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Readout of Epigenetic ModicationsDinshaw J. Patel1 and Zhanxin Wang 1, 21Structural Biology Department, Memorial Sloan-Kettering Cancer Center, New YorNew York 10021; email: [email protected] Laboratory of Cell Proliferation and Regulation Biology of the Ministry of EdBeijing Normal University, Beijing 100875, China; email: [email protected]

Annu. Rev. Biochem. 2013. 82:81118

The Annual Review of Biochemistryis online at biochem.annualreviews.org

This articles doi:10.1146/annurev-biochem-072711-165700

Copyright c 2013 by Annual Reviews. All rights reserved

Keywordshistone PTMs, multivalent readout, binding pockets, PTM cross tahistone mimics, drug discovery

Abstract This review focuses on a structure-based analysis of histone posttrantional modication (PTM) readout, where the PTMs serve as docksites for reader modules as part of larger complexes displaying matin modier and remodeling activities, with the capacity to chromatin architecture and templated processes. Individual topicsdressed include the diversity of reader-binding pocket architecturescommon principles underlying readout of methyl-lysine and metarginine marks, their unmodied counterparts, as well as acetyl-lyand phosphoserine marks. The review also discusses the impact of tivalent readout of combinations of PTMs localized at specic gensites by linked binding modules on processes ranging from gene scription to repair. Additional topics include cross talk between hisPTMs,histone mimics, epigenetic-based diseases, and drug-based thapeutic intervention. The review ends by highlighting new initiaand advances, as well as future challenges, toward the promise ohancing our structural and mechanistic understanding of the readouhistone PTMs at the nucleosomal level.

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ChIP-Seq:a technique thatcombines chromimmunoprecipit(ChIP) with maparallel DNA sequencing to idthe binding sitesDNA-associatedproteins in vivo

Histone mimicsnonhistoneposttranslationamodications (Pthat are recognireader modules recognition prinin common withhistone PTMs

Surface groovepocket: a widemore accessiblepocket, with themethyl-lysine sichain positioneda surface groovegreater tolerancmethylation stat

Cation- interactions:electrostatic

stabilization of tdiffuse positive of themethylammoniugroup throughstacking with a negative chargering of an aromresidue

readout by paired reader modules, the crosstalk between marks, and their impact onDNA-templated processes ranging from genetranscription to DNA replication, recom-bination, and repair. The contribution of histone mimics is outlined and so is the topicof dysregulation of PTM readout, which can

result in aberrant gene expression patternsand/orgenomic alterations, thereby facilitatingthe onset of disease. This in turn has stimulatedresearch on the development of epigeneticdrugs targeted to reader modules, thereby providing an approach to disease-specictherapeutic intervention. The review includesa supplemental Initiatives and Challenges sec-tion (at http://www.annualreviews.org ) that lists some recent technological developmentsand outstanding challenges in the eld. Severalexcellent reviews written earlier are availableon readout of histone PTMs (37).

METHYL-LYSINE ANDLYSINE READOUT There is no net change in charge on lysinemethylation, characterized by its relatively stable and multivalent qualities as reected inmonomethyl (Kme1), dimethyl (Kme2), andtrimethyl (Kme3) modications. Methylationof histone H3 lysine 4 (H3K4), H3K36, andH3K79 is linked to transcriptional activation, whereas methylation of H3K9, H3K27, andH4K20 is linked to repression. Of considerableinterest is the architecture of methyl-lysine-binding pockets in their respective readermodules, as well as the principles underlyingmolecular recognition of both the PTM andthe anking sequence, which account for thesequence-specic recognition. Conversely,reader modules have now been identiedthat recognize unmodied lysines, with theirinteractions perturbed on methylation. Aninformative review has been published onthe earlier structure-function aspects of thereadout of methyl-lysine (Kme) marks (8).

Royal Family Modules The Royal family modules, which include thechromodomain, Tudor, and PWWP domains,

provided the rst insight into the principlesunderlying Kme recognition by aromatic cagepockets (reviewed in Reference 9). The initialstructural studies focusedon the chromo (chro-matin organizer modier) domain of HP1 (10,11) and on Polycomb (12), known repressorsthat contribute to epigenetic silencing. Here,

we focus on the binding of the H3K9me3 mark to the HP1 chromodomain (Kd = 2.5 M);the crystal structure of the complex is shown inFigure 1 a (10). The peptide binds in anextended -strand conformation between two -strands of HP1, thereby completing anantiparallel -barrel architecture. The direc-tionality of the bound peptide reects inter-molecular contacts involving three amino acidspreceding and one amino acid following theK9me3 mark (Figure 1 b). The methyl-lysineside chain inserts into a surface groove pocket lined by three conserved aromatic amino acids, where it is stabilized by cation- interactions(Figure 1 a) (13). Mutation on any of the cage-forming aromatic amino acids results in a 20-fold lossin binding afnity.Structural studiesof additional chromodomains have demonstratedthat the sequence context anking the methyl-lysinePTMdetermines thesequence specicity of recognition (reviewed in Reference 7).

The same aromatic cage capture recogni-tion of methyl-lysine has been observed forH3K36me3 bound to the Tudor domains of PHF1( Figure 1 c ) andPHF19 (1417), thereby facilitating intrusion of the PRC2 (polycombrepressive complex 2) into active chromatin re-gions to promote gene silencing, as well asH3K36me3 binding by Kme3 cage capture tothe PWWP domain of Brf1 ( Figure 1 d ) (18).Ineach case, theH3K36me3 peptidewas bound with directionality (Figure 1 c ,d ), andmutationof the cage-lining amino acids resulted in lossof binding afnity.

PHD Fingers The plant homeodomain (PHD) nger is com-posed of a two-stranded -sheet supplementedby one or more helical segments whose crossbrace topology is stabilized by a pair of Zn ions

www.annualreviews.org Epigenetic Posttranslational Modications 83

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Q5

S10Q5

S10

H3K9me3

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R40S31

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R40

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Tudor (PHF1) PWWP (Brf1)

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c d H 3 K 3 6

m e 3

Figure 1Structures of single Royal family modules bound to methyl-lysine-containinghistone peptides. (a) The 2.4- A crystal structure of the complex of the HP1chromodomain bound to the H3(115)K9me3 peptide, Protein Data Bank (PDB) reference 1KNE. The bound K9me3-containing H3 peptide can betraced from Gln5 to Ser10 (Q5 to S10). (b) Details of the antiparallel alignment of the -strand of the bound H3K9me3-containing peptide sandwichedbetween the -strands of the HP1 chromodomain, resulting in generation of athree-stranded antiparallel -pleated sheet on formation of the complex.(c ) The 1.85- A crystal structure of the complex of the PHF1 Tudor domainbound to the H3(3140)K36me3 peptide complex (PDB: 4HCZ). The boundK36me3-containing H3 peptide can be traced from Ser31 to Arg40 (S31to R40). (d ) The 1.5- A crystal structure of the complex of the Brf1 PWWPdomain bound to the H3(2242)K36me3 peptide complex (PDB: 2X4W). Thebound K36me3-containing H3 peptide can be traced from Ser28 to Arg40 (S28to R40). Abbreviations: C, C terminus; N, N terminus.

coordinated to a Cys4-His-Cys 3 segment (19). The function of the PHD nger was poorly un-derstood until structural studies of H3K4me3recognition by the PHD ngers of BPTF (thelargest subunit of the nucleosome-remodelingfactor, NURF) (20, 21) and ING2 (inhibitorof growth 2) (22, 23) identied it as a readerof methyl-lysine marks. In this regard, earlier

studies had shown that the H3K4me3 mark associated with nucleosomes near the promoers and the 5 ends of highly transcribed gen(24, 25).

