Binding of different marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2)

Chao Xua,1, Chuanbing Biana,1, Wei Yangb,1, Marek Galkac, Hui Ouyanga, Chen Chend, Wei Qiua, Huadong Liuc, Amanda E. Jonesb, Farrell MacKenziea, Patricia Pana,e, Shawn Shun-Cheng Lic,2, Hengbin Wangb,2, and Jinrong Mina,f,2

aStructural Genomics Consortium, University of Toronto, 101 College Street, Toronto, ON, Canada M5G 1L7; bDepartment of and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Building Room 430, 720 South 20th Street South, Birmingham, AL 35294; cDepartment of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1; dSamuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; eDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9; and fDepartment of Physiology, University of Toronto, Toronto, ON, Canada M5S 1A8

Edited by Tony Pawson, Samuel Lunenfeld Research Institute, Toronto, Canada, and approved September 14, 2010 (received for review June 23, 2010)

The polycomb repressive complex 2 (PRC2) is the major methyltrans- tional silencing through an unknown mechanism (11). In addition ferase for H3K27 , a modification critical for maintain- to silencing Hox genes, the polycomb group complexes are also ing repressed gene expression programs throughout development. involved in X-inactivation, germ-line development, stem cell It has been previously shown that PRC2 maintains histone methyl- pluripotency and differentiation, and cancer metastasis (2). ation patterns during DNA replication in part through its ability to PRC2 complex contains four core components: EZH2, EED, bind to . However, the mechanism by which PRC2 recog- SUZ12, and RbAp46/48. The catalytic activity of PRC2 is con- nizes H3K27me3 is unclear. Here we show that the WD40 domain of ferred by the suppressor of variegation [Su(var)3-9], enhancer EED, a PRC2 component, is a methyllysine histone-binding domain. of zeste [E(z)], and trithorax (SET) domain in EZH2, but PRC2 The crystal structures of apo-EED and EED in complex respectively exhibits robust methylation activity only as a complex because in with five different trimethyllysine histone peptides reveal that EED vitro enzymatic assays indicate that EZH2 has virtually no histone binds these peptides via the top face of its β-propeller architecture. methylation activity on its own (6). Biochemical and genetic The ammonium group of the trimethyllysine is accommodated by studies suggest that both EED and SUZ12 are required for the an aromatic cage formed by three aromatic residues, while its ali- integrity of PRC2 and PRC2-mediated H3K27 methylation be- phatic chain is flanked by a fourth aromatic residue. Our structural cause mutations in either protein lead to EZH2 to destabilization data provide an explanation for the preferential recognition of the and deficient methylation of H3K27 (12–14). Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and EED contains seven WD40 repeats at its C terminus (residues H1K26 marks by EED over lower methylation states and other his- 81–441) preceded by a small N-terminal domain (residues 1–80). tone methyllysine marks. More importantly, we found that binding Studies of the Drosophila EED orthologue extra sex combs (ESC) of different histone marks by EED differentially regulates the activ- revealed that the N-terminal region interacts directly with the ity and specificity of PRC2. Whereas the H3K27me3 mark stimulates core domain of histone H3 and that this interaction is essential the histone methyltransferase activity of PRC2, the H1K26me3 for E(Z)-dependent trimethylation of H3K27 (15). Furthermore, mark inhibits PRC2 methyltransferase activity on the . although an N-terminally truncated ESC can form a complex Moreover, H1K26me3 binding switches the specificity of PRC2 with E(Z) when expressed in stable S2 cell lines, this complex from methylating H3K27 to EED. In addition to determining the cannot carry out trimethylation of histone H3 (15). A similar molecular basis of EED-methyllysine recognition, our work provides mechanism may regulate PRC2 function in vertebrates because the biochemical characterization of how the activity of a histone the ESC/EED–histone H3 interaction is evolutionarily conserved methyltransferase is oppositely regulated by two histone marks. (15). The WD40 repeat is a structural motif of approximately ∣ ∣ methyllysine-binding domain WD40 repeat-containing protein 40 amino acids, which forms a four-stranded antiparallel β-sheet X-ray crystallography and often contains a Gly-His dipeptide at the end of the fourth strand and a Trp-Asp dipeptide at the end of the third strand (16). he polycomb (PcG) and trithorax groups of proteins act in Most WD40 repeat proteins contain seven or eight WD40 repeats Tconcert to maintain the transcriptional state of developmental and adopt a β-propeller architecture. WD40 proteins are involved Hox control genes such as the genes. These patterns are estab- in diverse functions such as transcription regulation, signal trans- lished during early embryonic development and are heritable through many generations of cell division (1–3). Polycomb group proteins are transcriptional repressors that maintain target genes Author contributions: C.X., C.B., W.Y., M.G., S.S.-C.L., H.W., and J.M. designed research; in an inactive state, whereas trithorax group proteins are tran- C.X., C.B., W.Y., M.G., H.O., W.Q., H.L., A.E.J., F.M., and P.P. performed research; C.X., C.B., W.Y., M.G., H.O., C.C., W.Q., H.L., S.S.-C.L., H.W., and J.M. analyzed data; and J.M. scriptional activators that maintain their target genes in an active wrote the paper. state. This system of cellular memory is conserved in nearly all The authors declare no conflict of interest. multicellular eukaryotes (4). This article is a PNAS Direct Submission. PcG proteins often function as complexes, and the two best- characterized complexes are the polycomb repressive complex Freely available online through the PNAS open access option. 1 (PRC1) and 2 (PRC2). PRC2 exhibits histone methyltransferase Data deposition: The crystallography, atomic coordinates, and structure factors have – been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3JZN, 3K26, activity on H3K27 (2, 5 7) and weakly on H1K26 (8). Studies 3K27, 3JZG, 3JZH, and 3JPX). Drosophila in have shown that PRC1 contains a chromodomain 1C.X., C.B., and W.Y. contributed equally to this work. protein, polycomb, which preferentially recognizes the histone 2To whom correspondence may be addressed. E-mail: [email protected], [email protected], or jr. H3K27me3 mark (9, 10). One hypothesis is that H3K27 methyl- [email protected]. ation by PRC2 creates a binding site recognized by the chromo- This article contains supporting information online at www.pnas.org/lookup/suppl/ domain of polycomb of PRC1, which in turn maintains transcrip- doi:10.1073/pnas.1008937107/-/DCSupplemental.

