© 2016 Nature America, Inc. All rights reserved. M I C U 1 ulationevents or pathology is not understood. Further highlighting However, the role of this diversity in the context of specific gene reg has become clear that a large number of diverse PRC1 complexes exist. repression of underlying the genes and methylationDNAcompaction, chromatin with associated turn BMI and RING1B RING1A, PRC1, of subunits ligase ubiquitin E3 the by (H2AK119ub) H2A onLys119 of ination monoubiquit the allowing H3K27me3, complexto the targetingby repression transcriptional PRC1-mediated facilitate to thought are mannerexclusivemutually a in PRC1 intoincorporate CBX8) complex repressor multiprotein the and CBX7 CBX6, chromodomainsCBX4, PRC1 The(CBX2, PRC1. of component reader Kme chromodomain-containing the by recognized then is and PRC2 by In the ‘canonical’ Polycomb-signaling EED model, reader H3K27me3 H3K27me3 is the deposited and SUZ12 are PRC2 of components EZH2 of Polycomb repressive complex 2 (PRC2). The other two core repressive signal that is installed by the methyltransferases EZH1 and lysine modified the flanking acids amino the with interactions through achieved is ity marily by cage andgeometry composition, sequence while specific methylation states (mono-, di- and trimethylation) is conferred pri Waalsder vanand static interactions chainand engages methylated the amine through cation- side lysine hydrophobic the accommodates that cage through aromatic an PTM this recognize that readers, Kme termed proteins, to confer agrowth advantage, whereas UNC4219, amethylated negative control compound, hasnegligibleeffects. overexpressionCBX7 of ability known the with proliferation,consistentcell PC3 Finally,inhibits cells. UNC3866 cancer prostate PC3 in dependent isoform is PRC1 incorporationinto UNC3866, EED that and of PRC1 intact engagesderivative UNC3866 demonstratethat to biotinylated used was a UNC4195, tail. H3 methylated the of those mimic closely chromodomains CBX the with mainswhilebeing highly selective over >250other protein targets. X-ray crystallography revealed thatUNC3866’s interactions CBX7 most potently, with a and CBX4 of chromodomains the binds UNC3866 chromodomains. their Polycombvia H3K27me3 targeting of sites byto (PRC1) 1 expression complexrepressive gene regulate proteins CBX Polycomb chromodomains. of families CDY and CBX Polycomb the of function reading (Kme) methyllysine the of antagonist potent a UNC3866, of characterization and design the report We Cheryl H S Fengling Li S Jacob I chromodomains of A nature CH *e-mail: U C nstitute ofMentalHealth anada. SA. niversity of tephanie HCholensky amantha G enter for Notwithstanding this simplistic model of sequential PRC action, it Trimethyllysine 27 on histone 3 (H3K27me3) is a transcriptionally cellular chemicalprobe targeting the 5 D epartment of epartment 3 i D n oiiain PM poie a okn st fr effector for site docking a provides (PTM) modification regulation chromatin to utes contrib critically histoneson residues lysine ofethylation E g epartment of epartment I S MIC ntegrative e r N tuckey m A orth orth A a 2 rrowsmith L BIOLOGY n , Xi- @ P C e arolina at attenden 1 m P , C 3 harmacology, 1 . a P hemical , Bradley M E i l ing Huang . pigenetics andMolecular P u

| Adv sychoactive n c . e C d a K B 2 hapel Hill, u nce online public , Lindsey IJames iology and d or of ~100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodo 1 1 , Wolfram , MasoudVedadi U s 7 niversity of v , 8 f . D r 4 y 1 rug Screening . Selectivity between different between Selectivity . D e , 5 C @ , Bryan L D 1 hapel Hill, ickson , e 2 rug Ti post-translational This . m a i D N l . a u C iscovery, T orth orth tion | n 6 arcinogenesis, empel . H2AK119ub is in in is H2AK119ub . c 1 N . , e P C orth orth d N rogram, R arolina at u www.nature.com/naturechemicalbiology P 1 ancy Cheng , oth * D & C 2 olycomb repressive complex 1 ivision of , 2 arolina, π , P 4 , electro , S U S eter JBrown , 5 niversity of tephen VFrye u Qin C U , Brandi MBaughman 5 hapel HillMedical School, niversity of and and U C SA. hemical 4 ------.

pu 2 2 , KatherineGHuber Structural Genomics 1 , Yanli Liu N bli esized esized that antagonism of the H3K27me3 reading function of PRC1, intrinsicpontineglioma diffuse ofmodels lymphoblastic leukemia acute in growth-suppressivepropertieshave also UTX and JMJD3 currently in clinical trials mutationsareactivatingandlymphomas with in levels H3K27me3 aberrant targeting at effective are EZH2 methyltransferase PRC2 the of Inhibitors interest. pharmaceutical much garnered recently for oncogenesis, example H3K27demethylase the JMJD3(ref. in functions contradictory have to shown been tion to CBX7, other developmental Polycomb factors have similarly pressorlung,in pancreatic, colon and thyroid tissues cancer types and CBX8 have also been shown to enhance proliferation in various throughknockdown of (refs.CBX7 tate, gastric, leukemia and lymphoma cell lines that can be inhibited pros proliferativein advantagea confers CBX7 ofOverexpression types cancer numerous to contributing as implicated been has reader Kme antagonism may require ligands that target multiple PRC1 CBX proteins. through regulation chromatin of modulation Therefore,redundant. functionally partially be may proteins these genomeoverlapping the toof regions PRC1 target to proteins CBX of ability However, the PRC1. of targeting genomic the affects potentially that diversity of point another CBX componentis variable The mark. same the for readers Kme potential two complexes PRC1 CBX-containing of component a the EED, that reported H3K27me3 recentlyreader once believed to participate only in PRC2, was is also it PRC1, of complexity the orth orth T s B ouain f 32m3 inln va ml mlcls has molecules small via signaling H3K27me3 of Modulation and studied best the is CBX7 chromodomains, CBX the Of exas M h iology andMedicinal ed C 2 , Mark arolina at o 1 , D * n Anderson li 20– 2

ne: 25Januar , Jacqueline L 2 2 C C . Interestingly, CBX7 functionsalso as a tumor sup T hapel Hill, hapel HillMedical School, C Bedford onsortium, onsortium, 1 , Guillermo C ancer C 2 2 5 hemistry, 4 and robust antitumor activity in xenograft 1 . Inhibitors of the H3K27me3 demethylases N , Cari orth orth C y U enter, Smithville, 2016 | 3 niversity of , Jinrong Min C N arolina, UNC S orris

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© 2016 Nature America, Inc. All rights reserved. article that results information of Kme-reading the aromatic cage induced-fit, an via CBX7 engage contrast, substrates,Histonechallenge.in significant peptide a CBX7 of tion por this targeting mimetics Kme small-molecule of discovery the structure apo ( the in unformed is CBX7 of cage aromatic the that observed 2K1 discovery efforts. We compared the hit- apo our CBX7 NMR guide structure (PDB to information structural existing the to turned we hits Therefore,interaction. tractable peptide CBX7-H3 the inhibiting of identifying capable in unsuccessful proved approach This 1 Fig. Supplementary 30 to up concentrationsat teins ammonium–bindingquaternary other prowith interact to known ligands available commercially of set a screened we Additionally, assay competition AlphaScreen established an using transferases largely designed for domainsKmereader ecules andlysinemethyl ligand characterization.full tativeprofiling versuschromodomainsother required forwouldbe tion informa structural existing is there because discovery ligand for target initial an as CBX7 selected we mind, in this Withwell. as mains to have affinity for other Polycomb chromodo likely is them of one binds that that molecule any suggests H3, with interact that regions the in CBX particularly PRC1, of chromodomains the between similarity of degree high extraordinary been has impact their yet and were family, BET the within antagonists selective not bromodomain reported initially Forexample, the families. target gous homolo highly within achievable tools the best be pragmatically, may, and utility great of also are profiles multitarget well- characterized with probes desirable, often is probes chemical monospecific of use the Although CBX antagonist Molecular dynamics–baseddesignofa RESU PC3 prostate cancer cells. in chromodomains CBX of function reading bio the H3K27me3antagonizing of the effects logical into investigation initial our along with chromodomains PRC1 of antagonist active cellularly and potent characterization a as UNC3866 of and design the report types. We cancer different in genes target CBX of roles precise the understanding and and UTX, JMJD3 of and EZH2 of inhibitors with investigating potential pharmacologic synergy therapeutic potential of CBX chromodomains, the biology,determining PRC1 Kme3- explicating in the inhibit reading capacity of thatPRC1 will be valuable tools probes Chemical probe. chemical active high-quality,cellularly criteria stringent the meet chromodomains that reported been have PRC1 molecules reports no for other ligands been of have there Although potential. therapeutic have also may PRC2, downstreamimmediatelyof occur to thought 2 consensus binding sequence is A(R/I/L/F/Y/V)Kme3(S/T)

Previous work using peptide arrays revealed that the overall CBX7 mol small of library targeted epigenetic our screened Wefirst 3 B , but with an understanding that quanti that understanding an with but , ) with the H3K27me3-CBX7 NMR structure (PDBstructure NMR H3K27me3-CBX7 the with ) LT S Fig. 1 a ). The absence of a preformed aromatic cage makes 29– 3 1 for classification as a as classification for fr opud ae ad structures). and names compound for , μ M (see (see M 3 2 The . 27 Supplementary Results Supplementary β , 2 8 -sheet-like interaction -sheet-like - - - - - ,

ab c N terminus label refers to the reverse sequence represents the corresponding associative path of with that well. Frames “ M T ( of Figure 1|Moleculardynamics–guidedinhibitor design. PBD Kme3 he Closed D CB Extensive hydrogen-bond S studies.Arrows are drawn from potential energy wells to representative structures associated K Kme3 nature

X7 inthe d 2 represents theaverage ofthree replicates network formed L 1 2L1 B 3 3 , lightgray, Kme3shown ingreen) conformations of . In fact, . I 2 ch ) and) N e MR structure oftheapo( Nat mic 3 N 3 - - - - , . terminus of a (apo) Phe11 l (bound) I