Here, we focus on readout of the H3K4memark by the PHD nger of BPTF becausstructure-function studies on the BPTF PHD-

bromo cassette have been undertaken at bothe peptide and nucleosome level. IndeeNURF-mediated ATP-dependent chromatinremodeling is directly coupled to trimethyltion at H3K4, so as to maintain HOX geexpression patterns during development (21 The BPTF PHD nger bound the H3K4me3peptide with a Kd equal to 2.7 M, with a som what weaker afnity (Kd = 5.0 M) for H3K4me2 peptide counterpart, while discriminating against monomethylated and unmoded counterparts. The crystal structure of thH3K4me3peptide-BPTF PHDngercomplexisshownin Figure2 a,b,withthepeptideboun with directionality on the surface of the PHnger through -sheet formation (Figure 2(20). The K4me3 mark is positioned througa surface groove recognition in a preformpocket composed of four aromatic residues astabilized by cation- interactions. Sequenspecicity of recognition is associated with termolecular recognition of the N terminuand the positioning of Ala1 in a hydrophobpocket, as well as by insertion of the side chaof R2 and K4me3 in the adjacent surface groovpockets separated by a tryptophan ring that part of the aromatic cage (Figure 2 a,b). Globloss of H3K4me3 resulted in loss of chromatassociation with BPTF, and the binding afnity was dramatically reduced on mutation of thcage tryptophan, consistent with developmental defects associated with this mutation (2 The preference of the BPTF PHD nger fothe H3K4me3 mark over the H3K4me2 marcould be reversed following replacement ocage tyrosine by a glutamic acid, thereby facitating a hydrogen bond between the dimethylammonium proton of Kme2 and the carboxlate of glutamic acid (Figure 2 d ,e) (26).

Parallel structure-function studies H3K4me3 binding to the PHD nger of ING2

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PHD-bromodom(PHD-bromo)cassette: theformation of a dreader module blinkage of a PHnger and abromodomain cof simultaneoustargetingmethyl-lysine anacetyl-lysine ma

have highlighted how, in response to DNA damage, recognition of H3K4me3 by the PHDnger of ING2 stabilizes the mammalian Sin3atranscription regulatorhistone deacetylase1(mSin3a-HDAC1) complex at the promotersof proliferation genes (22, 23). Furthermore,disruption of the binding interactions in the

complex impairs the ability of ING2 to induceapoptosis in vivo. Other studies have evaluatedthe consequences of somatic mutations in ING PHD ngers on solid tumors (reviewed inReference 27).

The PHD nger of RAG2, an essentialcomponent of the RAG V(D)J recombinasethat mediates antigen-receptor gene assem-bly, couples H3K4me3 readout with V(D)Jrecombination (28). Structure-function studiesof H3K4me3 binding to the PHD nger of RAG2 evaluated the effects of methylationof Arg2 (29), whereby both the depletionof the H3K4me3 mark as well as mutationsabrogating intermolecular recognition severely impacted V(D)J recombination in vivo. Strik-ingly, the Trp that is bracketed by the R2 andK4me3 marks in the RAG2 PHD-H3K4me3complex is mutated in patients with immunode-ciency syndromes, providing the rst insight into how disruption of methyl-lysine readout impacts an inherited human disease (28).

Structure-function studies have examinedthe consequence of fusing the C-terminalH3K4me3-bindingPHD nger of JARID1A (a jumonji domain lysine demethylase) to NUP98(nucleoporin 98), thereby generating potent oncoproteins that arrest hematopoietic differ-entiation and induce acute myeloid leukemia inmurine models (30). The aromatic cage of the JARID1A PHD nger is composed of two or-thogonally positioned Trp residues; the muta-tionof either Trpabrogates H3K4me3 bindingand also abolishes leukemic transformation.

Two recent comprehensive reviews haveprovided further details regarding the recog-nition of methyl-lysine marks on histone tailsby PHD nger reader modules, emphasizingthe contribution of plasticity in the recogni-tion process (reviewed in References 31 and32).

In addition to recognizing methyl-lysinemarks, the PHD nger also targets unmodiedlysines on histone tails. The unmodied H3(110) peptide binds to the PHD nger of BHC80 with a Kd equal to 30 M (33). The high-resolution structure of the H3 peptide-BHC80PHD nger complex is shown in Figure 2 f .

The peptide binds with directionality to thePHD nger with an unmodied K4 hydrogenbonded to a backbone carbonyl group andan acidic side chain, with methylation of K4 abrogating complex formation. Complexformation is stabilized by interdigitation of the side chains involving the H3 peptide (R2and K4) and the PHD nger (Met and Asp)(Figure 2 f ). Functionally, RNAi-mediatedknockdown of BHC80 results in derepressionof the LSD1 target genes, with BHC80 andLSD1 depending reciprocally on each other toassociate with chromatin (33).

The same conclusion regarding readout of unmodied lysines by reader modules hasalso been observed for the ADD domains of DNMT3A (34) and ATRX (35, 36), as wellas for additional examples that are discussedbelow.

BAH DomainsBromo-adjacent homology (BAH) domains areprotein folds associated with gene regulation,but like PHD ngers, their function was poorly understood. Here, we focus on the BAHdomain from the mammalian ORC1 (origin of replication protein 1), which has been shownto adopt a conserved -sheet core from whichemerge loop and short helical segments (37).Recently, structure-function studies have es-tablished that the BAH domain of mammalianORC1 is a reader of the H4K20me2 mark (Kd = 5.2 M) and discriminates against itsKme1 and Kme3 counterparts (38). ORC1, which mediates pre-DNA replication licensing(39),hasbeenshowntocontributebothtoORC(a six-subunit origin of replication complex) as-sociation and regulation of origin activity. Thestructure of the H4K20me2 peptide bound tomouse ORC1 is shown in Figure 2 g , where the


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peptide binds with directionality and insertsthe K20me2 side chain into an aromatic cage, with the dimethylammonium proton hydrogenbonded to a Glu side chain (Figure 2 g ,h).Functionally, mutation of the aromatic cageresidues of the ORC1 BAH domain impairsORC1 occupancy at replication origins, ORCchromatin loading, and cell-cycle progression(38). Other mutations in the ORC1 BAHdomain have been linked to Meier-Gorlinsyndrome, a form of primordial dwarsm

(40). Aromatic cage mutants of ORC1, unli wild-type ORC1, fail to rescue the growretardationof orc1morphants,andzebrash depleted of H4K20me2 marks exhibit diminishebody size (38). These studies have identiedirect link between the H4K20me2 mark anthe metazoan DNAreplication machinery, mediated by ORC1, with a primordial dwarssyndrome.

In addition to ORC1, BAH domains arobserved in a number of proteins associat

R8 H3K4H3K4R2

A1

N

H3K4me3 H3K4me2

Glu

BAH (ORC1)

G14R23 H4K20me2H4K20me2

N

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G14R23

PHD (BPTF)

PHD (BPTF)

H3K4m

e3

H 3 K 4 m

e 3 R2R2

T6

A1R2R2

Trp T r p

H3K4m

e3

H 3 K 4 m

e 3

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A1

H3K4me3

T6

PHD (BHC80)

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d e f

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GluGlu

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Cavity insertionpocket: a deepmore restrictedpocket, with themethyl-lysine ininto and buried a deep binding with size-sensitiselection capabi

with epigenetic inheritance processes (reviewedin Reference 41). The BAH domain is oftenanked by other reader modules of histonemarks, such as PHD and chromodomains, sug-gesting the potential for a combinatorial read-out. In this regard, we discuss below the com-plex of H3K9me2 peptides bound to both the

BAH and chromodomains of the plant CMT3DNA methyltransferase (42).