19266–19271 ∣ PNAS ∣ November 9, 2010 ∣ vol. 107 ∣ no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1008937107 Downloaded by guest on September 30, 2021 duction, vesicular trafficking, cytoskeletal assembly, cell cycle selective binding of EED to trimethylated lysine by de- control, and apoptosis (16). A common function of these WD40 termining the crystal structure of apo-EED and its complexes repeat proteins is to serve as a scaffold for protein–protein or with five trimethylated histone peptides, respectively. protein–DNA interactions and to coordinate downstream events. Since the first WD40 repeat structure Gβ was solved (17, 18), tens Results and Discussion of WD40 structures have been reported, which, together, show The WD40 Repeat Domain of EED Selectively Recognizes Trimethylated that the WD40 β-propeller is capable of recognizing a diverse Histone Peptides. PRC2 methylates histone lysine residues, mainly group of ligands, including proteins (17, 18), phosphopeptides on H3K27 (2, 5–7), and also binds to the H3K27me3 mark (32). (19–21), and nucleic acids (22), mainly through the small top sur- Efficient binding requires a ternary complex of EZH2, EED, face. For example, WDR5 uses the top surface of its β-propeller and SUZ12 but is independent of the catalytic SET domain of to recognize histone H3K4 peptide (23–26) or a mixed lineage EZH2 (32). However, which component in PRC2 is responsible leukemia (MLL) fragment (27–29). WDR5 recognizes a peptide for binding to H3K27me3 is unclear. Aided by the crystal struc- through an arginine residue that inserts into the central pore ture of EED in complex with a short N-terminal fragment of of the β-propeller and anchors the peptides into a shallow groove EZH2 (30), we identified an aromatic cage on the top face of the at its top face. EED β-propeller structure (Fig. 1A), raising the possibility that WD40 proteins can also bind their ligands through the larger EED may mediate direct binding of PRC2 to H3K27me3 through bottom surface. For example, the structure of the WD40 repeat this aromatic cage. To test this hypothesis, we measured the domain of EED in complex with an N-terminal fragment binding of EED to a panel of peptides that represented the of EZH2 revealed that the peptide bound to the WD40 repeat histone marks H3K4, H3K9, H3K27, H3K36, H3K79, H4K20, domain of EED on the larger bottom surface (Fig. 1A) (30). and H1K26 in different methylation states (Fig. 1 B and C). Mutations in the WD40 region of the ESC perturbed its binding We found that EED preferentially bound trimethylated histone to E(Z) in vitro and abolished its silencing function in vivo (31). peptides, with higher affinities to the , H1K26me3, Recently, it was shown that the EZH2–EED–SUZ12 trimeric and H3K27me3 peptides than the other trimethylated histone PRC2 complex binds to and colocalizes with the H3K27me3 mark peptides. Our EED-histone peptide binding data agree with at sites of active DNA replication and in the G1 phase of a cell the result of Margueron et al. that was published while this manu- cycle (32). However, it is unclear which component is responsible script was under review (38). for recognizing the H3K27me3 mark. An examination of the top surface of the EED structure revealed an aromatic cage consist- H3K27me3 Stimulates Whereas H1K26me3 Inhibits the Methyltransfer- ing of residues Phe97, Tyr148, Trp364, and Tyr365 (Fig. 1A). An ase Activity of PRC2 on Nucleosomal Histone H3K27. The binding of aromatic cage is a common feature of methyllysine-binding EED to trimethylated lysine marks on histones suggests a me- motifs (33) such as the chromodomain (9, 10, 34, 35) and the chanism for PRC2 to propagate and spread the H3K27me3 mark malignant brain tumor repeats (36, 37). This finding led us to to daughter strands during cell division. To investigate how propose that EED may possess a methyllysine-binding activity methylated histones may regulate the function of PRC2, we mea- that is important for PRC2 function. Here, we provide evidence sured its histone methyltransferase activity on nucleosome in the in support of this notion. Specifically, we show that the WD40 presence of different histone peptides. Our results showed that