Phe11 UNC3567 to increase potency. of optimization further justified andantagonists PRC1 active larly cellu of goal our toward progress encouraging represented ment develop This calorimetry). titration isothermal by determined as consensus original ( the topeptide CBX7 ( UNC3567 peptide, a in resulted context this in residue diethyllysine a with Kme3 the ing replac that find Weamine.to encouragedquaternary were the for varying size, of hydrophobicity amines and basicity were secondary incorporated andas replacements tertiary which in screened and permanent positivethe werecharge. ofpreparedpeptides variety A demethylases lysine towardinstability potential Kme3: with associated liabilities the address to was priority next Our purification. in aid and licity lipophiincrease the cap benzoyl N-terminal towaschosen further permeability, cell anfacilitate andto leucine with Kme3 the ceding pre arginine the replaced first We peptides. for challenge known improveto permeability,as cell so charge polaritydecreasingand a while potency enhancing of goal the with ligands peptide-based of 110 and fiiy hn h crepnig 32m3 etd ( peptide H3K27me3 corresponding the than affinity greater22-fold RGFALKme3STHGwith sequence CBX7 binds the biology ” to “ ure recognition III (apo) Open Kme3 N terminus (bound) ” represent one possible dissociative path of H3 from Closed

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O 2K1 ± H N s.d.( II B b , darkgray) andH3K27me3-bound 0.6 1 iolog ), that was approximatelyforequipotent was that ), O 3 c Teeoe w iiitd optimization initiated we Therefore, . ) Free energy plot from the ( Hydrogen -bond N H a a 0.8 D tion | ) , root meansquare deviation. formation C CBX7 omparison of the aromatic cage O Ph CB y UNC3567 ( www.nature.com/naturechemicalbiology 1 H N K X7. ( d CB

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© 2016 Nature America, Inc. All rights reserved. Nat UNC3866 selectivity studies interaction with CBX7at concentrations up to 100 gen bond, and, consistent with our simulations, UNC4219 shows no the cellular in use studiesthrough methylation ofalanine the residue at Wegeneratednegative a controlalso compound, ( UNC4219 1 Table Supplementary a with CBX7, for reported ligand UNC3866 ( relationshipactivity studiessynthesisultimatelyof(SAR) the to led interactStructure-favorablyofCBX7. and L53 R52 D50, V13, with zoylglycine of UNC3567 that were less flexible and were predicted to CBX7 around UNC3567, thereby increasing ligand affinity. CBX7 would facilitate the hydrogennecessary bonding for closure of esized that optimizing the N-terminal interactions of UNC3567 with hypoth and UNC3567 with CBX7 of association the for model a as formation of the aromatic cage. We used this pathway for H3 binding work, this also orients Phe11 of CBX7 to contact Kme3 and facilitates addition to causing the formation of an extensive hydrogen bond net ‘zip’ bonds the hydrogen backbone formed, is turn the Once folding. critical the forming to analogous D50, is association during L53) V13, and R52 residues (namely CBX7 and III) Q5; (residue peptide H3 the of portion N-terminal the between contacts ate 2 ( Fig. CBX7 of 8–13 residues by β antiparallel two to analogous is and path exit this of reversal microscopic the of loss contacts.these the following occurs of CBX7 dissociation and H3 Complete III). (frame and L53 R52 D50, hydropho V13, with made through contacts bic primarily engaged CBX7 H3 leaving with III), to II (transition frame cage from aromatic the with contact loses Kme3 the Subsequently, II). frame to I frame from (transition H3 with contact tial par losing byopens CBX7 ofterminus N the in the shown as state from bound Starting UNC3567. to analogous most is which 4–10), (residues H3K9me3 of favored when we in considered a truncated form shown pathway the and structure, CBX7–H3K9me3 the from paths 2L1 (PDB CBX7 to bound H3K9me3 of structure simulations NMR published previously the dynamics using (MDS) molecular biased ( cage aromatic the form to Trp35 and Trp32 to proximity enough close in Phe11 position and histone the around close CBX7 residues of 7–13 whereby CBX7, conformational of rearrangement the drive that CBX7-H3 interactions critical the understand better to implied by prior structural studies nature CH associate its cellular effects with the modulation of Knowledgea of the specific activity profile molecular of a molecule is essential in order to i. 1 Fig. -strand is formed by the histone and the other Weof replacements series synthesizeda forben N-terminal the simply is H3 and CBX7 of Association recognition induced-fit the of Because 2 β N . e optd osbe itn exit histone possible computed We ). -hairpin into the fully folded state folded fully the into -hairpin α . hrfr, salsig h appropri the establishing Therefore, ). ure position ( position a . e hrfr tre t adaptively to turned therefore We ). E MIC 2

) ( ) chem A Fig. 2 L BIOLOGY β Fig. 2 Fig. β srns I ti cs, one case, this In -strands. -hairpin folding between between folding -hairpin a ), which is, to our knowledge, the most potent i a c β

| Adv ). This modification disrupts a key hydro key a disrupts modification This ). for key SAR data that led to UNC3866). to led that data SAR key for tr drn hairpin during -turn al Figure 1 Figure a Supplementary Supplementary nce online public b iue 1 Figure Fig. 1 Fig. iolog 3 , we sought K c , frame I, frame , d of 97 of c , frame frame , 36 c , was 3 7 y . In the case of CBX7, in in CBX7, of case the In .

± 3 d - - - 5 2.4 nM ( nM 2.4

oi a tion | : 10.1038/n on amicroarray containing 96epigenetic reader proteins across multipledomaintypes. derivative of (kcal mol baseline hasbeensubtracted. ITC replicates compound, UNC3866. Figure 2| ab d c μ M ( www.nature.com/naturechemicalbiology . T op graph shows raw titration heatvalues ( Fig. 2 Fig. Fig. 2 O -1 O O IT ) ofinjectantplotted against molarratio. ( ± ch ( N H N H s.d.)and UNC N H C studies ofUNC3866andUNC4219withCBX7 andselectivity profiling of a 3 UNC b ) Structures of e b ), for), ; see see ; O Ph mbio ). Single methylation Ph O O Ph 4219 (blue).( H N UNC4219 ( UNC3866 ( 3866. ( Me N H N - - - -

O .200 UNC O O N H not associated with PRC1. The array also revealed binding to three tobinding revealed also array The PRC1. with associated not HP1 as known (also CBX6 and CBX8. We did not detect CBX4, binding CBX2, orthologs, to PRC1 other CBX1, the and CBX3 CBX7 with or observed CBX5 ofchromodomains. positivea As binding expected, interaction was families two to binds UNC4195 that revealing streptavidin, tagged 3a Fig. duplicate in ( membrane spotted nitrocellulose-coated a chromodomains, onto distinct 28 and proteins teins, including at least 19 known H3K9me3 or H3K27me3 binding to evaluate binding to 96 purified chromatin-associated effector pro ( forCBX7 affinity high lated derivative of UNC3866, UNC4195 ( over other the nine members of Kme this reader panel. AlphaScreen by (IC determined as interaction, CBX7-H3 the of nist ( ITC or AlphaScreen by Tudorfamilies and MBT,PHD the from proteins encompassingreaders Kme of set small againsta selectivity itsated epigeneticrangeandnon-epigenetic ofboth targets.We evalu first Accordingly, we evaluated the selectivity of UNC3866 against a wide targets molecular potential of panel broad a against activity its of understanding in-depth an upon dependent is probe chemical a as molecule a of classification and use the Thus, interest. of target(s) N H N H d 3 7 4219 (nointeraction upto 100 2 ) O To facilitate further selectivity studies, we generated a biotiny a generated we studies, selectivity further facilitateTo ) 50 ) O UNC O H N UNC = 66 = UNC4195 ( H N H N b B . idn itrcin wr vsaie wt fluorescently with visualized were interactions Binding ). ) ottom graph shows normalized integration heatvalues O 4195 selectively interacts with two subfamilies of chromodomains C 3866 (black)andthestructurally related negative control upeetr Tbe 2 Table Supplementary O N O N H omparison of ± N N H N N H 1.2 nM), and is more than 100-fold selective for CBX7 CBX7 for selective 100-fold thanmore is and nM), 1.2

OH O 4 ) H N OH O OH O OMe OMe O β 11 μ , HP1- ,

UNC Amount of injectant cal/s) plotted against theintegration timeafter −1 Titration heat ( cal s-1) K (kcal mol ) µ − − − − − H N − 0.40 c d 0.60 0.20 0.50 − 0.30 − − − 0.10 = 220 = ) Structure of 4.0 O 8.0 6.0 2.0 3866 ( 0 0 γ and HP1- and 01 μ 0 M) bindingto 02 K ± . N36 i a oet antago potent a is UNC3866 ). d 22 nM). WenM). derivative22 this used =97 S 0.5 H 03 HN Time (min) Molar ratio 04 H UNC NH ± 1.0 α O 2.4 nM,average ofthree 05 4 , respectively), which are which respectively), , i. 2 Fig. ) ( ) 4195, abiotinylated 06 CB Fig. 2 1.5 07 X7 asmeasured by c article 08 , 2.0 c Supplementary Supplementary ), which retains), which 0 CBX8 chromo CBX7 chromo CBX6 chromo CBX4 chromo CBX2 chromo CDYL2 chromo CDYL1b chromo CDY1 chromo

K UNC3866 ( no interactio UNC4219 ( d =0.097 3 µ 2 2 n ); M ) 9 3 - - - - .