MBT Repeats Malignant brain tumor (MBT) repeatcontaining proteins impact on diverse pro-cesses, ranging from regulation of mitosis andtumor suppression to maintenance of cellularidentity and body pattern development (43). The early structure-function studies focused onthree MBT repeats containing L3MBTL1 (44)and two MBT repeats containing DrosophilaSCML2 (Sex comb on midleg-like 2) (45)proteins. The MBT repeat is composed of acore fold and extended arms with interdigita-tion between adjacent MBT repeats, resultingin a three-leaved propeller-like architecturefor L3MBTL1, with each color-coded MBTrepeat containing an aromatic-lined pocket located on the same face of the triangulararchitecture (Figure 3 a). Binding studies

established that L3MBTL1 exhibited littlesequence specicity for Kme-containingpeptides, although it showed a preference forlower-methylation (Kme1 and Kme2 modica-tions) states (26). Structural studies establishedthat the side chains of Kme1/2 insert deep intoaromatic pocket 2 of L3MBTL1 ( Figure 3 a,b),

and a proline ring from a proline-serine seg-ment from the C-terminal tail of an adjacent L3MBTL1 protomer inserts into a shalloweraromatic pocket 1 (Figure 3 a,c ). The methy-lation state specicity is readily understoodbecause pocket 2 is both deep and narrow,thereby serving as a size-selective lter, withthe gating and caging loops restricting accessto the larger Kme3 group. Such a cavity insertion pocket mode of methyl-lysinerecognition is distinct from the surface groovepocket recognition mode outlined in the previ-ous sections. A parallel study of the H4K20me2peptide bound to L3MBTL1 came to the sameconclusions,but the investigatorsalso proposedan unanticipated mode of peptide-mediateddimerization leading to a model of L3MBTL1-mediated chromatin compaction (46). Moreimportantly, structure-function studies havedemonstrated that L3MBTL1 compactsnucleosomal arrays containing lower lysinemethylation marks at H1K26 and H4K20,

Figure 2Structures of the PHD nger (BPTF) and BAH domain (mammalian ORC1) bound to methyl-lysine-containing histone peptides.(a) The 2.0- A crystal structure of the complex of the BPTF PHD nger bound to the H3(115)K4me3 peptide, Protein Data Ban(PDB) reference 2F6J. The PHD nger (as part of a PHD-bromo cassette) in a ribbon representation is shown ( blue), with twostabilizing bound Zn ions ( silver balls ). The bound peptide from Ala1 to Thr6 (A1 to T6) is in yellow with the trimethyl group ofshown as dotted magenta balls. The residues forming the aromatic-lined cage pocket are orange. ( b) Positioning of Arg2 (R2) anLys4me3 (K4me3) side chains in the adjacent open surface pockets (surface groove mode), separated by the indole ring of an inv Trp in the complex. The PHD nger and peptide in the complex are shown in surface and space-lling representations, respectiv(c ) Details showing the antiparallel alignment of the -strands of the bound H3K4me3-containing peptide and PHD nger, result

in generation of an antiparallel -pleated sheet on formation of the complex. Note that the positively charged N terminus is anchin its own pocket. (d ) Positioning of the K4me3 group within the aromatic-lined cage in the complex. ( e) Positioning of the K4mgroup into an engineered pocket containing a Glu residue that replaced the Tyr residue in panel d (PDB: 2RIJ). ( f ) The 1.43- Astructure of the complex of the PHD nger of BHC80 bound to the H3(110) peptide (PDB: 2PUY). The bound H3 peptide can traced from Ala1 to Ser10 (A1 to S10). ( g ) The 1.95- A crystal structure of the complex of the mouse ORC1 BAH domain boundH4(1425)K20me2 peptide (PDB: 4DOW). The bound K20me2-containing H4 peptide can be traced from Gly14 to Arg23 (G14R23). (h) Details of the alignment of the K20me2-containing H4 peptide from G14 to R23 positioned on the mouse ORC1 BAHdomain in the complex. The dimethylammonium group of H4K20 inserts into an aromatic-lined pocket in the BAH domain. Abbreviations: C, C terminus; N, N terminus.


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Interfacial compositepocket: a compositebinding pocket at theinterface of twointeracting modules, with methyl-lysinerecognition dictatedby shapecomplementarity

such that combinatorial readout of these marksby HP1 and L3MBTL1 results in chromatincondensation at Rb-regulated genes (47).

ADD Domains The ATRX-DNMT3A-DNMT3L (ADD)domain is found in the mammalian DNA methyltransferase DNMT3A/DNMT3L andin ATRX, a protein whose mutated form is as-sociated with X-linked mental retardation. Thestructure of the ADD domain, which containsadjacently positioned zinc-coordinated GATA and PHD ngers, adopts a scaffold where thetwo domains and a C-terminal -helix pack against each other to form an overall glob-ular fold (48). Half of the disease-associatedmissense mutations, many of them related topancreatic endocrine tumors (49), are clustered within the ADD domain. The structure of thecomplex between H3K9me3 peptide and the ADD domain (Kd = 0.5 M) solved at very high resolution (0.9 A) is shown inFigure 3 f , with intermolecular contacts outlined inFigure 3 g (35, 36). Molecular recognitioninvolves recognition of the N terminus,unmodied K4, and K9me3 (Figure 3 g ),readily explaining why binding is promotedby H3K9 trimethylation but inhibited by

H3K4 methylation, given that in the latter

there is no room to accommodate methylatiomarks. Unexpectedly, the K9me3 side chainpositioned in an interfacial composite pockeinvolving van der Waals contacts associat with a high degree of surface complementar(Figure 3 h), supplemented by a set of atypicarbon-oxygen hydrogen bonds with th

K9me3 group (Figure 3 i ). Functionally, bothe H3K9me3-binding pocket and ATRX syndrome mutants are defective in both H3K9mebinding and localization at pericentric hetrochromatin (36). In addition, the readout ounmodied H3K4 and H3K9me3 marks by th ADD domain of ATRX was shown to be facitated by recruitment of HP1, a reader moduthat independently recognizes the H3K9memark, resulting in tripartite recognitio with the potential for spanning neighborinnucleosomes (35).

Tandem Repeats Many reader modules occur as tandem repeaas discussedabove forMBT repeats, with repemodules showing diversearchitecture andprinciples for recognition of methyl-lysine markIn this section, we rst consider molecurecognition by a tandem chromodomain repeaandthenhighlightrecognitiondiversityfor tan

dem Tudor repeats.