domain of EED is a histone methyllysine-binding motif that pre- the H3K27me3 peptide stimulated the enzymatic activity of BIOCHEMISTRY ferentially recognizes Ala-Arg-Lys-Ser (ARKS) motif-containing PRC2 by approximately three folds (Fig. 2A). These data are con- trimethylated H3K27, H3K9, and H1K26 peptides and that the sistent with a recent report showing that H3K27me3 peptide sti- nucleosome methylation activity of PRC2 is enhanced by binding mulates the PRC2 activity (38). With the exception of H1K26 to the H3K27me3 histone peptide via EED but inhibited by peptide, we did not observe any significant effects of other H1K26me3. We further investigated the molecular basis for the peptides on the activity of PRC2. Addition of the H1K26me3 A B

Fig. 1. Preferential binding of EED to trimethylated C lysine histone peptides. (A) The top surface of EED har- bors an aromatic cage consisting of Phe97, Tyr148, Trp364, and Tyr365, distinct from the EZH2-binding site shown in the crystal structure of EED in complex with an EZH2 fragment (30). The aromatic cage residues on the top surface of EED are displayed in a stick model. EED and the EZH2 peptide are colored in green and blue, respectively. (B) Binding of different trimethy- lated histone peptides to EED measured by fluores- cence polarization. (C) Tabulated binding affinities

(Kd) of different histone peptides for EED, measured by fluorescence polarization binding assays. The target lysines are colored in red. NB, no detectable binding; ND, not determined.