© 2016 Nature America, Inc. All rights reserved. article f B7 ( CBX7 of atoneleast hydrogen backbonebond the with in participates UNC3866 of amide backbone understanding of its binding interactions. Each 5EP cocrystal (PDB CBX7 to bound X-rayUNC3866 of structure an solved additionally We UNC3866 cocrystallization studies to chromodomainspecific antagonism. consequences that biological the determining indicate in control negative valuable UNC4219 a be will UNC4219 and UNC3866 6 Figs. V and NK2 the for antagonism weak profiles in these follow-up assays, with both compounds having very 5 Figs. ( control)(negative UNC4219 and UNC3866 both 10 atbinding radioligand assays tional PDSP) panel, which includes 22 additional GPCRs. Follow-up func (NIMH Program Screening Health’s Drug Mental proteins Psychoactive of transporter 3 and channels ( ion 5 (GPCRs), receptors eral pharmacology panel (Cerep) consisting of 49 G protein-coupled Supplementary Tables 3 ( case each in demethylases inactive be lysine to found and 7 and methyltransferases protein 33 mains, a ‘known unknown’ of use inthe UNC3866. antagonizing these proteins in a cellular context challenging. This is CBX proteins, which makes assessing the potential consequences of Polycombof that than characterized well less is function biological CDYproteinsof biochemistry explore the todone been has work some Although CDYL2. and CDYL1bover CBX7 and CBX4 for selective 9-fold and CDY1 over CBX7 and CBX4 for selective 65-fold is UNC3866 Additionally, respectively. CBX8, and CBX6 CBX2, over CBX7 and CBX4 for selective 12-fold and 6- 18-, whereas is it CBX7, to similar most is which CBX4, for equipotent is ( CBX7 of that relativeto main chromodo each of identity sequence percent the with correlated of UNC3866 for CBX2, CBX4, CBX6 and CBX8 is surprisingly well ing to each of these chromodomains. We also found that the affinity 3b Fig. for affinity byUNC3866 the ITC( fied positive binding showed to UNC4195 thaton the proteinsarray and subsequently all quanti purified and expressed We CDYL2. and CDYL1b CDY1, chromodomains: of family CDY the of members 4 L53 and R52 D50, of chains side the contacts tert K CD CD CD CB CB CB CB CB Chromodomain CDY chromodomains by T Supplementary Table 5 Table Supplementary

d able 1|Quantitative analysis ofUNC3866bindingto CBX and values are reported astheaverage ofatleasttwo replicates N36 ws lo vlae aant pnl f 8 bromodo 48 of panel a against evaluated also was UNC3866 X8 X7 X6 X4 X2 btlezy cp f N36 primarily UNC3866 of cap -butylbenzoyl Y Y Y1 J ) at a resolution of 1.6 Å to improve ourimprove to Å 1.6 of resolution a at ) L L 2 1b – ). In agreement with the microarray data, we detected bind detected microarraywe data, the agreementIn with ). 8 ). Both UNC3866 and UNC4219 showed nearly identical nearly showed UNC4219 and UNC3866 Both ). and i. 3a Fig. 39 8a , 4 0 o tres ipaig 5% niiin f control of inhibition >50% displaying targets for . h smlr rfls of profiles similar The ). , b ), while the N-terminal N-terminal the while ), and ) as well as against the National InstituteNational the against as well as ) μ IT M UNC3866 were also performed with performed also were UNC3866 M C 4 ). UNC3866 was tested against a gen Supplementary Fig. 3c Fig. Supplementary 1a Table Table receptors ( receptors Supplementary Fig. 4 Fig. Supplementary UN ± 0.097 s.d. 0.610 0.094 0.85 C3866 0.91 1 1.2 1.2 6.3 6.3

1.8 and and ± ± ± ± ± ± ± 0.021 pocket in formed by UNC resolvedonly partially by electron density). ( ( Figure 3|Structural studies ofUNC3866withCBX7 andCBX8. a ± 0.0078 0.076 0.0024 0.21 0.92 0.076 PDB Supplementary Supplementary Supplementary Supplementary Supplementary 0.017 K d ( nature 3866. (

 ). UNC3866 ). 5 EP M) J CB ; surface shown) withthe CB 3 8 c X7 and , their their , ch ) X7 residues and and I nteractions ofthe e Nat mic ------

CB a l

X8 ( enhancingvan Waalsthe der interactions withUNC3866. Thissin Supplementary Fig. 9b and CBX4 in ( leucine a and valines contrast,two of up made is pocket this CBX7 In group. methyl alanine UNC3866 the binds that pocket hydrophobic open relatively a form chains side whose alanine,and leucine valine, consistsa ofCBX8 and CBX2 in a of result pocket alanine the The pocket. this in differencesignificant, yetsubtle, be might CBX8 and CBX2 over CBX7 and CBX4 for UNC3866 of potency improved the in that revealed UNC3866 protein each of alanine the for pocket binding the of inspection chromodomain( each to fashion similar a in binds UNC3866 thatillustrates clearly structures these ( UNC3866 of code (PDB CBX2 to bound interactions with D50,R52and L53. this through of molecule the ability around CBX7 the of closure nucleate in to moiety improvement an of result the is UNC3866 the the for of UNC3567 substitution of cap upon benzoylglycyl observed increase that affinity suspect we 70-fold studies, MDS the our of basis the On embedded. is the which within L53 with D50, R52 andside chain this rigidified forms a hydrophobic groove ( density is comparatively weak for CBX7 ( C the a adopting as modeled is structures, side chain of UNC3866, when bound to CBX proteins in our crystal all- extended, fully adoptinga as eled mod been has side-chain H3K27me3 the Whereas structures. our CBX chromodomains and the diethyllysine of UNC3866 revealed by to aid of design ligands. inthe selective understanding complete more a obtain to needed are in-depth studies SAR and structural Further CBX7. and CBX4 for UNC3866 of selectivity modest the contributeto mayvariation sequence this importance of this interaction in the binding of UNC3866 to CBX7, CDY the proteins.in motif (N/H)-C-E andemonstrated Given the the engages that ily ( microarray ( 9f Fig. sequences the of Alignment proteins. over CDY CBX7 and the CBX4 for UNC3866 of selectivity the explain to directly with UNC3866. interact that acids amino the withinvariations significant other no are there as CBX7, and CBX4 for UNC3866 of affinity higher the determines that factor predominant the be to likely is residue gle we model the side chain in the the in chain side the model we biology ure Fig. 3 Fig. b D diinly w sle Xry rsa srcue o UNC3866 of structures crystal X-ray solved we Additionally, We were also interested in the interactions between the Polycomb challenging more is it structures, cocrystal of absence the In PDB 50, R52 and γ 5EQ -C

c ) of the CBX and CDY proteins that bind UNC4195 on the the on UNC4195 bind that proteinsCDY and CBX the of ) chem

). The guanidinium moiety of R52 forms a salt bridge with bridge salt a forms R52 of moiety guanidinium The ). | Adv 5 δ E tert bond in 0 Q i odr o etr nesad h slciiy profile selectivity the understand better to order in ) C 0 a -butylbenzoyl capof Fig. 3 Fig. -terminal portion of -terminal portion ) (top andbottom panels,respectively). nce online public i. 3 Fig. L 53. ( i c a b Figure 3 Figure al ) indicates that the key motif in the CBX fam CBX the in motif key the that indicates ) tert ) d d ) I nteraction of and and 5EP C – -butyl of UNC3866 (D-X-R) is replaced by replaced is (D-X-R) UNC3866 of -butyl omparison ofthe b c f ), which mold more tightly to the alanine, iolog K a upeetr Fg 9 Fig. Supplementary ASP-50 ), CBX4 (PDB CBX4 ), . Although the corresponding electron tert a LEU-53 tion | UNC UNC gauche btlhnl ru o UNC3866 of group -butylphenyl Supplementary Fig. 9a Fig. Supplementary CB 3866 (green; serinemethyl ester 3866 withthehydrophobic groove y www.nature.com/naturechemicalbiology X7 withthe ARG-52 ( anti

a d conformation based on the the on based conformation gauche ) UNC oi I Supplementary Fig. 10a conformation nteraction of tert : 10.1038/n 5EP 3866 alanine-binding d btlezy cp of cap -butylbenzoyl ofrain about conformation N L CBX8 CBX7 -terminal portion of -terminal portion ) and CBX8 (PDB CBX8 and ) . lgmn of Alignment ). Supplementary Supplementary ch CB e 3 VAL-13 X7 ALA-13 , the lysine the , mbio ). A closer closer A ).

Fig. 3 Fig. .200 d ), 7 - - - ,

© 2016 Nature America, Inc. All rights reserved. Nat in UNC of intact chemiprecipitates lysates andchemiprecipitates additional selectively chemiprecipitates Figure 4|UNC4195 pulldown studies inPC3cells. the the PC3 lysate input; however, UNC4195 pulled down only EED3 malian isoforms and of EED (EED1, EED3 and EED4) of UNC4195 to pull down EED in PC3 cells. Three of the four mam CBX-containing in PRC1 complexes participate intrigued us can EED that finding recent The I the disrupting without PRC1 complex’s core ‘canonical’ composition. extension engages by and UNC3866, UNC4195, that indicating HPH2, and BMI-1 ing three ‘canonical’ PRC1 subunits ( intact complex by probing for representative proteins of the remain investigated the ability of UNC4195 to chemiprecipitate PRC1 as an CBX6 interact with UNC4195 on the microarrays ( chromodomainsisolatedand purified, CBX2 of the that fact the by become inaccessible to pulldown with UNC4195. This is supported chromodomains their that way a such in complexes PRC1 partici in pate CBX6 and CBX2 CBX8, and CBX7 CBX4, to contrast in that, suggesting experiments, these in CBX6 and CBX2 down pull to We failed UNC4195. tochromodomains the of binding specific ( which was inhibited by the presence CBX6, of excess UNC3866 in CBX4, the lysate CBX2, of CBX7 and CBX8. presence We detected pulldown the of CBX4, CBX7 and forCBX8, probed we UNC4195, function CBX7 study to lization abundance high PolycombCBX ofeach protein priorand their uti relatively their for chosen were cells PC3 reagent.pulldown CBX a as utilityinvestigate its to order in UNC4195 with performed were lysatesprostateChemiprecipitation cell cancerPC3 in experiments UNC4195 engages ‘canonical’ PRC1 the than demanding endogenous sterically Kme3 by recognized domains. these more is which group, functional diethyl the accommodate to chromodomainsPolycomb other the and CBX7 of ability the explain may state apparent bound the this in Further,flexibility CBX7. to bound when flexible somewhat remains UNC3866 in chain side the that reflect may atoms these ( state the supports the electron density of Although the CBX8-UNC3866 structure more structure). strongly CBX7-UNC3866 the for Å as 1.6 to Å compared (1.2 structure cocrystal CBX8-UNC3866 higher-resolution nature CH of UNC3866 to the lysate ( EED4, both of which could be competed away through the addition soform-specific incorporation ofEEDinto PRC1 Fig. 4 Fig. Supplementary Figure 19 4195. Supplementary Fig. 10b Fig. Supplementary ure a ab PC ), confirming that successful pulldown is dependent on the the ondependent is pulldown successful that confirming ), UNC3866 E n 3 cells with MIC =3for allsamplesin

chem gauche A L BIOLOGY

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CBX2 HPH2 BMI-1 RING1b CBX8 CBX7 CBX6 CBX4 | Adv . 3866 for 24 hinhibitspulldown of al EED Fig. 4 CB a X4, 4 from 4 nce online public b ; therefore, we investigated the ability a – b ), the ambiguity in the position of position the in ambiguity the ), iolog c c CB ). Additionally, because EED binds . Full-sized western blotsare provided 1 UNC3866 EED4 EED3 EED1 0 . Upon chemiprecipitation with with chemiprecipitation Upon . X7 and Input PC P Fig. 4 R 3 cell lysates. ( UNC4195 pulldown C 1 components. ( [UNC3866]

