 1 H 2B ub 1

a

b

H2Bub1 H2Aub1 H3 H2A/H2B H4

a
1

WT 3 9 1

Asx22P4 3 9 Ratio of extract loading H2Aub1 H2A (WB: anti-H2A) H2Bub1 H2B (WB: anti-H2B) H2Aub1/H2Bub1 (WB: anti-ubiquitin) H3K4me3

1

2

3

c

Unmod.

Ubiquitinated mononucleosomes
Calypso– Calypso(C131S)– ASX(2–337) ASX(2–337)

Unmod.

Ubiquitinated mononucleosomes

kDa 25 17 12 25 17 12 1 2

kDa 25 17 12 25 17 12

BAP1

BAP1– ASXL1(2–365)

2.5 10 40 2.5 10 40 Time (min) H2Aub1 H2A 3 4 5 6 7 8 H2Bub1 H2B Drosophila PR-DUB

2.5 10 40 2.5 10 40 Time (min) H2Aub1 H2A 9 10 11 12 13 14 15 16 H2Bub1 H2B Human PR-DUB

C al yp C so al yp so –

d
kDa – 45 35 25 17 12 7.5

C

C

35 ub4 ub3 ub2 ub1 1 2 3 4 5 K63-linked ub-hexamers 25 17 12 7.5

C

C

al

kDa 45



al

al

y

o ps

(C

13

) 1S

yp so yp C so al – yp AS C so(C X(2 al 1 –3 y AS ps 31 37 X( o(C S) ) 2– 1 33 31 7) S)–

AS X( 2– al 33 y 7) AS ps o( X( C 2– 1 31 33 7) S)–

H3K27me3

ub4 ub3 ub2 ub1 7 8 9 10 11 K48-linked ub-hexamers

H3 1 2 3 4 5 6 hs-calypso(C131S)

b

No transgene

hs-calypso

Figure 3 | Recombinant Drosophila and human PR-DUB deubiquitinate H2A in nucleosomes in vitro. a, Cleavage of Ub-AMC by Calypso and Calypso–ASX(2–337) complexes. Reactions (n 5 4) contained 25 pmol UbAMC and 10 pmol of the indicated protein (complex); release of AMC was monitored by fluorescence spectroscopy at 436 nm; error bars indicate 6s.d. a.u., arbitrary units. b, Mononucleosomes were reconstituted with recombinant Xenopus histone octamers and were unmodified (lane 1), monoubiquitinated at H2AK119 (lane 2) (see Methods) or monoubiquitinated at H2BK120 (lane 3) (see Methods). The material was analysed on a 4–12% polyacrylamide gradient gel and histones were visualized by Coomassie staining. c, Drosophila and human PR-DUB deubiquitinate H2Aub1 in nucleosomes. Xenopus mononucleosomes (15 pmol) containing 30 pmol of either H2Aub1 (top gels left and right) or H2Bub1 (bottom gels left and right) were incubated without (lanes 2 and 10) or with 30 pmol of the indicated Drosophila PR-DUB complexes (lanes 3–8) or human BAP1 or PR-DUB complex (lanes 11–16), respectively, and deubiquitination was monitored at indicated time points by western blot analysis with anti-H2A (top gels left and right) or anti-H2B (bottom gels left and right) antibody (5 pmol nucleosome per lane). Unmodified mononucleosomes (lanes 1 and 9) served as a control. Comparable results were obtained with Drosophila mononucleosomes containing H2Aub1 (Supplementary Fig. 4). PR-DUB containing full-length ASX(1–1668) also specifically deubiquitinated H2Aub1 but not H2Bub1 in nucleosomes (Supplementary Fig. 4). d, K48- or K63-linked hexameric polyubiquitin chains (160 ng; corresponding to maximally 17.5 pmol ubiquitin linkage bonds) were incubated for 40 min with 10 pmol of the indicated protein or protein complex under the same assay conditions as in c, followed by western blot analysis with an anti-ubiquitin antibody.