Xu et al. PNAS ∣ November 9, 2010 ∣ vol. 107 ∣ no. 45 ∣ 19267 Downloaded by guest on September 30, 2021 A EED WT to EED, which is confirmed by our isothermal titration calorime- try (ITC) and peptide competition assays (Fig. S1 B and C). Con- sistent with its loss of binding, the H1K26me3A24E peptide was unable to inhibit PRC2 activity (Fig. 2C). Taken together, these H3 Radiograph data clearly demonstrate that H1K26me3 inhibition of PRC2

Coomassie activity on nucleosome is conferred by its direct binding to EED. It is therefore likely that, in vivo, the activity of PRC2 is regulated by both the H3K27me3 and the H1K26me3 marks that play opposing roles. This finding suggests a mechanism for dy- namic control of in that the recruitment of PRC2 by H3K27me3 enhances its activity and allows for pro- pagation of the H3K27me3 mark to sister chromatins during a cell division, whereas recruitment of the PRC2 complex to the H1K26me3 mark shuts down the methylation activity toward core histones. Whereas the addition of the H1K26me3 peptide into the in B EED WT + H1K26 vitro methylation reaction inhibited nucleosomal H3 methyla- 293T No peptide me0 me1 me2 me3 tion, it promoted methylation of another protein of approxi- EED Radiograph mately 50 kDa (Fig. S2A). This protein was subsequently identified as EED by liquid chromatography MS/MS. To further H3 Radiograph characterize the methylation of EED, we used multiple reaction 1 2 3 4 5 6 monitoring mass spectrometry to identify the specific sites that are methylated. We checked all 32 lysines in EED and obtained EED WT C 293T no peptide H1K26me3 H1K26me3A24E high quality data on 23 lysines, of which K66, K197, K268, and K284 were methylated (Fig. S3). Moreover, all three methylation H3 Radiograph states were detected for these four sites, and the most abundant methylation states are shown in Fig. S3. The identities of these methyl peptides were confirmed by both multiple reaction Coomassie monitoring and the corresponding MS/MS spectra collected in information-dependent acquisition mode. Of these methylation Fig. 2. The H3K27me3 peptide specifically stimulates, but the H1K26me3 sites identified in EED, the K66 site is located in a sequence motif peptide inhibits, the activity of PRC2. (A) Effects of different histone peptides of Gly-Arg-Lys-Ser that resembles the H3K27 sequence, ARKS. on the activity of PRC2 on histone H3 methylation. Top is an autoradiograph However, mutation of K66 to an alanine did not abolish the of the Middle (Coomassie blue staining). Quantification of two independent methylation of EED (Fig. S2B). Given the multiple sites that experiments is shown in Bottom. Error bars are standard deviations of the could be methylated in EED, this result is not unexpected. There- two experiments. (B) Effects of different methylation states of the H1K26 peptide on the methylation activity of PRC2. (C) The H1K26me3 peptide fore, our data indicate that, in contrast to H3K27me3 and other containing an A24E mutation lost its ability to inhibit PRC2 HMT activity histone peptides, the H1K26me3 peptide changes the substrate on nucleosome. preference of PRC2.