Figure 3Structures of malignant brain tumor (MBT, i.e., L3MBTL1) and ADD (ATRX) domains bound to methyl-lysine-containing histonepeptides. (a) The 1.66- A crystal structure of the complex of L3MBTL1 bound to the H1(2226)K26me2 peptide, Protein Data Bank (PDB) reference 2RHI. A C-terminal peptide from an adjacent L3MBTL1 inserts proline 523 (P523) into the aromatic-lined pocket othe MBT domain 1 ( pink). The dimethylammonium group of bound K26me2 inserts into the aromatic-lined pocket of the MBTdomain 2 (blue), with the K26me2-containing H1 peptide traced from Thr24 to Lys26me2 (T24 to K26me2). A polyethylene glycol(PEG) molecule inserts into the aromatic-lined pocket of the MBT domain 3 ( green). (b) Details of insertion (cavity insertion mode) the dimethylammonium group of bound K26me2 into the aromatic-lined pocket of the MBT domain 2. ( c ) Details of insertion of theP523 from an adjacent L3MBTL1 into the aromatic-lined pocket of the MBT domain 1. This pocket is shallower than the one shown

in panel b. (d ) Chemical formula of UNC669, a pyrrolidine-containing small molecule. ( e) Details of insertion of UNC669 into thearomatic-lined pocket of MBT domain 2 based on the 2.55- A crystal structure of L3MBTL1 bound to UNC669 (PDB: 3P8H).( f ) The 1.6- A crystal structure of the complex of the ADD domain of ATRX bound to the H3(115)K9me3 peptide (PDB: 3QLA). The GATA-like and PHD ngers are green and blue, respectively. Bound Zn ions are shown ( silver balls ). The K9me3-containing H3peptide is traced from A1 to S10. ( g ) Details of the intermolecular contacts involving the bound K9me3-containing H3 peptide in thecomplex, with the bound peptide traced from Ala1 to Ser10 (A1 to S10). (h) A surface and space-lling representation of thesurface complementarity between K9me3 and the walls of the composite pocket lined by the GATA-like and PHD nger domains inthe complex. (i ) Ribbon and stick representation of K9me3 positioned to interact with the GATA-like and PHD nger domains in thecomplex. Abbreviations: C, C terminus; N, N terminus.

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Tandem chromodomains that target the H3K4me3 mark are observed in theCHD (chromo-ATP/helicase-DNA-bindingdomain) protein, which is involved in regulat-

ing ATP-dependent nucleosome assembly andmobilization at sites of transcriptional activity.In the structure of the H3K4me3 peptidebound to CHD1 (Kd = 5 M), the juxtaposed

H3K9me

T24

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a

d

b

e

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4 m e 3

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e 3

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a

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N

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A1 H 3

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3N

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b c

d e

AspAsp

H4K20me2H4K20me2

Figure 4Structures of tandem Royal family modules bound to methyl-lysine-containing histone peptides. ( a) The 2.4- A crystal structure of thcomplex of double chromo domains of human CHD1 proteins bound to the H3(119)K4me3 peptide, Protein Data Bank (PDB)reference 2B2W. Tudor domains 1 and 2 are shown ( blueand green, respectively) with the connecting helix-turn-helix linker ( pink).

The bound K4me3-containing H3 peptide can be traced from Ala1 to Gln5 (A1 to Q5). ( b) The 1.7-

A crystal structure of the compleof tandem Tudor domains of 53BP1 bound to the H4(1524)K20me2 peptide (PDB: 2IG0). Tudor domains 1 and 2 are blue andgreen. The bound K20me2-containing H4 peptide can be traced for the Arg19-Lys20me2 (R19-K20me2) step. ( c ) The 2.1- A crystalstructure of the complex of tandem Tudor domains of JMJD2A bound to the H3(110)K4me3 peptide (PDB: 2GFA). Individual Tudodomains are blue and green. The bound K4me3-containing peptide can be traced from Ala1 to Ala7 (A1 to A7). (d ) The 1.26- A crysstructure of the complex of tandem Tudor domains of Sgf29 bound to the H3(111)K4me3 peptide (PDB: 3MEA). Tudor domains 1and 2 are blue and green. The bound K4me3-containing peptide can be traced from Ala1 to Lys4me3 (A1 to K4me3). ( e) The 2.7- A crystal structure of the complex of tandem Tudor domains of the Arabidopsis thalianaSHH1 protein bound to the H3(115)K9me2peptide (PDB: 4IUT). A bound zinc ion is shown ( silver ball ). Tudor domains 1 and 2 are blue and green. The boundK9me2-containing H3 peptide can be traced from Thr3 to Ser10 (T3 to S10). Abbreviations: C, C terminus; N, N terminus.

tandem chromodomains that are linked by a helix-turn-helix motif form a continuoussurface, with the peptide positioned betweenchromodomains ( Figure 4 a) (50). The K4me3side chain inserts into a pocket formed by orthogonal alignment of a pair of Trp rings,and the side chain of R2 is stabilized throughstacking (cation- interactions) over one of these Trp rings, with mutation of either Trp

impacting binding afnity. It is noteworththat owing to the unique inserts blockincanonical binding sites, CHD1 uses atypicbinding surfaces for peptide recognition.

The diversity of methyl-lysine marks reconition by tandem Tudor domains is shown bfour examples outlined below. Initial structurfunction studies focused on the complex the H4K20me2 peptide bound to the tandem

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Tudor domains of 53BP1 (p53-binding pro-tein 1), whereby histone lysine methylationfacilitated recruitment of 53BP1 and its relo-calization to DNA double-strand breaks uponexposure to DNA-damagingagents. The struc-ture of the H4K20me2 peptide-53BP1 tandem Tudor domain complex (Kd = 19.7 M) is

shown in Figure 4 b, with the side chain of K20me2 inserting into an aromatic cage pocket (four aromatic residues and an aspartic acid)of the rst Tudor domain, whose dimensionsprevent accommodation of a Kme3 modica-tion (51). Mutation of aromatic residues liningthe pocket resulted in loss of binding afnity in vitro and impacted on the targeting of 53BP1to DNA double-strand breaks in vivo.

In contrast to 53BP1, the tandem Tudordomains of JMJD2A (a jumonji histone lysinedemethylase) adopt a domain-swapped inter-digitated topology with a two-stranded -sheet separating the two domains, as shown for thestructure of the H3K4me3 peptide-JMJD2A tandem Tudor domain complex ( Figure 4 c )(52). In this hybrid complex, the side chain of K4me3 is inserted in the aromatic cage pocket of one Tudor domain, while intermolecularcontacts involve side chains of the other Tudordomain. An extension of these studies hasshown that the tandem Tudor domains of JMJD2A bind H3K4me3 (Kd = 0.5 M)and H4K20me3 (Kd = 0.4 M) peptides inopposite orientations with the intermolecularcontacts in the structures of the two complexesidentifying single-point mutations, whereby it was possible to inhibit recognition of H4K20me3 but not H3K4me3, and vice versa(53).

Another example of the diversity of molec-ular recognition by tandem Tudor domainsemerged following structural comparisons of complexes of methyl-lysine peptides boundto Sgf29 on the one hand and the UHRF1(ubiquitin-like with PHD and Ring nger 1)domain and SHH1 (Sawadee homeodomainhomolog 1) on the other. Thus, the tandem Tudor domain of Sgf29, a component of theSAGA (Spt-Ada-Gcn5 acetyltransferase) com-plex, binds to H3K4me2/3 peptides along one

surface of a tightly packed face-to-face dimericalignment (Figure 4 d ) (54). Interestingly, theside chain of Ala1 inserts into a pocket of one Tudor domain, and the K4me2/3 side chain in-serts into an aromatic cage pocket in the other Tudor domain. Functionally, the tandem Tu-dordomains of Sgf29 targetH3K4me2/3marks

to recruit the SAGA complex to its target sitesand mediate acetylation of H3 tails.By contrast, the H3K9me2 peptide is bound

with directionality within a channel betweenthe Tudor domains, thereby interacting withboth domains, in complexes with the tandem Tudor domains of mammalian UHRF1 (55)and plant SHH1 (56). The structure of theH3K9me2 peptide-SHH1 tandem Tudordomain complex (Kd = 1.9 M) is shownin Figure 4 e, with sequence specicity at-tributed to recognition of both unmodied K4and K9me2 (in an aromatic cage pocket) (56).Functional studies show that the SHH1 proteinacts upstream of the RNA-dependent DNA methylation (RdDM) pathway in plants toenable siRNA production from the most activeRdDM targets and that SHH1 is required forRNA polymerase IV (Pol IV) occupancy of these same loci. Because key residues withinboth K4 and K9me2 pockets of SHH1 arerequired to maintain DNA methylation in vivo,the results provide insights into the mechanismunderlying SHH1 recruitment of RNA Pol IV to RdDM targets in plants (56).