Anti-UBX GFP

GFP

Anti-Calypso

matin; the presence of H2Aub1 in some regions of a gene may be critical for repression but may be detrimental to it in others. Alternatively, H2A ubiquitination and deubiquitination may have to occur in a temporally regulated cycle to maintain repression, similar to what has been proposed for H2B ubiquitination and deubiquitination during transcriptional elongation25. Interestingly, calypso and Asx mutant embryos show not only derepression of HOX genes but also a partial loss of HOX gene expression in the central nervous system (Supplementary Fig. 8a). This loss of HOX gene expression seems to be restricted to the nervous system and we have not been able to detect a reduction of HOX gene expression in
246

Figure 4 | PR-DUB is required for H2A deubiquitination in Drosophila and its catalytic activity is essential for HOX gene repression. a, PR-DUB is required for H2A deubiquitination in Drosophila embryos. Serial dilutions (1:3:9) of histone extracts from 16–18-h-old wild-type or Asx22P4 homozygous embryos were separated on 4–12% polyacrylamide gels and analysed by western blotting with the indicated antibodies. H2Aub1 levels in lanes 3 and 4 are comparable, suggesting that H2Aub1 levels are almost tenfold higher in Asx22P4 mutants than in wild type. H2Bub1 levels in Asx22P4 mutants are less than threefold increased compared to wild type (compare lane 3 with lanes 5 and 6). The band detected by an anti-ubiquitin antibody represents the combined signal of H2Aub1 and H2Bub1. H3K4me3 levels appear very slightly increased in Asx22P4 mutants. b, Calypso deubiquitinase activity is required for HOX gene repression. Wing imaginal discs with clones of calypso2 homozygous mutant cells from animals that carried no transgene or the indicated hsp70-calypso transgenes. calypso2 mutant cells are marked by the absence of GFP and discs were stained with antibodies against UBX or Calypso protein, as indicated. In all cases, clones were induced 96 h before analysis and larvae were repeatedly heat-shocked for 1 h every 12 h over a 96-h period to provide a continuous supply of Calypso protein from the transgene. In the absence of an hsp70-calypso transgene, Ubx is misexpressed in most calypso2 mutant clones in the pouch of the disc but remains repressed in the notum and hinge (left). Wild-type Calypso protein rescues repression of Ubx in mutant clones (middle), whereas the Calypso(C131S) protein fails to rescue (right), even though both transgeneencoded proteins are expressed at comparable levels and show nuclear localization like endogenous Calypso protein (bottom row).

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the embryonic epidermis or in imaginal disc cells (Supplementary Fig. 8b). Thus, even though PR-DUB is primarily required for repressing PcG target genes outside their expression domains, it might also be needed to fine-tune expression levels within these domains in certain tissues, perhaps by preventing repressive hyper-ubiquitination of H2A by PRC1 or dRAF complexes. It will be interesting to determine whether the mammalian complex has a similar prominent role in PcG repression during development and for maintenance of stem cell pluripotency, and to explore how the tumour suppressor activity of BAP1 (ref. 13) relates to the H2A deubiquitinase activity of human PR-DUB.
METHODS SUMMARY
Mapping and molecular cloning of the calypso gene. Detailed information can be found in the Methods. ChIP assays and genome-wide Calypso and ASX profiling. X-ChIP in Drosophila imaginal disc cells, quantitative PCR (qPCR) analysis of immunoprecipitates and genome-wide profiling using Affymetrix whole genome tiling arrays were performed as described17,19. Tandem-affinity purification of Calypso complexes. The a-tubulin1-TAP-calypso transgene fully rescued the viability and fertility of calypso2/Df(2R)Exel6063 animals. TAP was performed as described26. Baculovirus expression of recombinant proteins. Flag-affinity purification of proteins was carried out as described26 with the only modification that protein complexes were reconstituted by infecting cells with individual viruses and then mixing cell lysates and incubating them for 2–3 h at 4 uC under mild agitation before Flag-affinity purification. Recombinant mononucleosomes containing H2Aub1. Recombinant Drosophila and Xenopus octamers were assembled on a 59 biotinylated 288base-pair (bp) DNA fragment (601) by stepwise salt-dialysis. Nucleosomal H2A was ubiquitinated as described24. H2A-ubiquitinated nucleosomes were purified by binding to Streptavidin-coupled Dynabeads (Sigma), followed by washes and released by endonucleolytic cleavage with EcoRV. See the Methods for details. Deubiquitination assays. Reactions were carried out at 25 uC in deubiquitination buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM ZnCl2, 1 mM dithiothreitol (DTT)) and were stopped by the addition of SDS sample loading buffer and incubation at 95 uC for 5 min. Analysis of PR-DUB function in Drosophila. Induction of homozygous mutant cell clones in imaginal discs of Drosophila larvae, rescue experiments with hsp70calypso transgenes and immunofluorescence staining were performed following previously established protocols27. Generation of H2Bub1-containing mononucleosomes. Xenopus octamers containing H2Bub1 were generated as described28 and mononucleosomes were assembled as described earlier.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Received 7 July 2009; accepted 25 February 2010. Published online 2 May 2010.
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18. 19.