peptide into the reaction inhibited nucleosomal histone H3K27 Overall Structure of Apo-EED and EED-H3K27me3. To investigate the methylation (Fig. 2A). Although histone H1 inhibition of PRC2 molecular basis for the selective binding of EED to trimethylated methyltransferase activity on nucleosome was previously shown histone peptides, we determined the crystal structure of apo-EED (8), the strong inhibitory effect of a methyllysine peptide on and its complex with the H3K27me3 peptide (Fig. 3). As shown previously (30), the WD40 repeat domain of EED folds into a PRC2 activity has not been reported to date. To investigate β A whether the methylation status of H1K26 affects the inhibitory seven-blade -propeller structure (Fig. 3 ). The seven blades are activity, we carried out the in vitro methylation assay in the pre- symmetrically arranged around a pseudosymmetry axis, and a narrow channel runs through the middle of the β-propeller struc- sence of the H1K26 peptide with varying methylation states. In- ture. The crystal structures of apo-EED and the EED– terestingly, H1K26 peptides of lower methylation states exhibited H3K27me3 peptide complex were almost identical, with an rmsd a weaker inhibitory effect compared to the H1K26me3 peptide α of ∼0.2 Å for all aligned C atoms. As predicted from the struc- (Fig. 2B). PRC2 has been shown to methylate H1K26 weakly tural analysis, EED utilizes the smaller top surface of the β-pro- (8). Presumably, H1K26 will exhibit full inhibition effect only peller to recognize H3K27me3. Since the first WD40 repeat when it is trimethylated because lower methylated H1K26 pep- structure Gβ was solved (17, 18), many WD40–ligand complex tides have much weaker binding affinity for EED (38). structures have been determined, most of which use the smaller To further investigate whether the inhibitory effect of top surface to accommodate a ligand with a small interface area H1K26me3 is mediated by its direct binding to EED, we carried (Figs. S4 and S5). out a peptide competition assay. Our results show that the The histone-binding mode of EED is reminiscent of WDR5, H1K26me3 peptide binds to EED directly and that the binding which also binds histone and other arginine-containing peptides can be competed off by the H3K27me3 peptide, suggesting that through its top surface (23–29). WDR5 binds histone and MLL H1K26me3 and H3K27me3 bind to the same site on EED peptides mainly via an arginine residue of the peptide that acts (Fig. 1C and Fig. S1). We next investigated whether impairment like a pin and inserts into the central pore of the β-propeller to of H1K26me3 binding to EED affects the inhibitory effect of form a large network of hydrogen bonds and cation-π interactions PRC2 complex on nucleosome. On the basis of our EED–histone with WDR5 (23) (Fig. 3E). In the EED–H3K27me3 peptide com- peptide complex structures, which will be discussed later, the re- plex structure, the histone peptide is anchored to EED through sidue at the P-2 position relative to the methyllysine at the histone the trimethyllysine, which is accommodated by a binding pocket peptides is important for complex formation. Thus, an Ala to Glu formed by four aromatic residues (Fig. 3 B and C). The side chain mutation at the P-2 position in the conserved ARKS motif of of the trimethylated lysine is inserted into an aromatic cage H3K27me3, H3K9me3, and H1K26me3 would impair its binding formed by F97, Y148, and Y365. The aromatic rings of these

19268 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008937107 Xu et al. Downloaded by guest on September 30, 2021 Fig. 3. Structure of EED in complex with a trimethylated H3K27 peptide. (A) Top view of the EED WD40 domain in complex with a trimethylated H3K27 peptide. EED is shown in a cartoon representation and colored green, and the H3K27me3 peptide is shown in a stick model. (B) The com- plex structure of EED and H3K27me3. EED is shown in a sur- face representation with positive electrostatic potential

denoted in blue and negative potential by red. The BIOCHEMISTRY H3K27me3 peptide is depicted in a stick model. (C) Detailed view of intermolecular interactions between EED and H3K27me3. EED residues in contact with the H3K27me3 peptide are shown in sticks. Hydrogen bonds are marked with black dashed lines. (D) Superposition of the crystal structures of EED in complex with different trimethylated histone peptides. Methyllysines are shown in a stick model. (E) Top view of WDR5 in complex with an H3K4me2 peptide (23). The H3K4me2 peptide and the H3R2-binding residues in WDR5 are shown in stick models. Hydrogen bonds are shown in red dashed lines.

three residues make close contact and are oriented roughly per- constituted PRC2 was severely diminished when these methylly- pendicular to each other, forming the three sides of the methyl- sine-binding residues in EED were mutated (Fig. S7B). Besides lysine-binding pocket and establishing van der Waals and cation-π providing biochemical evidence to support the structure, these interactions with the trimethylated ammonium group. The ex- data suggest that the function of PRC2 is dependent on EED, tended aliphatic chain of the methyllysine lies against the planar in particular its WD40 repeats. Our data agree with that obtained ring of W364, further enhancing the hydrophobic interaction for the Drosophila EED orthologue, ESC (31), in which certain between the protein and the methylated lysine. This interaction point mutations in the WD40 repeats of ESC perturbs its binding mode is a unique methyllysine-binding feature for EED. The four to E(Z) (31). aromatic residues identified in the EED–H3K27me3 complex are In addition to the interactions of methyllysine with the aro- highly conserved in EED orthologues (Fig. S6). The trimethylly- matic cage, other interactions may also help stabilize the complex. sine-binding mode of EED is similar to that of other classical tri- These include a backbone hydrogen bond between Trp364 in methylated histone-binding proteins, such as HP1 and polycomb EED and H3R26 in the peptide, a hydrogen bond between the (9, 10, 34, 35). side chain of EED Trp364 and the backbone of H3A25, and The importance of the methyllysine-binding residues in EED the side chain of EED Arg414 and the backbone of methyllysine was verified by subsequent mutagenesis experiments. Fluores- (Fig. 3C). Interestingly, Arg414 is absolutely conserved in all cence polarization measurements showed that mutating F97, orthologues of EED and forms a salt bridge with another Y148, W364, or Y365 to alanine, or W364 to leucine, completely conserved residue Asp430 (Fig. 3C). The Arg414–Asp430 pair abolished binding of the resulting EED mutant to the H3K27me3 may therefore play an important role in binding methyllysine. Re- peptide (Fig. S7A). We next reconstituted the PRC2 complex sidues C-terminal to the methyllysine in the peptide form virtually with the EED mutant W364A or Y365A and examined its histone no interactions with the protein, which explains why EED binds K μ lysine methyltransferase (HKMT) activity. The activity of the re- its ligands more weakly ( d: 108 M with H3K27me3) than do the