Paired ModulesG9a is a euchromatin-associated methyltrans-ferase, which represses transcription followingthe writing of H3K9me1/2 marks by the C-terminal methyltransferase SET domain and,in addition, reads the same marks using N-terminal ankyrin repeats. The structure of theH3K9me2 peptide bound to the G9a ankyrinrepeats (Kd = 6 M) is shown in Figure 5 a. The bound peptide is sandwiched with speci-city solely between the -turns and helicesof the fourth and fth ankyrin repeats, withthe K9me2 side chain inserted into an aro-matic cage pocket, and the complex is stabilized


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Ankyrin (G9a)

L22

H18H18

H 4 K20 m e 1 H 4 K 2 0 m e 1

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H 3 K 9 m e 2

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e2

H 3 K 9 m

e 2

BAH-MTase-chromo (ZMET2)

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d e

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S10H3K9me3H3K9me3

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A7

H3K4me2

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c

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G13

H 3 K 9 m e 2

PHD (Pygo) + HD1 (BCL9)

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MTase

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Tudor

PHD

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Figure 5Structures of linked binding and accessory modules involved in multivalent readout of methyl-lysine-containing histone peptides.(a) The 2.99- A crystal structure of the complex of the ankyrin repeats of G9a bound to the H3(115)K9me2 peptide, Protein DataBank (PDB) reference 3B95. The aromatic-lined binding pocket bound by K9me2 is positioned between the fourth and fth ankyrinrepeats of G9a. The bound K9me2-containing H3 peptide can be traced from Ala7 to Gly13 (A7 to G13). ( b) A superposed view of crystal structures of the complex of the BAH domainmethyltransferase (MTase) domainchromodomain of maize ZMET2 bound tothe H3(132)K9me2 peptide (2.7 A, bound to BAH domain) (PDB: 4FT4) and bound to the H3(115)K9me2 peptide (3.2 A, bound chromodomain) (PDB: 4FT2). The chromodomain, BAH, and methyltransferase domains are green, blue, and beige, respectively. The

bound K9me2-containing H3 peptides can be traced from Gln5 to Thr11 (Q5 to T11) when bound to both the BAH andchromodomains. (c ) The 2.9- A crystal structure of the complex of the tandem Tudor-PHD nger cassette of UHRF1 bound to theH3(113)K9me3 peptide (PDB: 3ASK). The tandem Tudor domains are shown in the lower segment ( cyanand green), and the PHDnger is shown in the upper segment (blue). The bound H3(113)K9me3-containing peptide can be traced from Ala1 to Ser10 (A1 toS10). (d ) The 1.7- A crystal structure of the ternary complex of the Pygopus PHD nger ( blue) bound to the H3(17)K4me2 peptide inthe presence of the homology domain 1 (HD1) domain of BCL9 ( pink) (PDB: 2VPE). The bound K4me2-containing H3 peptide canbe traced from Ala1 to Ala7 (A1 to A7). (e) The 2.35- A crystal structure of the complex of MSL3 chromodomain bound to theH4(931)K20me1 peptide in the presence of duplex DNA ( surface representation) (PDB: 3OA6). The bound K20me1-containing H4peptide can be traced from His18 to Leu22 (H18 to L22). Abbreviations: C, C terminus; N, N terminus.

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by intermolecular recognition of H3 residuesfrom positions 9 to 11 (57). Although mutationsof the aromatic cage residues perturb complexformation with H3K9me2 peptide, they haveno impact on G9as SET domain-mediatedmethyltransferase activity.

A notable example of recognition of the

same methyl-lysine marks by two domains ina protein has been highlighted from structure-function studies of plant chromomethylase3 (CMT3; ZMET2 in maize). CMT3,an H3K9me2-dependent plant-specic DNA methyltransferase responsible for methylationat CpHpG sites, is composed of an N-terminalBAHdomainanda DNAmethyltransferase do-main, within which is embedded a chromo-domain. The structure of the H3K9me2 pep-tide bound to ZMET2 is shown in Figure 5 b, with H3K9me2 peptides bound with direction-ality to both chromodomain and BAH domainpositioned at the corners of a triangular archi-tecture, and complex formation is accompaniedby insertion of K9me2 side chains in aromatic-lined pockets in both domains (42). Comple-mentary functional studies demonstrated a per-fect correlation between H3K9me3 andCMT3genome locations and also established that CMT3 is stably associated with H3K9me2-containingnucleosomes.In addition, triplearo-maticcage mutants ofeither thechromodomainor BAH domain disrupted CMT3 binding tonucleosomes and further showed a completeloss of CMT3 activity in vivo (42).

The UHRF1 protein contains an N-terminal tandem Tudor domain followed by aPHD nger. The H3K9me3 peptide binds tothe isolated tandem Tudor domain with a Kdequal to 1.75 M and to the isolated PHD n-gerwithaKdequalto1.47 M. By contrast, theH3K9me3 peptide binds to the UHRF1 tan-demTudor-PHDcassette with a 1:1stoichiom-etry and with a Kd equal to 0.37 M, indicativeof multivalent readout of dual marks by linkedbinding modules. The structure of H3K9me3peptide bound to the UHRF1 tandem Tudor-PHD cassette is shown in Figure 5 c (58, 59). There is minimal contact between the PHDnger and the tandem Tudor domains, with the

17-residue linkerpacked betweenthePHDand Tudor domains. The compactly folded boundH3K9me3-containing peptide is positioned within a central hole in the protein scaffold.Key intermolecular contacts involve unmodiedH3R2 recognition by the PHD nger througha network of intermolecular hydrogen bonds,

with methylation at this position resulting inreduced binding afnity, whereas H3K9me3recognition involves insertion into an aro-matic cage pocket of the rst Tudor domain(Figure 5 c ). Interestingly, phosphorylation of a serine within the UHRF1 linker segment resulted in a 30-fold loss in binding afnity,suggesting that such a modication could act as a switch between the potential regulatory pathways utilized by UHRF1 during mainte-nance of DNA methylation and transcriptionalrepression (58).

Accessory Modules Pygopus , together with its cofactor BCL9, reg-ulates -catenin-mediated transcription and iscritical for Wnt signaling during development. The interacting components of this complex arethe PHD nger of Pygopus and homology do-main 1 (HD1) of BCL9. Structural studies havebeen undertakenof the binary complexof Pygo- pus PHD nger and HD1 (60), as well as of itsternary complexwith theadded H3K4me2 pep-tide (60,61).The H3K4me2 markand HD1 arepositioned on opposite sides of the PHD ngerin the ternary complex (Figure 5 d ), with ef-cient H3K4me2 mark recognition requiringas-sociation of the Pygopus PHD nger with HD1,suggesting that HD1 binding to the PHD n-ger triggers an allosteric transition, thereby fa-cilitating optimal recognition of the H3K4me2mark through aromatic cage pocket capture.