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24. 25.

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Nijman, S. M. B. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005). 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). Amerik, A. Y. & Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta 1695, 189–207 (2004). 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). 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). 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). 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 A resolution. EMBO J. 16, 3787–3796 (1997). Ji, H. & Wong, W. H. TileMap: create chromosomal map of tiling array hybridizations. Bioinformatics 21, 3629–3636 (2005). 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). Gambetta, M. C., Oktaba, K. & Muller, J. Essential role of the glycosyltransferase ¨ Sxc/Ogt in Polycomb repression. Science 325, 93–96 (2009). Papp, B. & Muller, J. Histone trimethylation and the maintenance of ¨ transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20, 2041–2054 (2006). Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004). Lagarou, A. et al. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22, 2799–2810 (2008). Weake, V. M. & Workman, J. L. Histone ubiquitination: triggering gene activity. Mol. Cell 29, 653–663 (2008). 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). 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). 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). 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). Beuchle, D., Struhl, G. & Muller, J. Polycomb group proteins and heritable silencing ¨ of Drosophila Hox genes. Development 128, 993–1004 (2001). 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).

Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements 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. Muller 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. 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. Author Information The microarray data have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under the accession number E-TABM-908. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to J.M. (juerg.mueller@embl.de).

247
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doi:10.1038/nature08966

METHODS
Molecular identification of the calypso gene. We previously mapped the calypso mutations to the right arm of chromosome 2 (2R)8. Complementation tests with a set of chromosomal deficiencies uncovering much of chromosome arm 2R allowed us to locate the calypso mutations to a short interval in the 52F-53A region. In particular, calypso1 failed to complement Df(2R)Jp4, Df(2R)Jp5, Df(2R)Jp6, Df(2R)Jp7, Df(2R)Jp8 or Df(2R)Exel6063 but complemented all other tested deficiencies on chromosome arm 2R. calypso2 also failed to complement Df(2R)Jp4 and Df(2R)Exel6063. Because Df(2R)Jp4 complemented mutations in Khc8 (provided by P. R?rth), the calypso mutations were thus mapped to a chromosomal interval extending from the centromere-proximal end of Df(2R)Exel6063 and the Khc locus. Moreover, we found that calypso1 and calypso2 both complemented csulRM (provided by B. Mechler), a viable but female-sterile P-element insertion and csulRL, a small chromosomal deletion that removes csul, Khc and fidipidine. Sequencing of the CG8445 open reading frame on the FRT40 FRT42 y1 calypso1 and FRT40 FRT42 y1 calypso2 chromosomes revealed a C-to-T transition that was present in both alleles and changed the CAA codon for Gln 41 into a TAA termination codon; in contrast, the wild-type CAA codon for Gln 41 was present on the isogenic FRT40 FRT42 y1 chromosome on which these mutations had been induced by ethylmethanesulphonate (EMS)8. We note that even though calypso1 and calypso2 contain the same Gln 41.stop mutation, the two alleles had been isolated in two independently performed mutagenesis experiments8. No other mutations were detected in the CG8445 open reading frame, but we note that the FRT40 FRT42 y1, FRT40 FRT42 y1 calypso1 and FRT40 FRT42 y1 calypso2 chromosomes all contain a TCA codon (serine) at residue 17 of the CG8445 open reading frame, whereas the database genomic sequence and the SM6b balancer chromosome contain a GCA codon for alanine at this position. Molecular analysis of ASX alleles. Asx22P4 homozygous embryos fail to express detectable amounts of ASX protein (Supplementary Fig. 5). We have not been able to find a lesion in the ASX open reading frame on the FRT40 FRT42 y1 Asx22P4 chromosome8. Asx27J6 homozygotes express a truncated ASX protein that migrates with an apparent molecular mass of 58 kDa (Supplementary Fig. 5). Sequencing of the ASX open reading frame on the FRT40 FRT42 y1 Asx27J6 chromosome8 showed a deletion of the cytosine in the CGT codon for Arg 433. This mutation results in a frameshift that is predicted to terminate the open reading frame after 45 nucleotides, that is, the predicted Asx27J6 open reading encodes ASX(1–432) fused to 15 further amino acids. Immunostaining of Drosophila embryos and imaginal discs. Staining of Drosophila embryos and larval imaginal discs was performed following standard protocols. Induction of homozygous mutant cell clones in imaginal discs of Drosophila larvae and the rescue experiments with hsp70-calypso transgenes shown in Fig. 4 were performed as previously described27. Generation of H2Bub1-containing mononucleosomes. Xenopus octamers containing H2Bub1 were chemically generated as described28. Mononucleosome assembly was performed by stepwise salt dialysis as described earlier and the nucleosomes were directly used for deubiquitination assays. We note that the H2Bub1 isopeptide bond can be hydrolysed by UCH-L3 under appropriate experimental conditions29. Antibodies. The following previously described antibodies were used in this study: anti-UBX30, anti-ABD-B31, anti-H2A (Millipore, 07-146), anti-H2B (Millipore, 07-371), E6C5 (Millipore, 05-678, lot: DAM1514075), antiUbiquitin P4D1 (Santa Cruz Biotechnology, sc-8017), anti-Flag (Sigma, F7425), anti-HA (Sigma, H3663), anti-H3 (Abcam, AB1791), anti-H3K4me3 (provided by B. Turner), and anti-H3K27me3 (Upstate, 07-449). For this study, antibodies against Calypso(1–471) and ASX(210–336) were raised in rabbits. In both cases, the epitopes for antibody production were expressed as 63His-tagged fusion proteins in E. coli and purified under denaturing conditions. As shown in Supplementary Fig. 6a, the E6C5 antibody (Millipore/Upstate) recognizes a multitude of protein bands in nuclear extracts from Drosophila embryos. For the western blot analyses shown in Supplementary Fig. 6b and c, H2Aub1-containing Drosophila mononucleosomes were generated as described earlier. K48-linked ubiquitin pentamers (Boston Biochem, UC-216) and K63linked ubiquitin pentamers (Boston Biochem, UC-316) were used as a control. ChIP assays. X-ChIP from Drosophila larval imaginal discs and qPCR analysis to determine protein binding at specific chromosomal locations were performed as described19 with the following modifications. Washes of immunoprecipitated material on protein A-sepharose beads were performed for 10 min at 4 uC with 1 ml of the following buffers: five washes with RIPA buffer (350 mM NaCl (ASX ChIP)/140 mM NaCl (Calypso ChIP), 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM phenylmethylsulphonyl fluoride (PMSF)), followed by one wash with LiCl buffer (250 mM

LiCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and two washes with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA); each time, wash solution was removed after centrifugation for 2 min at 3,000g at 4 uC. ChIP-chip in Drosophila using Affymetrix whole genome microarrays. Chromatin was prepared from imaginal disc (wing, haltere and third leg) and central nervous system tissues of third instar Drosophila larvae and ChIP was performed as described17. To determine regions bound by PR-DUB, three ChIP assays were performed with the anti- Calypso(1–471) antibody and three with the anti-ASX(210–336) antibody, using independently prepared batches of chromatin. The immunoprecipitated material was amplified by ligation-mediated PCR, fragmented, labelled and hybridized to Affymetrix whole genome microarrays as described17. Data analysis was done as described in ref. 17 and later. Genome assembly and annotation. The third Drosophila melanogaster genome assembly (UCSC dm3 or Flybase 5.x) and the gff export of Flybase 5.23 for genome annotation was used. Affymetrix probes re-mapping. The Dm35b_MR_v02-3_BDGPv4h.new.bpmap file from the CisGenome website (http://www.biostat.jhsph.edu/,hji/cisgenome/) was used. This file contains the re-mapped location to genome version 5 (that is, UCSC dm3) of all the original Affymetrix 25mer sequences but lacks probes that cannot uniquely map to the genome. Detection of regions that are significantly bound by PR-DUB. A quantile normalization32 was applied to normalize all six ChIP hybridizations (three anti-Calypso and three anti-ASX ChIP assays, see earlier) to three genomic DNA hybridizations. Regions that were significantly enriched for PR-DUB binding were identified using TileMap16 with the Hidden Markov Model (HMM). A TileMap score where 50% of the previously identified 237 regions bound by PhoRC and Ph in larva17,18 were recovered was used as threshold for considering significantly bound regions. In addition, the top 5% of regions below cutoff exhibiting overlap with Pho17, dSfmbt17, Ph18 or Sce-bound regions (K.O., L. Gutierrez and J.M., unpublished observations) were rescued and included in the final set of high-confidence PR-DUB binding sites. We thus identified 879 regions (560 scoring above cutoff, plus 319 rescued) bound by PR-DUB in larval CNS and imaginal disc cells. Venn diagram counts were obtained as follows. Two or more regions that overlap with at least one base were merged and