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.200 UNC3866 does not bind directly to EED ( EED nottobind directly does UNC3866 that confirmed wedomain, reading Kme WD40 its via H3K27me3 bination of pulldown and ITC experiments that the corresponding the that experiments ITC and pulldown binationof com a through confirmed we therefore, esterases; intracellular by hydrolysis to susceptible is ester methyl C-terminal the if prodrug K compound concentrations for cellular studies relative to the ( low be to it found and assay Caco-2 a using UNC3866 of permeability peptide inhibitors often have poor cellular activity,As we context.evaluated the cellular a in UNC3866 of utility the assessed Wenext UNC3866 antagonizes PRC1 chromodomains incells offunction isoform this inPC3cells. and partners binding the about questions raises PRC2 and PRC1 forms on PRC1 function. Furthermore, the absence of iso EED1 these in both of effects the examine to work future for basis the vides EED incorporates into PRC1 in an isoform-dependent fashion pro the (ref. PRC2 dictate of substrate can histone PRC2 into EED of isoforms different of ( (ref. UNC2399 reagent pulldown biotinylated EZH2 and EZH1 the using PRC2 in forms iso EED of presence the investigatedWe next PRC1. incorporation into EED of through mediated is pulldown the that indicating rtis s eemnd y loecne eoey fe photo after recovery bleachingexperiments (FRAP) fluorescence by determined as these proteins CBX of mobility of cellular the chromodomains on effects the significant has of proteins deletion nor mutation neither Further, proteinsreadout. unreliable an H2AK119 CBX monoubiquitinationof contain not do that complexes PRC1 cal challenging. proteins these activity, ligase AlthoughE3 PRC1has presence ofthe noncanoni of engagement cellular onstrating main targets incells. chromodo its of functions antagonize the and engage to ability its evaluate to possible is it that stable and permeable cell sufficiently is UNC3866 Overall, conditions. these under limited is UNC3866 of hydrolysis that indicating UNC4007, of concentration cellular intracellular UNC3866 The intra the than approximately higher concentrationwas seven-fold concentration. extracellular the of ~5% is UNC3866 concentrationof intracellular the that indicating 1.4 concentrationof intracellular an in resulted h 24 for 30 ensure sufficient with accumulation of cells intracellular UNC3866. Treatment PC3 incubated we permeability, byLC-MS/MS ( fied the intracellular concentration of both UNC3866 and UNC4007 chromodomains ( ( UNC4007 acid, C-terminal CBX7 incells at concentration. this ± 30 with treated completelyUNC4195waswith inhibited lysatesin frompre cells chemiprecipitationEncouragingly,CBX7 lysed. and trypsin with and compound were removed and the cells were washed, harvested medium excess which after h, 24 for UNC3866 concentrationsof block wouldchemiprecipitation. Whole werecells PC3 incubated varying with UNC3866 with pretreatment that with expectation engagement, the target cellular demonstrate to used be experiments could chemiprecipitation competition-based that esized (CETSA) assay shift thermal cellular the via engagement target cellular of evaluation precludes which protein, full-length the ofrelatively (~25%) a portion prises small ( lysates cell in UNC3866 by CBX7 of thermostabilization detect previously been appliedhas chromatinto readers SupplementaryFig. 12 Supplementary Fig. 15 0.3 d 7 . Additionally, we noted the potential for UNC3866 to serve as a as serve to UNC3866 for potentialAdditionally, the . noted we h nnnyai ntr o crmdmis ae dem makes chromodomains of nature nonenzymatic The Supplementary Fig. 13 Fig. Supplementary μ M, Fig. 4 Fig.

Supplementary Table 6 Supplementary μ c Supplementary Fig. 14 M UNC3866 (intracellular concentration = 1.4 = concentration (intracellular UNC3866 M ), indicating that UNC3866 effectively engages effectively UNC3866 that indicating ), 2 ). It). shown has been that incorporation the ), likely because the chromodomain com 4 . neetnl, e eetd ny EED4 only detected Interestingly, we ). ), indicating a need for relatively high relativelyhigh for need a indicating ), 5 ), is still capable of engaging PRC1 engaging of capable still is ), 4 3 4 , ruling out technique,ruling , this which 2 ), and hence our discovery that discovery our hence and ), 4 ). Owing to the lowto the Caco-2 Owing ). 4 Teeoe w hypoth we Therefore, . and Supplementary Fig. 11 SupplementaryFig. 20 ). Next, we quanti article μ 32 UC86 to UNC3866 M , 3 3 . We. to failed ± 0.3 6 n vitro in makes makes in vitro μ M, ), 5 ------

© 2016 Nature America, Inc. All rights reserved. article to knockdown of CBX7 by shRNA (>60% mRNA reduction) in PC3 expressionof the analyzed Nonetheless,we capacity proliferative enhance necessarily not does locus this of regulating negatively without controversial capacity proliferative is enhance to shown locus been has this CBX7 example, regulating in role actual its the of regulation through liferation capable is of inducing senescence. knockdown CBX7 that observed study same the (HPrEC) Furthermore, cells epithelial prostate human in senescence rep licative bypassing of capable genes for screen genetic a in identified originally was CBX7 because observation intriguing an was This for positive cells SA- of number the in increase dramatic a observed senescence-associated senescent of presence become had they that ( gesting morphology flattened and enlarged an exhibited genetic manipulation studies is striking CBX7 from data published previously with result this of cordance chromodomainsout,con the byboundruled cannot UNC3866 be other from phenotype antiproliferative this to contribution some nM ~100 sured mea the given CBX7 and CBX4 engage fully to adequate is which thereforeis cellsnM, PC3 340 estimated be to EC itsat concentration lularUNC3866 of an in EC resulting cells, UNC3866-treated for only count cell PC3 in reduction significant a observed we and UNC4219, and UNC3866 for dependency revers dose examined Wenext ible. partially least at are effects pound’s ( d 3 after UNC3866 with replenished not and washed proliferating when resumed cells that tial cell growth ( exhibitedDMSOexponenor UNC4219 with treated cells while d, 6 and 3 after count cell in change no almost in resulting treatment, UNC3866 uponproliferation cell in decrease cells PC3 in ity ability of CBX7 to enhance proliferative capac ( cells in CBX7 engageeffectively toshown was tration 30 at UNC4219, control, negative the and UNC3866 with treated cells were PC3 proliferation, on effects assess To UNC3866toxicity noncancerousa in line. cell evaluate to used were cells HEK293T range. concentration same the within toxicity able 16 Fig. ( cells HEK293T and PC3 in CellTiter-Glo by assessed as effects, toxic any 100 treatmentto up UNC3866 with that to confirmed prelude we studies, a these As proliferation. cell PC3 on UNC3866 of effects investigate the to us aged cells these in a advantage confer growth to overexpression CBX7 of ability demonstrated the with coupled cells, PC3 in proteins CBX with of levels cells high The treating UNC3866. of consequence logical 6 sion. Further, evaluation of the effects of UNC3866 on expression of cells and did nota statistically observe significant change in expres Fig. 5

e et oue o etbihn a bio a establishing on focused next We Although it has been suggested that CBX7 controlsCBX7 thatsuggestedprocellular been hasit Although UNC3866 with treated cells PC3 that observed also We 50 β of 7.6 of gl olwn tetet ih 30 with treatment following -gal a ). Similarly, UNC4219 had no observ no had Similarly,UNC4219 ). , dotted line), indicating that the com Fig. 4 Fig. ± 2.5 c ). Consistent with the known the with Consistent ). Fig. 5 14 K , μ d 4 5 values against these targets ( targets these against values M ( M w osre a dramatic a observed we , a ). Further, we observed Fig. 5 Fig. μ μ M, as this concen this as M, M did not induce not did M b Supplementary Supplementary Ink4a ). The intracel The ). 10 , 4 β 5 / Ink4a encour , ARF glcoiae (SA- -galactosidase 17 , μ 1 9 8 50 / , and negative regulation negative and , UC86 ( UNC3866 M . ARF 4 in 6 Ink4a . We checked for the the for checked We . ------( Fig. 3 Fig. CDKN2A / bars are equalto 60 microscopy (40×). using phase-contrast microscopy (40×). SA- ( ( replicates withtwo technical replicates each). is >76 off-target effects, whichwere notevident atconcentrations below 100 replicates). biological replicates withthree technical replicates andonebiologicalreplicate withtwo technical inhibits representative ofatleastfour biologicalreplicates withvarying platingdensities.( of eachdatapoint( experiments (dotted line), inhibits cell proliferation. Figure 5|Cellular ofUNC3866. effects b a ARF d c

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). Arrows indicate examples ofpositive SA- Normalized cell count d 100,000 i. 5 Fig. 10,000 ) (% of DMSO control) b 1,000 nature 100 UNC in response in 50 ). Although ). 100 μ 0 β PC M, whichisthelower boundofthe95% confidence interval ( 9 gl and -gal) i. 5 Fig. ) locus ) , 02 11 3866 induces asenescent-like morphologyin c 3 cell proliferation exposure inadose-dependentmannerafter for 6d( T , , sug ), 1 01 8 he ch For . e EC d Nat mic 1 1 1 ). 4 8 8 ------50 log[compound] . , . T n

isreported asthe95% confidence interval. a μ he images are representative ofthree biologicalreplicates, andtheblackscale =3,singlebiologicalreplicate withthree technical replicates). l m. Time (d)

h ue f N49, e eosrtd ht N36 bns in binds UNC3866 that demonstrated we through UNC4195, of Further, use profile. the selectivity characterized thoroughly a recognition Kme CBX7 and CBX4 inhibitorof molar date to reported antagonist CBX7 potent most the UNC3866, of design the enabled CBX7 by recognition H3 of dynamics molecular the investigationsinto Our D ments is currently under investigation. permeability. cell The potential utility poor of UNC3866 at forhigher doses its of because intra engagement for target required levels cellular circulating high the by limited be may UNC3866 of use the compound, peptidic a for promising are results pharmacokinetic these Although clearance. moderate and ( UNC4007 time to all relative at tested, plasma points in species predominant the is UNC3866 that eritoneal injection (10mg/kg) Swiss inmale albino miceand found in PC3cells with 5-AzaC, suggesting that this gene may be irreversibly repressed PC3 Ink4a in repressed deeply hypermethylation is DNA through cells locus this that indicating evidence 17 Fig. ( UNC3866 antiproliferativeof the effects in lation changeshowedno at30 locus this biology ure 30 UNC I 46 SCUSS Finally,followingUNC3866intrapstabilityof the evaluatedwe UNC µ M upeetr Fg 18 Fig. Supplementary / 4219 treatment hasnoeffect on ARF