MSL3, a subunit of the MSL complex, was shown to be necessary for dosage com-pensation on Drosophilamale X chromosomes(62). Recently, it has been shown that the MSL3 chromodomain targets lower methy-lation states of H4K20 only in the presenceof guanine-adenine-rich (GA-rich) DNA (63). A preassembled complex of MSL3


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chromodomain and GA-rich DNA that boundH4K20me1 peptide with a Kd equal to15 M discriminates against unmodied andtrimethylation states. The structure of theternary complex formed by the MSL3 chro-modomain, GA-rich DNA, and H4K20me1peptide is shown in Figure 5 e with the chro-

modomain packed against the minor groove of the DNA and the K20me1 side chain insertedinto an adjacent aromatic amino acidlinedpocket (63). It was speculated that the potentialcorecognition of the H4K20me1 mark andthe DNA of two adjacent nucleosomes couldcontribute to the in vivo targeting of the MSLcomplex.

METHYL-ARGININE AND ARGININE READOUT

There is also no net change in charge on argi-nine methylation.This mark is characterizedby its relatively stable and multivalent qualities asreected in monomethyl (Rme1), symmetricaldimethyl (Rme2s), and asymmetrical dimethyl(Rme2a) modications, with their major sitesat H3R2 and H4R3. Of considerable interest is the architecture of methyl-arginine-bindingpockets in their respective reader modules,as well as the principles underlying molec-ular recognition of both the PTM and the

anking sequences that account for their

sequence-specic recognition. Converselreader modules have now been identied threcognize unmodied arginines with theinteractions perturbed on methylation. An informative and comprehensive review has bepublished on the structure-function aspects othe readout of methyl-arginine PTMs (64).

Expanded Tudor DomainsBoth canonical (65) and expanded Tud(6668) domains are involved in readout methyl-arginine marks. Because there is structural study of methyl-arginine-containinhistone targets, we discuss, below, exampof methyl-arginine recognition on nonhistone targets. Several germ lineexpand Tudor-containing proteins contain repeatof 60amino acid canonical Tudor domaianked by N- and C-terminal extension segments. Such expanded Tudor domains targemethyl-arginine marks located within the Nterminus of PIWI proteins, thereby contributing to the regulation of the PIWI-interactingRNA biogenesis pathway in the germ line asilencing transposable elements in the gerline during early development. The structurof a symmetrically dimethylated R15me2containing an N-terminal PIWI peptide boundto the extended Tudor domain of SND

(staphylococcal nuclease domain-containi

Figure 6Structures of expanded and paired modules bound to methyl-arginine and unmodied arginine-containing peptides. ( a) The 2.8- A crystal structure of the complex of the extended Tudor module of SND1 bound to R15me2s-containing N-terminal PIWI peptide,Protein Data Bank (PDB) reference 3NTI. The core fold of the Tudor domain is shown in blue, and the extensions are shown in green The R15me2s-containing N-terminal PIWI peptide can be traced from Arg11 to Arg17 (R11 to R17). ( b) Positioning of R15me2s inthe aromatic-lined cage pocket of the Tudor domain in the SND1 complex. ( c ) The 1.8- A crystal structure of the complex of the PHnger of UHRF1 bound to the H3(19) peptide (PDB: 3SOU). The bound H3 peptide can be traced from Ala1 to Arg8 (A1 to R8).Note the network of hydrogen bonds involving Arg2 (R2) and residues on the PHD nger. ( d ) The 1.5- A crystal structure of thecomplex of the WD40 motif of WDR5 bound to the H3(19)K4me2 peptide. The bound K4me2-containing peptide can be traced

from Ala1 to Arg8 (A1 to R8) (PDB: 2H6N). (e) Intermolecular hydrogen-bonding interactions stabilizing insertion of Arg2 (R2) intothe central channel of the WD40 motif in the H3(19)K4me2-WDR5 complex. ( f ) Insertion of symmetrical Arg2me2 (R2me2s) intthe central channel of the WD40 motif in the H3(115)R2me2s-WDR5 complex solved at 1.9 A (PDB: 4A7J). ( g ) The 3.18- A crystastructure of the complex of the chromodomains and ankyrin repeats of Arabidopsis thalianacpSRP43 bound to an Arg-Arg-Lys-Arg(RRKR)-containing peptide (PDB: 3UI2). The side chains of Arg536 (R536) and Arg537 (R537) of the bound RRKR-containingpeptide from Gln528 to Lys540 (Q528-K540) are positioned in adjacent pockets at the interface between the fourth ankyrin repeat anthe second chromodomain in the complex. ( h) Position of Arg536 (R536) of the RRKR-containing peptide within an aromatic-linedcage pocket in the complex. (i ) Positioning of Arg537 (R537) of the RRKR-containing peptide in a pocket lined by a Trp and twoacidic side chains in the complex. Abbreviation: C, C terminus.

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1) is shown in Figure 6 a, with the N- andC-terminal extensions combining to adopt anoligonucleotide- and oligosaccharide-bindingfold (67). The peptide binds with direction-ality within a wide and negatively charged

groove and in so doing inserts the planardimethyl-guanidinium group of R15me2s intoan aromatic-lined cage pocket (Figure 6 b), while making intermolecular contacts withresidues anking the R15me2s mark. Both

N

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H3K4me2H3K4me2

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T6 T6H3R2me2sH3R2me2s

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PHD (UHRF1)

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mutations in the aromatic residues and anasparagine lining the aromatic cage, along withtruncations of the canonical Tudor domain andanking N- and C-terminal extensions, impact on binding afnity. The same structure wasobserved for the complex of the R4me2s PIWIpeptide with the extended Tudor domain

of SND1, which had the binding afnity (Kd = 10 M) dropping by twofold for theR4me1 mark, fourfold for the R4me2a mark,and ninefold for the unmodied R4 mark.Functional studies have identied an intricateinterplay between the writers andreaders of theRme2s mark and the mark itself, impacting ontheregulation of transposon silencingandgermcell development (reviewed in Reference 64).

PHD Finger DomainsSimilar to recognition of unmodied lysines,reader modules have been identied that rec-ognize unmodied arginines on histone tails.It has been shown recently that the abil-ity of UHRF1 to suppress the expression of target genes is dependent on the PHD n-ger of UHRF1 binding to unmodied H3R2,thereby linking this recognitionevent involvingUHRF1 to the regulation of euchromatic geneexpression (69). Thestructuralbasis underlyingthis functional observation emerged on solv-ing the structure of H3(19) peptide bound tothe PHD nger of UHRF1 (Kd = 2.1 M), whereby the guanidinium group of R2 partici-pates in an extensive intermolecular hydrogen-bonding network ( Figure 6 c ), with methylationof H3R2, but not H3K4 or H3K9, disruptingcomplex formation (69).