Xu et al. PNAS ∣ November 9, 2010 ∣ vol. 107 ∣ no. 45 ∣ 19269 Downloaded by guest on September 30, 2021 K μ HP1 chromodomain ( d:4 M with H3K9me3) and the JMJD2A H3K9 is not a substrate of PRC2, and, even though H3K9me3 K μ tudor domains ( d: 0.4 M with ) to their respective can localize with PRC2 in cells, it does not form a feedback loop peptide ligands (10, 39). that helps spread the H3K9me3 mark. The structures of the holoenzyme in complex with these peptides would help clarify Preferential Recognition of the ARKme3S Motif by EED. Besides the the distinct effects of different histone peptides on PRC2. complex structure of EED with the H3K27me3 peptide, we also The ability of EED to bind to the product of PRC2 determined the crystal structures of EED complexed with the (H3K27me3) provides an effective means for the propagation H3K4me3, H3K9me3, H3K79me3, and H4K20me3 peptides, and/or spreading of the H3K27me3 repressive mark. In quiescent respectively. In all five complex structures, the backbone orienta- cells, recruitment of PRC2 via EED to an H3K27me3 mark may tions of the peptides are almost identical, suggesting that the help propagate the mark to neighboring . In postmo- interaction of methyllysine with the aromatic cage drives the com- totic cells, targeting of PRC2 to the H3K27me3 mark associated plex formation (Fig. 3D). However, the five histone peptides bind with the parent DNA strand may help spread this mark to the to EED with varying affinities, and EED preferentially binds nascent sister strand. This mode of dual function for a methyl- H3K9me3, H1K26me3, and H3K27me3, which share a conserved transferase or coexistence of catalytic and effector functions in ARKS sequence motif. the same protein complex is not without precedent. For instance, We compared the five EED complex structures to understand Clr4 is a histone H3K9 methyltransferase (40, 41) that also con- the structural basis for the preferential binding of the H3K27me3, tains a chromodomain at its N terminus, which binds specifically H1K26me3, and H3K9me3 marks by EED. This comparative to the H3K9me3 mark it creates. The ability of Clr4 to generate analysis identified two residues preceding the methyllysine in a and bind H3K9me3 is essential for the spreading of heterochro- peptide (referred to as the P-1 and P-2 positions) as key determi- matin silencing (42). Similar modes of regulation may underlie nants of such specificity. In the EED-H3K27me3 complex, the the function of other histone-modifying enzymes. P-2 residue alanine (H3A25) is accommodated in a relatively In summary, our work identifies EED as a central player hydrophobic and shallow pocket consisting of Y308, C324, and in PRC2 function. The unique ability of EED to bind multiple W364 (Fig. 3C). H3K9me3 also contains an alanine residue histone marks, which leads to distinct outcomes, may play a (H3A7) at the same position, so this binding mode is conserved pivotal role in controlling the activity of PRC2 and its function in the EED–H3K9me3 complex (Fig. S8A). In contrast, a histi- in propagating and spreading repressive histone marks in the nu- dine residue is found at the corresponding position in the cleus. By mediating a series of interactions—binding the histone H4K20me3 peptide. The histidine side chain would be too bulky H3 core domain via the N-terminal fragment, the N-terminal to be inserted into the P-2 pocket without causing significant ster- motif of EZH2 via the bottom surface, and the H3K27me3/ ic hindrance. Indeed, in the corresponding crystal structure, this H1K26me3 mark via the top surface of the EED WD40 do- histidine residue (H3H18) points to the solvent and makes a hy- main—EED may function as an adaptor and/or conformational drogen bond with the backbone carbonyl oxygen of D362 in EED switch that controls the integrity, targeting, activity, and specifi- (Fig. S8B). This alternative mode of P-2 recognition could explain city of PRC2. It should be noted, however, that the affinity of why the H4K20me3 peptide has comparable binding affinity to EED for H3K27me3 is significantly weaker than that of HP1 EED. In the EED–H3K79me3 complex structure, the corre- and Pc chromodomains