23 ). This was not entirely surprising as there is considerable is there as surprising entirely not was This ). chem 3866 was notreplenished atday 3.

| Adv IC UNC3866 IC UNC4219 IO is unaffected by both DNMT knockdown and inhibition 50 50 washout 30 30 30 DMSO 47– a =7.6 =>76 N µ µ µ nce online public 4 M UNC3866 M UNC3866 M UNC4219 9 ( . 8 a i ± µ ) c 2.5 M T β al reatment of β -gal expression was assessed usingbright-field µ E -gal expression. M rror bars represent thes.e.m. ofeachdatapoint. b d c iolog 27 , n sos 5 boviaiiy and bioavailability 25% shows and ), , PC 2 a 8 PC tion | UC86 s n qioet nano equipotent an is UNC3866 . 3 cells ( 3 cells with30 DMSO DMSO μ PC C y M, ruling out ruling M, www.nature.com/naturechemicalbiology UNC ell morphologywas assessed 3 cell proliferation. For washout

47– upeetr Tbe 7 Tables Supplementary d c E ) andexpression ofSA- oi rror bars represent thes.d. 5 n 4219 was usedto control for 0 . Further, methylation of methylation Further, . =6,three biological : 10.1038/n μ M. T μ he M Ink4a UNC EC 30 30 Supplementary Supplementary b n µ ch 50 in vivo µ =8,two ) T M UNC3866 M UNC3866 in vitro in for UNC 3866 he dataare e / mbio ARF UNC 3866 experi in vivo in β regu , with , .200 -gal 4219 – 9 7 ­ - - - -

© 2016 Nature America, Inc. All rights reserved. Nat 7. 6. 5. 4. 3. 2. 1. R bound, 5EP Accessioncodes. version o the in available are references associated any and Methods M published online25January2016; R understanding Polycomb signaling. in generally, more and, determination fate cell in probing assistfurther in thatrole will the of PRC1 chromodomains tools of set new relatedderivativesprovideitsa and UNC3866 and modomains play a pivotal role in regulating cellular differentiation inhibitors. In EZH2 addition to their role in different cancers, with PRC1 chro combination in or alone oncology, in therapeutics as further investigation of the use of PRC1 chromodomain antagonists supports proliferation cell PC3 inhibit to UNC3866 of ability The biochemical role of the the different EED isoforms in Polycomb-mediated into signaling. investigation future for basis the providing into PRC1 and PRC2 in an isoform-dependent manner in PC3 cells, Polycombincorporatedis thatEED revealed readershasKme CBX our to and, readers Kme knowledge, any Kme3 reader. of family CBX Polycomb the both for probe chemical high-quality first the as UNC3866 validatecontrol negative a as UNC4219 of use the and UNC3866, of engagement target cellular demonstrated the proteins, >250 versus UNC3866 lished estab well is utility their yet and family BET the within selective not bromodomainantagonistswere reported initially the example, for probes: chemical first-in-class for unusual not is This targets. other these from affects possible assess to dependency dose of tion evalua and activities these of considerationrequire will probe cal proteins and the CDY family of chromodomains. Its use as a chemi PolycombCBX other for affinity possesses it CBX7, and CBX4 for potent most is UNC3866 Although interest. of target(s) molecular specific a of modulation with effects cellular its associate to order quaternary amine is not required for inhibition of Kme3 readers. a that signifies broadly,more this and, ligand, active cellularly a of development the in achievement key a was mimetic amine tertiary the quaternary amine of the native peptide ligand with an unnatural toxicity.associated proliferationno replacementwith ofassaysThe cellular and pulldown competition in potency cellular micromolar exhibits UNC3866 Finally,‘canonical’PRC1. intact, of context the nature CH eceived 1May 2015; accepted 25November 2015;

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, 7 , © 2016 Nature America, Inc. All rights reserved. article from work at performed Argonne National Laboratory, Center Biology at Structural the using content high the imaging microscope. Results shown report inthis are derived antibodies, respectively. We (UNC) for thank N.Sciaky also help with counting cell labsthe of T. Magnuson (UNC) and D. Margolis (UNC) for providing EEDand BMI-1 (UNC) for providing PHF1, PHF19, PHF23 and JARID1A protein constructs. We thank screening and J.R. Walker structures. We for of review the crystal the thank G.Wang screening, A.Tumber (SGCOxford) for support with lysine the demethylase selectivity manuscript, O. Fedorov (SGCOxford) for support with bromodomain the selectivity We thank K.Pearce (UNC Chapel Hill) for discussions useful and careful reading of the A 50. 49. 48. 47. 46. 45. 44. 43. 42. 41. 8

cknowledgments

differentiation. cell stem embryonic during complexesrepressivepolycomb of dynamics H3. histone nucleosomal or H1 histone of methylation target complexescontaining methylation. histone PRC2-dependent for required motifs EED of mapping functional and 7 invasiveness. cell PC3 derived cancer prostate increases and methylation DNA specific locus affects silencing migration. and growth cellular represses and methylation, DNA locus-specific effects cells PC3 in DNMT3b CancerChromosom.Genes cancer.prostate metastatic and primary in (p16/MTS1) CDKN2 of aging. and cancer locus. p16 the at 9 lysine assay.shift thermal cellular the using tissues Mirochnik, Y.Mirochnik, F.Abbas,DNMT1 & S. Farooq, R., Qazi, S.A., Qureshi,Yaqinuddin, A., F.of Abbas,Down-regulation & R. Qazi, S.A., Qureshi,Yaqinuddin, A., D.F.Jarrard, in senescence of Regulation H. Zhang, & Ramkumar,C. Y.,H., Kong,Cui, Q. Li, D.Molina, Martinez and distributions the in T.K.ChangesKerppola, & C. Vincenz, X., Ren, EZH2- Different T.,Jenuwein,D.Kuzmichev,Tempst, A., P.Reinberg, & T.Magnuson,Molecular Montgomery, Yee,& Montgomery,N.D.,D., S.A. , e31052 (2012). e31052 , et al. et Polycomb CBX7 directly controls trimethylation of histone H3 at H3 histone of trimethylationcontrols directly PolycombCBX7 Mol. Cell Mol. et al. et J. Mol. Biol.Mol.J. et al. et Mol. Cell. Biol. Cell. Mol. Deletional, mutational, and methylation analyses methylation andmutational, Deletional, J. Aging Res. AgingJ. Androgen receptor drives cellular senescence. senescence. cellular drives receptor Androgen et al. et

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Competing financialinterests studiesdesigned results; and discussed J.I.S., L.I.J. and S.V.F. wrote paper. the ments; J.I.S., B.D.M., S.G.P., J.M., B.L.R., M.V., P.J.B., M.T.B., C.H.A,L.I.J. and S.V.F. B.M.B. and performed analyzed AlphaScreen assays; EEDITC G.S.performed experi F.L. methyltransferase performed assays; X.-P.H. assays. GPCRfunctional performed pulldownK.G.H. performed studies; C.S.and K.B. protein performed array experiments; UNC3866 with various the CBXproteins; J.L.N. and S.H.C.expressed proteins; purified and cellular proliferation assays; Y.L., W.T. and S.Q. structures of solved X-ray the crystal dynamics studies and assisted incompound CellTiter-Glo N.C.performed design; assays studies and culture; cell all performed B.M.D. adaptively performed molecular biased studies and pulldown studies, rendered images, structural assisted with AlphaScreen J.I.S. and designed compounds synthesized all and related analogs, ITC performed A Anderson Cancer Center. and by Centre the for Environmental and Molecular Carcinogenesis at MD the iswhich supported by Cancer the Prevention Research Institute of Texas (RP130432) Takeda and Wellcome Trust Grant 092809/Z/10/Z. M.T.B. directs Proteinthe Array Core, Foundation, of Ontario the Economic Ministry Development and Innovation, Pfizer, Institute Grant GlaxoSmithKline, OGI-055, Janssen, Novartis Canada,the Lilly Research Canadian Institutes of Health Research (CIHR),Genome Canada,Ontario Genomics from AbbVie, Ingelheim, Boehringer CanadaFoundation the for Innovation (CFI),the Foundation (G-1847).The SGCis aregistered charity (no. 1097737)that receives funds Cancer Research Fund, University of North Carolina at Chapel Hill, and Welch the Health (NIH,grant Carolina R01GM100919),the Partnership and University the by National the Institute of Medical USNational General Sciences, Institutes of contract no. DE-AC02-06CH11357. The research here was supported described Facility operated of by for Science Argonne Office DOE the National Laboratory under Advanced Photon of User Source, Science Office aUSDepartment of(DOE) Energy requests for materials should addressed to be L.I.J. and S.V.F. available online at available inthe Any supplementary information, chemical compound information and source data are A The authors declare no competing interests. financial biology ure uthor contributions dditional Information

chem

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d oi : 10.1038/n l . Correspondence and ch e mbio