defined as a ‘common’ region. PR-DUB-bound regions, target gene assignment and Gene Ontology (GO) slim analysis. For the data shown in Supplementary Table 2, the relative distance of the midpoint of each PR-DUB-bound region with respect to the closest transcription start site (TSS) was computed. Target genes were assigned based on TSS-proximal location. Assigned genes to each data set were tested for enriched GO slim term annotations. Quantitative PCR to determine binding at specific chromosomal locations. qPCR analysis was done as described19, using the following primers. Ubx-1: forward, 59-TGGGATTGCGATAGTGTTGGTC-39, reverse, 59-CGCAGCCATT ATGAAACCTCCT-39 (237.7 kb); Ubx-2: forward, 59-ATTTGGTCCGCAGT CGAGTGCATTT-39, reverse, 59-CCACATGCACATCCTGGATGCCAAT-39 (232.5 kb); Ubx-3: forward, 59-GCAGCATAAAACCGAAAGGA-39, reverse, 59-CGCCAAACATTCAGAGGATAG-39 (230.9 kb); Ubx-4: forward, 59-TAGT CTTATCTGTATCTCGCTCTTA-39, reverse, 59-CAGAACCAAAGTGCCGATA ACTC-39 (229.8 kb); Ubx-5: forward, 59-AAGGCGAAAGAGAGCACCAA-39, reverse, 59-CGTTTTAAGTGCGACTGAG-39 (229.6 kb); Ubx-6: forward, 59-GCACGCACTAAACCCCATAA-39, reverse, 59-TCCACCTCCTCTTCCTCT CTC-39 (229.0 kb); Ubx-7: forward, 59-GGTCAAAGGCCATACAATTCCA-39, reverse, 59-ATCTGTGAGAATGCGGCATCTAA-39 (216.0 kb); Ubx-8: forward, 59-ATCGGTAGCTTGTTGCAGCA-39, reverse, 59-GGCTACTTGGACA GGTGTGAGC-39 (22.4 kb); Ubx-9: forward, 59-TCCAATCCGTTGCCATCGA ACGAAT-39, reverse, 59-TTAGGCCGAGTCGAGTGAGTTGAGT-39 (0 kb); Ubx-10: forward, 59-AATTGGTTTCCAGGGATCTGC-39, reverse, 59-ATCCAA AGGAGGCAAAGGAAC-39 (10.8 kb); Ubx-11: forward, 59-ATGATATCTCGTC TGGCACTAC-39, reverse, 59- AGACATCCAGCAAACTGCGATA-39 (18.0 kb); Ubx-12: forward, 59-GCCGTGGAGCAGTTCAAAGTA-39, reverse, 59-TCGTTG GTCGTGTCCTCTTAATT-39 (126.8 kb); Ubx-13: forward, 59-CCATAAGAAAT GCCACTTTGC-39, reverse, 59-CTCTCACTCTCTCACTGTGAT-39 (131.5 kb); Ubx-14: forward, 59-GTCCTGGCCAAGGCAAATATT-39, reverse, 59-CGAAAG GAGAACGGAGAATGG-39 (134.4 kb); Ubx-15: forward, 59-GGCATCTTCCAG GTTTTGAGT-39, reverse, 59-ACCAGCATTCGTCACTATTCG-39 (170.6 kb); Ubx-16: forward, 59-GCCGAGGGTCAGAGAGTTTA-39, reverse, 59-CTGCATC CGACCACTTACCT-39 (172.3 kb); Abd-B: forward, 59-GGAATACCGCACTG TCGTAGG-39, reverse, 59-GCAGCCATCATGGATGTGAA-39 (10.2 kb); Scr: forward, 59-GAAGTGCGCCACGTTCAAT-39, reverse, 59-TCCTCTCTCTCGCA CTCGTT-39 (10.2 kb); control-1: forward, 59-TCAAGCCGAACCCTCTAAA AT-39, reverse, 59-AACGCCAACAAACAGAAAATG-39 (212.5 kb); control-2: forward, 59-CCGAACATGAGAGATGGAAAA-39, reverse, 59-AAAGTGCCGAC