for the H3K9me3 or H3K27me3 peptide. sponding residue Asp (H3D77) is disordered, suggesting that it The recruitment of the PRC2 complex to the correct chromatin did not contribute significantly to binding (Fig. S8C). In the case location must therefore involve multiple sites of EED and, likely, of the H3K4me3 peptide, the long side chain of the H3R2 could multiple components of PRC2. Indeed, it has been shown that all not be accommodated by the shallow binding pocket and is also components of PRC2 are required for optimal binding (32). In disordered (Fig. S8D). addition, it has been shown that RbAp46/46 is capable of binding In all the EED–peptide complexes, the peptide residue imme- the first helix of histone H4 (43–45). diately preceding the methyllysine (P-1 position) protrudes out of the binding interface and points to the solvent. Therefore, a Methods Protein Expression and Purification. Two fragments of human EED (amino hydrophobic residue at this position would not be favored ener- – – getically, which is the case for (V35 at P-1) and acids 40 441 and 76 441) covering the WD40 repeats were subcloned into a modified pET28GST-LIC vector. The recombinant proteins were overex- H3K79me3 (F78 at P-1). Together, these structures explain pressed at 16 °C in Escherichia coli BL21 (DE3) Codon plus RIL (Stratagene) why EED preferentially binds H3K9me3, H3K27me3, and as N-terminal GST-tagged fusion proteins and were purified by affinity chro- H1K26me3 marks. matography on glutathione-sepharose (GE Healthcare). After cleavage using thrombin (Sigma Aldrich) on the column at room temperature for 4–5h,the Implication of EED-H3K27me3 Binding in the Transmission of the flow-through was collected and purified further by size exclusion chromato- H3K27me3 Epigenetic Mark. On the basis of our binding assays graphy (Superdex 200; GE Healthcare). The proteins were concentrated to and structural studies, we conclude that (i) EED binds to multiple 15 mg∕mL in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and trimethylated histone peptides, but preferably to repressive 1 mM DTT. Mutated EED cDNAs were made by using the QuikChange II marks, (ii) the recognition of the methyllysine residue by the aro- XL Site-Directed Mutagenesis Kit (Stratagene); mutations were confirmed – by sequencing complete cDNAs. Mutated proteins were expressed in bacter- matic cage plays a key role in the formation of an EED methyl- ial cells and purified as above. lysine peptide complex, (iii) residues at the P-1 and P-2 positions relative to the methyllysine in a peptide are responsible for the Histone Methyltransferase Assay. Lentiviral vectors encoding wild-type and differential binding affinities, and (iv) the H3K27me3, H3K9me3, mutant FLAG-EED were transfected into a 293T cell by using Effectene and H1K26me3 peptides all bind EED with comparable affinities reagents (Qiagen). Then 48 h after transfect ion, cells were harvested and but exhibit different outcomes: The H3K27me3 peptide stimu- lysed in nondenaturing lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, lates PRC2 HKMT activity, H1K26me3 inhibits PRC2 HKMT 1% NP-40, 1 mM EDTA) at 4 °C for 1 h. After brief centrifugation activity, and H3K9me3 has no effect. (16;100 × g for 10 min), cell lysates were subjected to immunoprecipitate with anti-FLAG M2 agarose beads (Sigma) at 4 °C for 3 h. PRC2 complexes were It has been proposed that H3K27me3 binding to PRC2 intro- 0 4 ∕ 0 4 ∕ duces a conformational change in PRC2 and activates the PRC2 eluted from the beads with . mg mL FLAG peptide ( . mg mL) and dialysed against BC50 before used for the histone methyltransferase assay. HKMT activity (38). It is likely that H1K26me3 changes the The histone methyltransferase assay was performed as described previously conformation of PRC2 in a different way that makes EED a pre- (46). Briefly, affinity purified PRC2 samples were incubated with 10 μgof ferred substrate for PRC2, whereas H3K9me3 binding to PRC2 oligonucleosome or mononuclesomes prepared from a HeLa-S3 cell (47), does not change its conformation significantly. Furthermore, 1 μL 3H-S-adenosylmethionine (NEN Life Science Products), and histone H3