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7 © 2016 Nature America, Inc. All rights reserved. (Cat. # NET155V250UC; Perkin Elmer; PerkinNET155V250UC;(Cat. # Elmer; 20 a in performed were Assays described previously(SPA)AssayProximity as Scintillation using strates of sub peptide of incorporation residues arginine or lysine the to group methyl tritium-labeled monitoring by SMYD3, assessed was SMYD2, DNMT1 SETD2, and BCDIN3D PRDM9, PRMT8, PRMT7, PRMT6, complex, tetra MLL3 PRMT5-MEP50complex,PRMT3, PRMT1, trimeric complex, EZH2 meric complex, trimeric MLL1 SETD7, SUV420H2, SUV420H1, transferase activity of G9a, EHMT1, SUV39H1, SUV39H2, SETDB1, SETD8, previously expression and purification of the constructs used in this assay was described The Elmer). (Perkin laser AlphaScreen HTS an with equipped reader label multi-EnVisionan on read were plates the incubation, temperature.After room at dark the in min 30 additional an for incubate to allowed were plates or Next, 2 pound concentration to 100 in 1× assay buffer was added by Multidrop (Thermo) to bring the comfinal Elmer). To these plates 9 and 1 and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen) were diluted in 1× assay buffer (20 mM TRIS pH 8.0, 25 mM NaCl, 2 mM DTT (1 was generally performed as previously described protein ratio to a one site compound:binding model. the of function a as binding of heats the fitting least-squares, data was analyzed using Software Origin (MicroCal Inc., USA) by nonlinear titration The deleted. routinely was point data initial The curves. binding from protein-compound the subtracted were then generated of heats dilution the determine the applicable, If to compound. the diluting conditions from arise any, that if signals, identical heat under buffer into compound each 8 of powerreference a and s 180 of spacing a with performed were Injections compound. of tions 200 intoa 0.2 single a includedexperiment typical A mass.on based prepared were solutions stock compound whereas method, Edelhoch the using established was solution stock protein the of concentration The concentrations:desired 50 the achieve NaCl,and mM2 pound stock samples were in the target buffer (25 mM Tris-HCl, pH 8, 150 mM com and protein All MA). Inc., (MicroCal AutoITC200microcalorimeter Schrödinger, LLC. 1.7.4 Version System, Graphics Molecular PyMOL the using simulations rendered the were from images All design. ligand inform could that intuition some get to simulations ns 128 five Weran steps. five every updated was list neighbor The Å. 1.6 of spacing grid a approachwith Ewald mesh particle the with treated were electrostatics Long-range cutoff. Å 10 a used interactions rescaling ensemble velocity canonical stochastic the in with out carried were atsimulations Production equilibration bar. isothermal–isobaric 1 of ns 5 included construction System salt. and water and adding after peptide atoms or23,710 in protein resulting the face, cube between nearest Å the 12 in of performed minimum a were with simulations box cubic All a simulation. the during trajectory and r.m.s. deviation of peptide. We used the Kabsch algorithm to align and the clasp,reference of deviationr.m.s. defined: were variables collective two structure, hyperdynamics of ples metadynamics for done been recently has as dynamics approximateforarguemay onetransitionThus,states. near state-to-statebias bias of choice parameters stable This floods states extremely slowly, Å. to minimize adaptation 1/4 of the was width Gaussian The step. time dynamics lar biasparametersThe were potential biasing Adaptive methods. Experimental O doi:10.1038/nchembio.2007 (1 concentrations close to the N μ Methyltransferase selectivity assays. assays. AlphaScreen reader Kme experiments. ITC μ α l at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) DMSO) in dilutions 10-point 3-fold, concentration; highest mM 10 at l M, 10 M, -GST acceptor beads (45 acceptor beads -GST LI μ NE ME l l was spotted into the wells of low-volume 384-well Proxiplates (Perkin μ l of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate donor and nickel-chelate l of a of 1:1 mixture streptavidin-conjugate 5 μ μ 6 . l cell filled with protein,with followedsubsequent 1.5 by26 filled cell l M, and 50 and M, T β H -mercaptoethanol)and diluted then same the in buffer to All ITC measurements were recorded at 25 °C with an with °C 25 at recorded were measurements ITC All O μ 5 DS 3 cal/s. Control experiments were performed titrating titrating performed were experiments Control cal/s. μ . Using the first frame of the CBX7+H3K9me3 NMR CBX7+H3K9me3 the of frame first the Using .

μ M) of UNC3866 were used in all selectivity assays. assays. selectivity all in used were UNC3866 of M) dpiey isd oeua dnmc simulations. dynamics molecular biased Adaptively b l of protein-peptide mix ( 3 = 0.8, = 5 μ K was implemented in GROMACS 4.5.5 (ref. 4.5.5 GROMACS in implemented was M and incubated for 30 min at room temperature. m μ values for each enzyme. Three concentrations concentrations Three enzyme. each for values g/ml in 1× assay buffer) were added and the the and added were buffer) assay 1× in g/ml c 5 μ 4 = 0.0005/ = Vn e Was n drc electrostatic direct and Waals der Van . l reaction mixture containing 3H-SAM 3H-SAM containing mixture reaction l The AlphaScreen assay (Perkin Elmer) (Perkin assay AlphaScreen The The effect of UNC3866 on the methyl μ www.perkinelmer.co M protein and 0.5 mM compound.mM 0.5 proteinand M δ t , where , 5 5 . In brief, compound plates plates . compound In brief, Supplementary Table 10 μ δ 5 l compound injection injection compound l 2 t following the princi the following = 2 fs is the molecu the is fs 2 = m ) at) substrate μ l injec l 5 1 5 ). 7 ------) .

The temperature was raised The with a was temperature raised step of 3 °C per minute from 25 °C respectively. to 96 °C nm, 590 and 465 to set were dye SYPRO-Orange the for filters added as a fluorescence probe at a dilution of 1:1,000. Excitation and emission 25 ofconcentration final a at 2 concentrationof final a at buffered in 10 mM HEPES pH were 7.5, 500 Proteins mM NaCl and(Stratagene). assayed machine in a PCR 96-well Time plate Real Mx3005p an using out was measured using a TopCount plate reader. centrifuged and removed. Another 70 Cat. # 6013611, Perkin Elmer; www.perkinelmer.com) was added to each well, 100 andcentrifugation. and Platesdried were (180 wash ethanol one washes, TCA 10% additional two by followed min, 2 for Inc.) Coulter, Beckman - X-15R (Allegra rpm 2000 at centrifuged plates (Millipore; cat.# MSFBN6B10; tic acid (TCA) was added, and the samples were mixed and transferred to filter- of reaction mixtures were incubated at 22 °C for 1 h, 100 DNMT3A/3L, and DNMT3B/3L, a filter-based assay was used. In this assay, 20 in each data set were defined as background (0%). were defined as 100% activity. In the absence of the enzyme, the CPM counts (Perkin Elmer). The CPM counts in the absence of compound for each data set for 1 a h incubated using TopCountand CPM the were measured plate reader http:// Elmer; Perkin SMP103; # (Cat. FlashPlate 96-well a to transferred then and followed by 180 Tostopguanidinehydrochlorideenzymatic M reactions, the 7.5 added,was and diluted to 500 control or 100 twice with PBS and lysed with 500 reaching ~80–90% confluency. Following trypsinization, the pellet was washed Protein concentrations were quantified using the Bradford protein assay. down and the supernatant collected and transferred to a clean Eppendorf tube. followed by incubation at RT on a rotator for 20 min. The samples were spun and (used at Benzonase 25 Samples U/ml). were incubated at 37 °C for 10 min, Cytobuster protein extraction reagent supplemented with protease inhibitors HEPES. Cells were trypsinized using 0.25% trypsin. Lysis was performed using using GIBCO DMEM/F12 (Ham) supplemented with cultured were Cells Facility.Culture Tissue Lineberger UNC the through II Version Book found Protocol at Assay PDSP NIMH the to according performed μ fit curve using GraphPad Prism 5. the no enzyme control and the IC plate reader (Excitation 680 nM / Emission 570 nM). Data were normalized to 2 h at room temperature and the assay plates were read in a BMG FS Pherastar pre-incubated AlphaScreen beads (5 ml). Detection was allowed to proceed for the of addition by detected was mark methyl product H3 histone of presence for and 1 the assay concentration) (4× h mark final antibody with anti-methyl pre-incubatedwere mg/ml) (0.08 Protein-Aconjugated andbeads acceptor mg/ml) (0.08 EDTADonor beads (30 mM) and Streptavidin NaCl mM). (800 The enzyme reaction was stopped by addition of 5 ml assay buffer containing to proceed for the requiredallowed time. The assayfinal DMSO concentrationwas was 1%. reaction enzyme the and concentration) assay final (2× tide pep substrate H3 histone and concentration) assay final (2× sulfate nium acid (100 mM), 2-oxoglutarate (2× final assay concentration), ferrous ammo reaction was initiated by addition of substrate (5 ml) consisting of enzyme The compound. of dilutions with min 15 for pre-incubated was ics) assayconcentration final (see Tween-20). In brief, 5 ml of assay atenzyme buffer containing demethylase 2× buffer assay in out (50 mM HEPES pH 7.5, 0.1% Albumin(w/v) Bovine Serum and 0.01% (v/v) carried were steps subsequent All (Labcyte). Dispenser Acoustic 550 ECHO an using performed were nl) (100 transfers compound performed in 384-well plate format using white proxiplates (Perkin Elmer) and and fluorescence readings were taken at each interval. oii rcpos ad V and receptors; -opioid Bromodomain selectivity assays. METTL21D, METTL21A, ASH1L, NSD3, NSD2, NSD1, DOT1L, For Pulldown studies. Pulldown lysis. and culture Cell functionalassays.PDSP NIMH Demethylase AlphaScreen assays. www.perkinelmer.co https μ ://pdspdb.unc.edu/pd M UNC3866 was added to an aliquot of 1,000 μ l of buffer (20 mM Tris, pH 8.0). The reactions were mixed mixed were reactions The Tris, 8.0). mM pH (20 buffer of l μ l with 20 mM Tris (pH 8)/150 mM NaCl/0.1% Tween-20 NaCl/0.1% mM Tris 8)/150 mM 20 (pH with l Cells were cultured on a T175 tissue culture flask until until flask culture tissue T175 a on cultured were Cells PC3 cells were obtained from ATCC (CRL-1435) (CRL-1435) ATCC from obtained were cells PC3 m 1A μ V , ). The reaction mixtures in Flash plates were were plates Flash in mixtures reaction The ). Supplementary TableSupplementary 11 M in 20 in M μ M. SYPRO Orange (Molecular Probes) was was Probes) (Molecular Orange SYPRO M. 1B 50 Thermal melting experiments were melting experiments carried Thermal The NK1 and NK2 receptors;NK2 and NK1 The and V determined from the nonlinear regression regression from the nonlinear determined μ The demethylase AlphaScreen assay was was assay AlphaScreen demethylase The spWeb/?site=bindin l of lysis buffer. When applicable, vehicle vehicle applicable, buffer. When l of lysis http://www.millipore.co μ μ l of MicroO was added and the CPM L volume. Compounds were added wereCompounds volume. L 2 receptor functional assays were were assays functional receptor μ l of MicroO (MicroScint-O; (MicroScint-O; MicroO of l nature CH l -glutamine -glutamine and 15 mM μ l of 10% trichloroace – g . 13 μ forassayspecif E g of total protein m MIC ). Plates were A Δ l L BIOLOGY -, -ascorbic κ -, and -, μ l), l), μ - - - - l