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doi:10.1038/nature08966

AATGCAGTTA-39 (23.1 kb); control-3: forward, 59-CAGTTGATGGGATGAAT TTGG-39, reverse, 59-TGCCTGTGGTTCTATCCAAAC-39 (112.4 kb). Tandem affinity purification of Calypso complexes. The a-tubulin1-TAPcalypso transgene in the Drosophila transformation vector pCaSpeR had the following structure: a 2.6-kb fragment of the a-tubulin1 gene containing promoter and 59-untranslated region sequences, followed by the N-terminal TAPtag33 was linked to a calypso cDNA fragment that contained the whole calypso open reading frame (detailed plasmid map available on request). Rescue function of the a-tubulin1-TAP-calypso transgene was tested by introducing the three independent transgene insertions into a calypso2/Df(2R)Exel6063 mutant background; in all cases we obtained w; calypso2/Df(2R)Exel6063; a-tubulin1-TAPcalypso/1 animals that were wild-type in appearance and fully viable and fertile. TAP was performed on embryonic nuclear extracts prepared from wild-type or a-tubulin1-TAP-calypso transgenic embryos, following previously described protocols26. Mass spectrometry, capillary LC–MS/MS analysis and protein identification. Silver-stained protein bands were excised and trypsin digested as previously described34. The samples were loaded and separated on a nano-flow 1D-plus Eksigent (Eksigent) HPLC system coupled to a qStar Pulsar i quadrupole timeof-flight MS (Applied Biosystems). The peptides were separated by a linear gradient that started at 100% mobile phase A and increased the mobile-phase composition to 50% B (0.5% acetic acid in 98% acetonitrile) over a span of 45 (single protein band) or 120 (complex samples) minutes at a constant flow rate of 200 nl min21. The mass spectrometer was operated in data-dependent positive-ion mode. MS spectra were acquired over m/z range from 350 to 1,300 for 1 s and one subsequent MS/MS spectrum from 60 to 1,800 m/z for 1.5 s. MS/MS data was extracted using the AnalystQS software (Applied Biosystems). Peptides were identified by searching the peak-list against the NCBI database using the MASCOT (Matrix Science) algorithm. Peptide tolerance was limited to 50 p.p.m., and peptides with a score below 15 were excluded. Protein identifications were accepted if a single peptide with an individual MASCOT score above 45 or at least two peptides with a summed MASCOT score of more than 40 were identified. A detailed list of peptide sequences obtained from mass spectrometry analysis of the protein bands shown in Fig. 1 is shown in Supplementary Fig. 1 and in Supplementary Table 1. Peptides identified by LC–MS/MS analysis of total purified material are also shown in Supplementary Table 1. Constructs for baculovirus production and purification of recombinant proteins. Baculoviruses expressing Flag–ESC, Flag–Sce, BMI1–Flag and Flag– RING1A have been described previously35–37. For this study we generated baculovirus vectors (pFastBac) that encode the following proteins: Flag–Calypso and Flag–Calypso(C131S), both containing the entire Calypso(1–471) open reading frame, HA–ASX, containing the entire ASX(1–1668) open reading frame, HA– ASX(2–337), Flag–BAP1(2–729) and HA–ASXL1(2–365). Detailed plasmid maps for all constructs are available on request. Flag-affinity purification of proteins and complexes was carried out as described26 with the only modification that protein complexes were reconstituted by infecting cells with individual viruses and then mixing cell lysates and incubating them for 2–3 h at 4 uC under mild agitation before Flag-affinity purification. Deubiquitination assay on Ub-AMC. Ub-AMC (25 pmol) (BostonBiochem) was coincubated with 10 pmol of protein or protein complex in 13 assay buffer (25 mM HEPES, pH 7.5, 0.25 mM EDTA, 0.05% Chaps, 10% dimethylsulphoxide (DMSO) and 10 mM DTT)38 at 25 uC. Fluorescence spectroscopy measurements were done 13/20 s at excitation wavelength 5 380 nm, and emission wavelength 5 436 nm. Generation of H2Aub1-containing mononucleosomes. As a template for assembly of mononucleosomes we used PCR to generate a 288-bp-long 59-biotinylated DNA fragment that included 87 bp of pUC19 DNA followed by a unique EcoRV restriction site and a 201-bp-long ‘601’ nucleosome positioning sequence39 (full fragment sequence available on request). Recombinant Xenopus or Drosophila octamers were prepared as described40 and mononucleosomes were assembled from the 59-biotinylated 288-bp DNA fragment by stepwise salt dialysis, as follows. Octamers and DNA were mixed in a ratio of 1.0 mg octamers:1.0 mg DNA (288 bp) in TE buffer containing 2 M NaCl (2 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, pH 8.0). The mixture was incubated at room temperature for 30 min, then stepwise dialysed against TE buffer containing 1.2 M NaCl, 1.0 M NaCl, 0.8 M NaCl and 0.6 M NaCl at 4 uC for 2 h each. The final dialysis step was carried out overnight against buffer C (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM ZnCl2, 1 mM DTT)24. Nucleosomal H2A was ubiquitinated using recombinant human E1, recombinant human E2 (UbcH5c) and recombinant partial human E3 complex (RING1B(1–159)–BMI1(1–109)) as described before24. (Recombinant E1, E2