WD40 Repeat Domains The WD40 repeat protein WDR5 withits toroidal -propeller fold is a commoncomponent of the SET1 family of histonemethyltransferases, with the latter playing acritical role duringproper HOX gene activationand vertebrate gene development. It has beenproposed that WDR5 associates with and isessential for H3K4 methylation and vertebrate

development (70). To this end, structures havbeen determined of the complex of H3K4mepeptide bound to WDR5 ( Figure 6 d ) (717Somewhat unexpectedly, it was the side chaof unmodied R2 rather than K4me2 thinserted into the narrow central channel (cavitinsertion mode) of the toroidal -propeller fo

of WDR5,whereby its alignment was stabilizeby stacking with staggered phenylalanine sichains and was oriented through direct an water-mediated hydrogen bonds ( Figure 6 More recently, the structure of the compleH3R2me2s peptide bound to WDR5 demonstrated that the dimethyl-guanidinium grouof symmetrical R2me2s, but not R2me2a, alinserts into the central channel of WDR(Figure 6 f ) (75). Comparison of the insertiocavities in the H3R2 (Figure 6 e) and tH3R2me2s (Figure 6 f ) complexes indicathat the methyls of the dimethyl-guanidiniumgroup are accommodated following displacment of one of the water molecules togeth with a protrusion of the dimethylated sichain toward the phenylalanine ring, therebforming enhanced hydrophobic interactionFunctionally, the H3R2me2s mark retaingenes in a poised state in euchromatin ftranscriptional activation following transfrom the cell cycle and differentiation (75).

Future challenges will be to identify potetial readers of Rme2a and Rme1 marks and understand the principles underlying optimselectivity as a function of the methylation staof arginine.

Chromodomains A very interesting mode of unmodiarginine recognition on a nonhistone protein has been recently reported betweethe cpSRP43( CD3) protein (composed ankyrin repeats anked by chromodomainbound to a peptide containing the RRKR motfrom cpsSRP54 associated with the chloroplasignal recognition particle in plants (76). Thstructure of this complex (Kd = 6.4 M)shown in Figure 6 g with the peptide bindinat the interface between the fourth ankyr

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repeat and the second chromodomain, suchthat the peptide is positioned through -sheet formation within a hydrophobic bindinggroove of the second chromodomain. The sidechain of unmodied Arg536 is inserted intoa threearomatic residuelined cage pocket (Figure 6 h), and the side chain of the adjacent

unmodied Arg537 is anchored throughhydrogen bonding in a pocket containing onearomatic and two basic residues (Figure 6 i ), with replacement of either arginine on thepeptide or aromatic amino acids lining thepockets, abolishing both complex formationand functional readout. This insightful contri-bution introduces the novel concept of readout of adjacent unmodied arginines by a twinnedaromatic cage and denes an unexpectednonnuclear function for chromodomains inposttranslational targeting.

ACETYL-LYSINE READOUT Interest in histone acetylation came to the fore-front with the realization that lysine acetyla-tion impacts both the topology of nucleosomesand internucleosome interactions responsiblefor the higher-order architecture of chromatin.Furthermore, the linkage of histone acetylationand transcription was established early on thebasis of the opposing activities of acetyltrans-ferases and deacetylases involved in transcrip-tional regulation. We outline below the resultsemerging from structure-function studies onreadout of histone acetyl-lysine marks, starting with the initial key discoveries and moving onto more recent contributions, including the ex-citing development of inhibitors against acetyl-lysine pockets (see Inhibitors of Kac-BindingBromodomains, below). An overview of acetyl-lysine readout can be found in a comprehensivereview on the subject (77).

Bromodomains As part of an effort toward understand-ing the molecular principles underlyingacetyl-lysine recognition, attention turned tobromodomains (bromo), which are a known

component of certain histone acetyltrans-ferases and chromatin remodeling complexesas well as helicases, histone methyltransferases,and transcriptional coactivator complexes.In a pace-setting contribution to the eld of epigenetic readout of histone PTMs, NMR studies determined the solution structure of the

bromodomain of the histone acetyltransferasecoactivator P/CAF (p300/CBP-associatedfactor) in the free state and bound to an H4peptide acetylated at K8 (H4K8ac) (78). TheP/CAF bromodomain adopted a left-handedantiparallel four-helix bundle with long ZA (connecting helices Z and A) and BC(connecting B and C) loops packed against each other to form a hydrophobic pocket at one end of the scaffold. The H4K8ac peptidebound the P/CAF bromodomain with modest afnity (Kd = 346 M), with the observedNMR chemical shift changes characteristicof insertion of the acetylated lysine into thehydrophobic binding pocket formed betweenthe four -helices and extended by the ZA and BC loops. Mutation of the hydrophobicresidues in the pocket, especially aromaticones, resulted in a loss of binding afnity.

Next, in an equally important contribution,a high-resolution (1.9- A) crystal structure of the complex of H4K16ac-containing peptidebound to the bromodomain of the histoneacetyltransferase Gcn5p identied the role of a key asparagine side chain and bound watermolecules in the binding pocket to acetyl-lysine recognition, as well as the contributionof anking amino acids to sequence-specicrecognition (79). The structureof theH4K16acpeptide-Gcn5p bromodomain complex isshown in Figure 7 a, with details of inter-molecular contacts dening context-dependent readout in Figure 7 b and the binding pocket in Figure 7 c . The overall structure andpositioning of the binding pocket (Figure 7 a)are common to both P/CAF and Gcn5pbromodomains. The peptide is positioned withdirectionality in a shallow depression betweenthe ZA and BC loops, such that the channelis lined with hydrophobic and charged aminoacids at opposite ends and with the complex


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T32 T32

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H4K16acH4K16ac

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R17R17luGlu

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Bromo (TRIM33) Bromo (TRIM33)Bromo (TRIM24)

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Figure 7Structures of bromodomains bound to acetyl-lysine-containing histone peptides. ( a) The 1.87- A structure the bromodomain of Gcn5p bound to the H4(1529)K16ac peptide, Protein Data Bank (PDB) reference1E6I. The peptide can be traced from Ala15 to Arg19 (A15 to R19). (b) Details of intermolecular recognitiobetween the peptide backbone and loops that project from the bromodomain scaffold. ( c ) Details of intermolecular contacts in the binding pocket of the Gcn5p bromodomain-H4(1529)K16ac complex. Theacetyl-lysine side chain inserts into a hydrophobic pocket and is anchored through hydrogen bonding withan asparagine side chain. Also note the extensive use of water-mediated intermolecular recognition in thepocket. (d ) The 1.9- A structure of the bromodomain of TRIM24 bound to the H3(1332)K23ac peptide(PDB: 3O34). The chain can be traced from Thr22 to Thr32 (T22 to T32), with the side chain of K23acinvolved in canonical recognition within the binding pocket. Interactions involving the side chain of Arg26(R26) of the bound peptide are also show in this panel. (e) The 2.7- A structure of the bromodomain of TRIM33 bound to the H3(122)K9me3K14acK18ac peptide (PDB: 3U5O). The chain can be traced from Ala1 to Leu20 (A1 to L20), with the segment from Arg17 to Leu20 (R17 to L20) shown in this panel. Thside chain of K18ac is involved in atypical recognition, in that it is not hydrogen bonded to an asparagine.Interactions involving the side chain of Arg17 (R17) of the bound peptide are also shown in this panel.( f ) Details of intermolecular contacts (using the structure at 1.95- A resolution) in the binding pocket of th TRIM33 bromodomain-H3(120)K9me3K14ac complex (PDB: 3U5N). The acetyl-lysine side chain insertinto a hydrophobic pocket and is anchored solely through water-mediated interactions. The extension of th -helix B ( B) in TRIM33 results in rotation of the asparagine residue away from the pocket such that it ino longer involved in recognition of the Kac side chain. Abbreviations: A, -C, -helix A and C; C, Cterminus; Kac, acetyl-lysine.

stabilized by intermolecular hydrogen bonds(Figure 7 b). The long acetyl-lysine side chainis inserted deep into the central pocket insidethe bundle of four helices; the hydrophobic

wall is required for stabilization of the aliphaside chain. Unique interactions in the bindinpocket involve a key intermolecular hydrgen bond between the amide nitrogen of

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conserved asparagine with the oxygen of theacetyl group of acetyl-lysine (Figure 7 c ). Inaddition, intermolecular contacts at the baseof the binding pocket are mediated through anetwork of water molecules hydrogen bondedto the backbone carbonyls lining the walls of the pocket (Figure 7 c ). The structure elegantly

explains why the deep binding cleft targetsacetyl-lysine in preference to the unmodiedamino acid and also explains the role of peptidesequence in dictating sequence-specic recog-nition on the basis of the diverse electrostaticsurfaces spanning either side of the entranceto the pocket.