© 2016 Nature America, Inc. All rights reserved. 2 bate for an additional 24 h. The media was then replaced with media containing (Ham) supplemented with DMEM/F12 (GIBCO media fresh with exchanged was mixture transduction (Mission pLKO.1-puro Non-Target shRNA, SHC016V control (Sigma)). After 20 Non-targeting h, or the TRCN0000019144RNA) (Sigma) SHCLNV CBX7, (Mission CBX7 Transducing targeting Units/well) shRNA(200,000 packaged was then exchanged for 2 ml of media containing polybrene (8 of10 was then loaded into an SDS page gel and analyzed by western blotting. Twenty microliters min. 3 for °C 95 to heated then were tubes The tube. each Ten was collected. supernatant to was of buffer added microliters 6× Laemmli followedcentrifugationbyat20,000 min turefor 3 nexus gradient qPCR machine. Tubes were then incubated at room tempera at from ranging 37 temperatures °C to 73 °C in 3° in increments a Mastercycle into 12 thin-walled PCR tubes (50 tube.The tubes were incubated at RT for 30min. The lysates were aliquoted with PBS + protease inhibitors. DMSO or 30 into two tubes (600 divided then was lysate The discarded. was pellet resulting the and collected suspension was centrifuged at 20,000 liquidN suspension was subjected to 3 consecutive freeze-thaw cycles performed with was re-suspended in 1,200 re-suspendedin was (Perkin Elmer) after 15 min at room temperature in dim light. were of CellTiter-Glo reagent. plates Luminescence was read on an Cell Envision plate reader (Titertek). 384 CO 5% and °C Multidrop 37 at h 48 for a incubated using well per cells 5,000 density a of at added immediately were #26140) (GIBCO Serum Bovine Fetal 10%supplemented andwith #31053) (GIBCO red phenolwithoutMedium sage, subconfluent HEK293T/PC3 cells grown in Dulbecco’s Modified Eagle’s pas low Twentyof microlitersSC). Charleston, (Nanoscreen, device dling tissue culture plates (Corning #3707) with a Multimek automated liquid han 5 then andcontrol) (vehicle at100 system (Promega #7573). Ten point, 1:3 dilution of curves compounds starting UNC4219 on cell viability was determined using a CellTiter-Glo ATP detection slope” equation in GraphPad Prism 5. was calculated using the “log[inhibitor] vs. the normalized response – variable cells was normalized to the average cell count of DMSO-treated cells. The EC UNC4219-treated or UNC3866- of count cell the studies, dose-response For Microscopy (Array Scan High Content Analysis, Thermo Fisher #NX10002L). cells were stained with DAPI (0.05 of the Nuclei PBS. with rehydrated and s. 30 for methanol ice-cold with fixed from 100 dose-response studies, the EC fresh For UNC4219. or UNC3866 DMSO, with containing media fresh with exchanged exchanged then was were media the 3, day On UNC4219. or FBS) UNC3866 DMSO, containing media 10% with media The supplemented overnight. adhere (DMEM to allowed were Cells #3524). (Costar plates of these proteins, 3% of sample input was used. was used for western blotting of all proteins except CBX8 and HPH2. For both (diluted in 1:10,000 antibodies rescent PBST). secondary One percent of input instrument appropriateandOdyssey the LI-COR using a and detected fluo appropriateblotting the using antibody( primary western via analyzed and a gel into loaded then was sample of the microliters 30 were washed 3× with 300–500 at 4 °C. The following morning, the depleted lysate was removed and the beads to or pre-bound had UNC4195 The been 2399. was mixture rotated overnight to an Eppendorf tube containing 30 200 with 3× werewashed beads the and removed then was reagent pulldown Unbound in. temperature room rotating 30 (TBST). Pulldown reagents were bound to Streptavidin M-270 Dynabeads by nature μ CBX7 knockdown and quantitative PCR. Cellular thermal shift assay (CETSA). CellTiter-Gloluminescentviabilityassay.cell Cell proliferation assay. g/ml of g/ml was complete puromycin as and to selection until allowed incubate μ l of 1× Laemmli sample buffer and heated at 95 °C for 3 min. Fifteen min. 3 for °C 95 at heated and buffer sample Laemmli 1× of l 5 cells/well in a 6-well plate and allowed to adhere overnight. The media media The overnight. adhere to allowed and plate 6-well a in cells/well μ M final concentration were diluted to 5× final concentration in PBS PBS in concentration final 5× to diluted were concentration final M ch 2 in orderFollowingcells.in freeze-thawthe the cycle, tolyse final the μ e M to 0.4 μ mic l of beads with a 20-fold excess of pulldown reagent for 30 min at a l

biology μ μ l) each. The volume of each tube was brought up to 650 M of UNC3866 or UNC4219. At day or 0, 3 were UNC4219. M or of 6, cells UNC3866 PC3 cells were seeded at 200 cells/well into 24-well 24-well into cells/well at 200 seeded were cells PC3 l -glutamine and 15 mM HEPES) and allowed to incu μ l of PBS completePBSof protease l with inhibitors. The μ 50 μ l were added to 384-well white, clear bottomclear white, 384-well to added were l was derived from a six-point titration ranging l of TBST. The beads were re-suspended with with l of TBST.were re-suspended beads The μ μ μ l of TBST.of l transferred then lysate was The l/tube). The tubes were incubated for 3 min g/ml) g/ml) and numerated using High Content μ g l of Streptavidin M-270 Dynabeads that Dynabeads M-270 l of Streptavidin for 20 min at 4 °C. The supernatant was A PC3 cell pellet consisting of 10 2 and then lysed with 25 microliters25 with lysed then and PC3 cells were seeded at a density density at a seeded were cells PC3 μ M UNC3866 M was to UNC3866 added each The effect of UNC3866 and and UNC3866 of effect The Supplementary TableSupplementary 14 g for 20 min at 4 °C. The The °C. 4 at min 20 for μ g/ml) g/ml) and pre- 7 cells μ 50 - - - - - ) l

rti epeso ad purification. and expression Protein Orca ER digital camera. an with equipped an with microscope IX81 Olympus microscopy bright-field using Wells imaged were recommendations. manufacturer’s the to according the Senescence After 3 days, the media was removed and the cells were fixed and stained using containing media 30 fresh with replaced then was The media to overnight. and adhere allowed at cells/well of a density 100–200 microscope equipped with an Orca ER digital camera. IX81 Olympus an and Volocitysoftware using imaged were Samples PBS. At day 6, cells were fixed with ice-cold methanol for 30 s. and 30 rehydratedor withDMSO containing media fresh with exchanged were 30 or DMSO containing media fresh exchanged withthen was supplemented(DMEMFBS) media 10% Thewith overnight.adhere to allowed were Cells #3524). (Costar plates 24-well into 2900HT instrument. Oligo sequences are available upon request. Universal SYBR Green Master Mix (Rox) in a 384-well plate format on the ABI for each PCR reaction. Quantitative PCR was performed using Roche FastStart facturer instructions. The resulting cDNA was diluted 1:10, and 2 (InvitrogenSynthesisStrandmanuSupermixaccordingFirst to #1172-050) transcription reaction was performed with 500 ng RNA and the Superscript III the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The reverse extracted using the RNeasy Plus Mini Kit (Qiagen) and RNA was quantified was on RNA transduced. not were that cells control all of death the by evidence 30 ml of lysis buffer (1× PBS, 5 mM DTT, 1× EDTA free protease inhibitor inhibitor protease free EDTA 1× DTT, mM 5 PBS, (1× buffer lysis of ml 30 cut-off (Merck Millipore, Carrigtwohill Co. Cork IRL). weight molecular concentratorsUltra-153,000 Amiconconcentrated using and pooled were protein pure containing those SDS-PAGE by and purity for a flow rate of 2 ml/min collecting 3-ml fractions. Peak fractions were analyzed NJ). Protein was eluted isocratically in sizing buffer over 1.3 column volumes at 2 mM DTT, 5% using glycerol) an ATKA (GE Piscataway,Healthcare, Purifier with 1.2 column volumes of sizing buffer (25 mM Tris, pH 7.5, 250 mM NaCl, preequilibrated been had that Piscataway, NJ) Healthcare, (GE column grade IRL). Concentrated protein was loaded onto a HiLoad 26/60 Superdex 75 prep Co. Cork Carrigtwohill (Merck Millipore, cut-off weight tors molecular 3,000 desired the protein were pooled containing and concentrated to fractions 2 ml in Peak Amicon Ultra-15 concentravolumes. column 20 over mM 500 imidazole) NaCl, mM 500 7.2, pH phosphate, sodium mM (50 buffer elution column to 100% in gradient and was eluted a protein linear buffer 15 of volumes binding with washed was column The NJ). Piscataway, Healthcare, (GE AKTAan usingimidazole) FPLC NaCl,30mM mM 500 phosphate,7.2, pH sodium mM (50 buffer binding of volumes column 10 been with had preequilibrated that NJ) Piscataway, Healthcare, (GE column FF HisTrap a onto pulse s followed 20 by a a 40 s ofrest. The CT) consisting cell lysate cycle was clarified Danbury, byeach centrifugation with and loaded cycles Ultrasonics, 12 for amplitude (Branson 40% at Sonifier 450 Digital Branson a with sonicationby ice on lysed were Cells culture.of liter per Indianapolis,IN)) Diagnostics, (Roche cocktail inhibitor protease free EDTA 1× imidazole, 30 ml of lysis buffer (50 mM sodium phosphate pH 7.2, 50 mM NaCl, 30 mM by harvested were Cells centrifugation and pellets were stored atovernight. −80 °C. shaking adding continuing by and induced IPTG mM was 0.5 expression and °C 18 to lowered the was time which temperature at ~0.6–0.8 reached OD600 the until shaking with °C EMD 37 at (Novagen, cells cells by growing competent induced was expression Protein CA). Diego, San Chemicals, BL21(DE3)pLysS Rosetta into formed with GST-tags N-terminal inpGEX4Texpression vector. 1–78 of NP_004815) and CDYL2 (residues 1–75 of NP_689555) were expressed CDYL1b(residues NP_733841), of chromodomains1–101 CDY1(residuesof The vector.expression pET30 a in NP_783640) His-tag C-terminal of a with 8–62 expressed was (residues CBX7 of chromodomain The vectors. expres sion pET28 in His-tags N-terminal with expressed were NP_065700) of 8–61 of (residues CBX8 and 8–65 NP_055107) of 8–65 (residues (residues CBX6 NP_003646), CBX4 NP_005180), of 9–66 (residues CBX2 of domains Senescence-associated contrastPhase microscopy images. GST-tagged proteins were purified by re-suspending thawed cell pellets in His-taggedproteins wereby re-suspendingpurified in thawed pellets cell purification. and expression Protein β -Galactosidase Kit (#9860) from Cell Signaling Technology Technology Signaling Cell from (#9860) Kit -Galactosidase β -Gal staining. -Gal PC3 cells were seeded at 200 cells/well200 at seeded werecells PC3 All expression constructs were transwereexpression constructs All PC3 cells were seeded in a 96-well plate