and E3 enzymes were a gift from G. Buchwald and T. Sixma.) For purification of ubiquitinated nucleosomes, the reaction mixture was incubated with streptavidin-coupled Dynabeads (Dynabeads M-280 Streptavidin, Invitrogen) for 1 h at room temperature, followed by removal of the supernatant and washes of the bead-bound nucleosomes with buffer C (4 3 2-min washes). Nucleosomes were released from beads by cleavage with the restriction enzyme EcoRV for 1 h at 37 uC. The supernatant containing H2Aub1-mononucleosomes was then used for deubiquitination assays. Deubiquitination assays on H2Aub1- or H2Bub1-containing mononucleosomes. Flag–Calypso, Flag–Calypso(C131S), Flag–Calypso–HA–ASX(2–337) or Flag–Calypso(C131S)–HA–ASX(2–337) were mixed on ice with H2Aub1or H2Bub1-containing mononucleosomes in an enzyme-to-substrate ratio of 1:1 (40 pmol Calypso and 20 pmol H2Aub1 or H2Bub1-containing nucleosome) in 50 ml deubiquitination buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM ZnCl2, 1 mM DTT) and reactions were incubated at 25 uC, 12.5 ml aliquots (that is, containing 5 pmol nucleosomes) were removed at indicated time points and reactions were stopped by the addition of SDS sample loading buffer and incubation at 95 uC for 5 min. Deubiquitination assays on K48- and K63-linked ubiquitin polymers. Flag– Calypso, Flag–Calypso(C131S), Flag–Calypso–HA–ASX(2–337) or Flag– Calypso(C131S)–HA–ASX(2–337) were mixed on ice with K48- or K63-linked ubiquitin hexamers (Boston Biochem) in a molar enzyme to substrate ratio of roughly 1:1 in which the molarity of substrate is calculated according to the number of cleavable bonds within the ubiquitin polymers. The reaction was carried out for 40 min at 25 uC in 50 ml deubiquitination buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM ZnCl2, 1 mM DTT) and was stopped by the addition of SDS sample loading buffer and incubation at 95 uC for 5 min. Total embryo extract preparation. Embryos (16–18-h old) were dechorionated and immediately incubated in SDS-gel-loading buffer for 5 min at 95 uC. The suspension was sonicated, debris was pelleted and the supernatant was analysed by SDS–PAGE. Embryos that were homozygous for PcG mutations were in each case hand-picked by selecting GFP-negative embryos from strains that contained the mutant chromosome and an appropriate balancer chromosome carrying a ubi-nGFP transgene. Acid-extraction of histones from Drosophila embryos. Embryos (16–18-h old) were dechorionated, homogenized and the homogenate was filtered through Miracloth tissue (Calbiochem). Nuclei were pelleted, resuspended in 10 volumes of 0.4 M HCl containing 10% glycerol, and incubated for 30 min on ice. Insoluble material was pelleted, and histones were precipitated from the supernatant with acetone and resuspended in SDS gel loading buffer. For isolation of histones from Asx22P4/Asx22P4 embryos, embryos were collected from a FRT40 FRT42D y1 Asx22P/CyO ubi-nGFP strain by selecting for GFP-negative embryos using an embryo sorter (COPAS, Union Biometrica).
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). 30. White, R. A. H. & Wilcox, M. Protein products of the Bithorax Complex in Drosophila. Cell 39, 163–171 (1984). 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). 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). 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). 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). 35. Muller, J. et al. Histone methyltransferase activity of a Drosophila Polycomb group ¨ repressor complex. Cell 111, 197–208 (2002). 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). 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). 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). ? ¨ 39. Thastrom, A. et al. Sequence motifs and free energies of selected natural and nonnatural nucleosome positioning DNA sequences. J. Mol. Biol. 288, 213–229 (1999). 40. Luger, K., Rechsteiner, T. J. & Richmond, T. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).

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