19270 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008937107 Xu et al. Downloaded by guest on September 30, 2021 peptides (40 mM) in histone methyltransferase buffer (20 mM Tris-HCl, pH MOLREP (49). The crystal structure of EED and EZH2 peptide was used as 8.0, 4 mM EDTA, 1 mM PMSF, 0.5 mM DTT) at 30 °C for 1 h. The reaction pro- the search model (Protein Data Bank ID code 2QXV). ARP/wARP was used ducts were resolved on 15% SDS-PAGE. After staining and destaining, gels for automatic model building (50). Graphics program COOT (51) was used 3 3 were treated with H enhancer (En hance; Perkin Elmer), dried, and exposed for model building and visualization. Crystal diffraction data and refinement – to X-film for 1 3d. statistics for these structures are displayed in Table S1.

Crystallization and Structure Determination. Purified protein EED (40–441) was Note. While this manuscript was under consideration, R. Margueron and mixed with histone trimethylated peptides by directly adding a twofold colleagues published similar work (38). molar excess of peptide to the protein solution and crystallized by using the sitting drop vapor diffusion method at 18 °C. The complex of GST-tag cleaved EED and H4K20me3 peptide was crystallized in a buffer containing ACKNOWLEDGMENTS. We thank Alexey Bochkarev and Guillermo Senisterra 3.5 M sodium formate, 10 mM Tris(2-carboxyethyl)phosphine (TCEP) chloride. for advice and technical assistance and Yanming Wang, Aled Edwards, For the other complexes, EED (76–441) was mixed with the histone peptides and Cheryl Arrowsmith for critically discussing and reading the manuscript. This work was supported by the Structural Genomics Consortium, a regis- by directly adding a twofold molar excess of peptides to the protein solution. tered charity (1097737) that receives funds from the Canadian Institutes All those complexes were crystallized by using the sitting drop vapor diffu- for Health Research, the Canadian Foundation for Innovation, Genome sion method at 18 °C and in a buffer containing 0.1 M 1,3-bis[tris(hydroxy- Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolins- methyl)methylamino]propane, 3.5 M sodium formate, 10 mM TCEP, and ka Institutet, The Knut and Alice Wallenberg Foundation, the Ontario Inno- – 15 20% glycerol. Prior to flash-freezing the crystals in liquid nitrogen, the vation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., crystals were soaked in a cryoprotectant consisting of 100% reservoir solution Inc., the Novartis Research Foundation, the Swedish Agency for Innovation and 20% glycerol. Systems, the Swedish Foundation for Strategic Research, and the Wellcome X-ray diffraction data were collected at 100 K by using the Rigaku FRE Trust. H.W. is a Sidney Kimmel Scholar and is supported by National Institutes High Brilliance X-Ray Generator with R-AXIS IV detector and Canadian Light of Health Grant GM081489. S.S.-C.L. holds a Canada Research Chair in Func- Source. Data were processed by using the HKL software package (48). All tional Genomics and Cellular Proteomics. Open access publication costs were structures were solved by molecular replacement by using the program defrayed by the Ontario Genomics Institute Genomics Publication Fund.

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