μ xrsin constructs. Expression M UNC3866. On day 3, the media the 3, day On UNC3866. M doi:10.1038/nchembio.2007 μ M UNC3866 or DMSO.orUNC3866 M μ h chromo The M UNC3866. UNC3866. M μ L L was used - - - - -

© 2016 Nature America, Inc. All rights reserved. consisting of 85% reservoir solution and 15% glycerol, and flash-frozen in in flash-frozen and liquid cryoprotectant nitrogen. glycerol, a 15% in and solution soaked reservoir were 85% of Crystals consisting CBX8. HEPES, for M 0.1 glycerol 5% citrate, 7.5, sodium pH M 1.4 M 0.2 and 3350, CBX7, PEG for 20% formate CBX4, for ammonium 5.5 pH cacodylate, sodium M 0.1 NaCl, M 0.2 3350, PEG 30% CBX2, for KSCN M 0.2 3350, PEG 20% were solutions by mixing 0.5 ratio and crystallized using the sitting drop vapor diffusion method at 18–20 °C Tris, pH7.5,150mMNaCl and 1 mMDTT. purified proteins were concentrated to 10 mg/ml in a buffer containing 20 mM over a Superdex75 column (GE Healthcare, Piscataway, NJ). For crystallization, byfiltrationgelproteins proteaseThepurified remove to tag. were further the thrombinor TEV with treated and (Qiagen), resinNi-nitrilotriacetate on phy chromatogra affinity by purified °C, 15 at (Stratagene) cells RIL plus Codon in overexpressed were proteins Recombinant proteins. fusion His-tagged N-terminal encode to vectors pET28a-Lic or pET28-MHL 8–65), (residuesCBX7 (residues 8–62) CBX4 and CBX8 (residues 9–62), 8–61) were subcloned (residues into modified CBX2 of chromodomains single of andpurification followed previousa description crystallography. X-ray β 25 mM Tris, into containing a mM 150 pH NaCl, 2 buffer 7.5, exchanged mM wasProtein IRL). Cork Co. Carrigtwohill Millipore,(Merck cut-off weight protein was concentrated using Amico Ultra-15 concentrators, 3,000 molecular the and by centrifugation sample the from removed was agarose Streptavidin and incubated with streptavidin agarose to remove thrombin from the sample. any protein column that still retained FF the tag. The column HisTrap flow through was a collected either over (His-tagged proteins) passed or a GSTrap then FF column was (GST-tagged proteins) to remove reaction cleavage The concentration of 1 unit thrombin per milligram final tagged a protein at forthrombin 16 h biotinylated at with 4 °C. incubated was protein purified Briefly, CA). Diego, San Chemicals, ufacturer’sEMD (Novagen,recommendations CBX6, CDY1, CDYL1b and CDYL2 by thrombin cleavage according to man Millipore, (Merck Carrigtwohill Co. Cork IRL). cut-off weight molecular 10,000 concentrators Ultra-15 concentratedAmiconandusing containing proteinpure pooled werethose SDS-PAGEandby purity for analyzed were fractions Peak fractions. 3-ml collecting ml/min 2 ofrate flow a atvolumes column 1.3 over buffer sizing in isocratically eluted was Protein Piscataway, NJ). Healthcare, buffer (GE FPLC sizing of volumes column 1.2 (25 mM Tris, with pH 7.5, 250 mM NaCl, 2 mM preequilibrated DTT, 5% glycerol) been using an ATKA had that NJ) Piscataway, Healthcare, (GE column grade prep 200 Superdex 26/60 Carrigtwohill Co. Cork IRL). Concentrated protein was loaded onto a HiLoad Millipore,(Merck cut-off weight molecular concentrators,10,000 Ultra-15 Amicon in ml 2 to concentrated and pooled were protein desired the taining con fractions Peak volumes. column 10 overglutathione) reduced mM 10 NaCl, mM 150 7.5, pH Tris, mM (50 buffer elution 100% in eluted was tein The column was washed with 10 column volume s of binding buffer and pro AKTAHealthcare,a using(GE Piscataway, FPLC DTT) 5mM PBS, NJ).(1x buffer binding of volumes column 10 with pre-equilibrated been had that lysate was loaded onto a GSTrap FF column (GE Healthcare, Piscataway, NJ) cell Clarified proteins. His-tagged for described as sonication by ice on lysed cocktail (Roche Diagnostics, Indianapolis, IN)) per liter of culture. Cells were doi:10.1038/nchembio.2007 -mercaptoethanol before use in ITC. Crystallization. Affinity tag removal. μ L of the protein with 0.5 Purified proteinPurified was mixed with compound at a 1:2 molar

The N-terminal affinity tag was removed from CBX2, CBX2, from removed was tag affinity N-terminal The rprto o poen fr crystallization. for proteins of Preparation μ L of the reservoir solution. Reservoir 3 . The coding DNA coding The . fragments . coli E. L1 (DE3) BL21 Expression - - - -

with preliminarily refined CBX7 coordinates. merging of data in space group further refined against data from an additional crystal and, subsequently, subsequently, and, crystal the current crystal. additional was an from model data The against modes. refined “map further building” in model used “automated was ARP/WARP and improvement” coordinates. CBX7 with replacement lar molecular replacement with coordinates from PDB entry further refined against data from the current crystal. was refinement model The “automated modes. building”andment” model replacement with CBX2 coordinates. ARP/WARP was used in “map improve for molecular replacement. model deposition in the Protein Data Bank and for publication. for data to compile were used software and CCP4 IOTBX as PHENIX, as well placement parameters were analyzed on the PARVATI server. dis AnisotropicPDB_EXTRACTresidues. the MOLPROBITY of 98% model’sleast at included plot Ramachandran respective the of regions Favored respectively. in COOT, and validation refinement REFMAC and building, MOLPROBITY, peptide selected for iterativeunderwent re-models Current jLigand. with crystallographic links restraints eLBOW/Mogul, with prepared were group 4- the as well as residue lysyl modified the for replacement was performed with the program PHASER XDS with integrated were sities or APS beamline 19ID (CBX4, CBX8; wavelength 0.979 Å). Diffraction inten CBX7) (CBX2, rotatingcopperanode a at refinement collected weremodel model refinement are given in refs. of InformationSupplementary tions, literature references to selected computer programs are provided in the 60. 59. 58. 57. 56. 55. 54. 53. 52. 51.

CBX8. CBX7. CBX4. Specifically: Data collection/structure determination and refinement. 658–674 (2007). 658–674 (2013). resolution? the is (2014). 12853–12858 cells. in activity methyltransferase SETD7 (2015). 53BP1. protein, binding methyl-lysine the proteins.binding methyl-lysine of antagonistsfor assay AlphaScreen-based an interactions:protein rescaling. events. 111 simulation. molecular scalable and load-balanced, efficient, highly for algorithms McCoy,A.J. what and data my are good Murshudov,P.R.HowEvans, & G.N. W.Kabsch, XDS. Barsyte-Lovejoy,D. M.T.Perfetti, T.J.Wigle, velocity through sampling Canonical M. Parrinello, & D.Donadio, G., Bussi, infrequent of Voter,dynamics molecular A.F.acceleratedHyperdynamics: dynamics. to metadynamics From M. Tiwary,P.Parrinello, & 4: GROMACS E. Lindahl, & D. Spoel, der van Kutzner,B.,C., Hess, , 230602 (2013). 230602 , The structure was first solved in the lower-symmetry The structure of an isomorphous crystal was solved by molecular molecular by solved was crystal isomorphous an of structure The The structure of an isomorphous crystal was solved by molecu by solved was crystal isomorphous an of structure The Phys. Rev. Lett.Rev.Phys. J. Chem. Phys. Chem. J. et al. et J. Chem. Theory Comput. Theory Chem. J. et al. et CBX2. et al. et Screening for inhibitors of low-affinity epigenetic peptide- epigenetic low-affinity of inhibitorsfor Screening Phaser crystallographic software. crystallographic Phaser Acta Crystallogr. D Biol. Crystallogr.Biol. Crystallogr.D Acta Acta Crystallogr. D Biol. Crystallogr.Biol. Crystallogr.D Acta Identification of a fragment-like small molecule ligand for ligand molecule small fragment-like a Identification of Coordinates from PDB entry Coordinatesentry PDBfrom et al. et

78

( 126 , 3908–3911 (1997). 3908–3911 , R )-PFI-2 is a potent and selective inhibitor of inhibitor selective and potent a is )-PFI-2 , 014101 (2007). 014101 , P Supplementary Table 15 41212, molecular replacement was performed 5 J. Biomol. Screen. Biomol.J. 8 and merged with AIMLESS with merged and 4–

4 , 435–447 (2008). 435–447 , 1 6 ). Details on diffraction data and and data diffraction on Details ). Proc. Natl. Acad. Sci. USA Sci. Acad.Natl. Proc. ACS Chem. Biol. Chem. ACS tert

nature CH 15 -butylbenzoyl protecting protecting -butylbenzoyl J. Appl. Crystallogr.Appl.J. , 62–71 (2010). 62–71 , 3H9

66 69 6 0 . Geometry restraints 4MN . Diffraction data for data . Diffraction Due to space restric , 125–132 (2010). 125–132 , , 1204–1214 , 1 (ref.

10 E 3 MIC Phys. Rev. Lett.Rev.Phys. , 1072–1081 , (ref. P 5 3 41 setting by 41 setting 9 . Molecular .

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