Since the earliest structural studies of bro-modomains (78, 79), there has been an out-pouring of structures of histone peptidebromodomain complexes, culminating recently in a large-scale comprehensive structural study of acetyl-lysine recognition by the human bro-modomain family (80). The binding afnitiesof acetyl-lysine (Kac) marks for bromodomainsspan a range from Kd 3 M to 200 M. One of the insights that emerged from this study high-lighted the contributions of anking PTMs foracetyl-lysine recognition.

Tandem Bromodomains Many bromodomain-containing proteins con-tain this reader module in tandem repeat withthe capacity to binda pair of acetyl-lysine marksseparated by appropriate spacing, thereby in-creasing the binding afnity as a result of bi- valent readout. Examples include the tandembromodomains of TAF1, the largest subunit of TFIID, where the bromodomains are alignedin an independent side-by-side arrangement, with Kac-binding pockets separated by approx-imately 25 A (81). The tandem bromodomainsof TAF1 bind di- and tetra-acetylated H4 pep-tides (acetylated with combinations of K5, K8,K12, and K16) with higher afnity than theirmonoacetylated counterparts.

By contrast, the tandem bromodomainsof yeast Rsc4p interact extensively with eachother to form a single structural unit, where thetwo Kac-binding pockets are oriented on the

same face and separated by a shortened (20- A)separation (82). In this case, the second bromo-domain bound the H3K14ac mark, withbinding disrupted on phosphorylation at nearby Ser10 on histone H3 (H3S10). Gcn5p-mediated acetylation of a fused construct linking the H3 tail to the bromodomains of

Rsc4p resulted in formation of H3K14ac andRsc4pK45ac marks. Interestingly, insertionof Rsc4pK45ac mark into the binding pocket of the rst bromodomain inhibited insertion of the H3K14ac mark into the binding pocket of the second bromodomain, thereby identifyingan autoregulatory mechanism to control Rsc4pactivity (82).

At the other extreme, the Polybromo (PB1)protein involved in chromatin remodeling con-tains six bromodomains in tandem, with indi-

vidual bromodomains exhibiting distinct pref-erences for specic acetyl-lysine marks (83).

TRIM Family BromodomainsHere, we expand on an unanticipated result whereby the PHD-bromo cassette of tripartitemotif 24 (TRIM24) reads out H3 peptidescontaining unmodied K4 and K23ac (84), whereas the PHD-bromo cassette of TRIM33reads out H3 peptides containing unmodiedK4, K9me3, and K18ac (85). Structural studiesestablish that K23ac recognition by the bro-modomain of TRIM24 involves both hydrogenbonding with the conserved asparagine and thenetwork of water-mediated interactions, withsequence specicity dened by the intermolec-ular hydrogen bonds with the anking residue Arg26 on the peptide, thereby accounting forits specicity for H3K23ac (Figure 7 d ) (84).By contrast, in the case of TRIM33, there isan insert in the sequence of the bromodomaincompared with TRIM24, which extends thelength of -helix B such that complex forma-tion favors a different sequence context. As aresult, K18ac inserts into the binding pocket with an intermolecular hydrogen bond to aanking residue (Arg17), stabilizing complexformation, and accounting for the specicity of TRIM33 for H3K18ac ( Figure 7 e) (85).


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More importantly, extension of the -helixB rotates the conserved asparagine out of thepocket, so that it no longer participates inhydrogen-bonding recognition of the acetyl-lysine (Figure 7 f ). Nevertheless, recognition isstill mediated by a network of water moleculesthat now spreads throughout the entire

binding pocket (Figure 7 f ), emphasizing theimportance of water-mediated recognition andstabilization to acetyl-lysine readout.

Tandem PHD Fingers The DPF3 protein, which contains C-terminaltandem PHD ngers, plays a key role in heart and muscle development. DPF3, associated with the BAF chromatin remodeling complex, was shown to bind acetylated histones H3 and

H4, with knockdown of DPF3 resulting incardiac, ventricular, and muscle ber defects(86). The potential of tandem PHD ngersof DPF3 for targeting acetyl-lysine marks wasunanticipated, and the molecular basis under-lying recognition emerged from the NMR so-lution structure of the DPF3 tandem PHD n-gers bound to theH3K14ac-containingpeptide(Kd = 0.5 M) (87). The two PHD ngers arepacked against each other to form a compact functional unit, thereby generating an extended

surface groove sharedbetweenthe two domainsfor peptide recognition ( Figure 8 a). The Nterminus of the H3K14ac-containing peptide

binds through -sheet formation to the seconPHDngerthrough recognition of unmodiedlysine (K4 and K9) and arginine (R2) residu while the side chain of K14ac inserts intohydrophobic pocket in the rst PHD nge with the acetylamide positioned within hydrgen bond distance to the side chains of an aspa

ticacid anda zinc-coordinatingcysteine residu(Figure 8 b). Complex formation, which is facitated by acetylation at H3K14, is inhibited methylation at H3K4. Notably, the H3K14acbinding pocket on the rst PHD nger is ondifferent surface of the PHD nger from binding pockets that target methyl-lysine marks.

Tandem PH Domains The H3K56ac mark has been implicated

the regulation of nucleosome assembly durinDNA replication and repair, as well as whthe nucleosome disassembles during gene trascription. The histone chaperone Rtt106 (regulator of Ty1 transposition 106), which contains tandem plextrin homology (PH) domaintoward its C terminus, contributes to the deposition of newly synthesized H3K56ac-labelH3-H4 tetramer on replicating DNA. A NMR-based chemical shift perturbation study idetied the tandem PH domains of Rtt106 a

readers of the H3K56ac mark with bindinafnity markedly increased on acetylation K56 (88). In the absence of a structure of t

Figure 8Structures of tandem PHD ngers (DPF3b) and bromodomains (BET family) bound to acetyl-lysine-containing histone peptides.(a) The NMR-based solution structure of the tandem PHD ngers of DPF3b bound to the H3(120)K14ac peptide, Protein Data Bank(PDB) reference 2KWJ. The rst and second PHD ngers are shown as green and blue, respectively. The H3 peptide was traced from Ala1 to Leu20 (A1 to L20). Interactions involving the side chains of Arg2 (R2), Lys9 (K9), and Thr11 (T11) of the bound peptide arshow in this panel. (b) Details of the alignment and interactions involving the side chain of K14ac with the rst PHD nger in theDPF3b tandem PHD nger-H3(120)K14ac peptide complex. ( c ) The 2.37- A structure of the rst bromodomain of Brdt bound to th

H4(120)K5acK8ac peptide (PDB: 2WP2). The bound peptide can be traced from Gly4 to Leu10 (G4 to L10). The side chain of K5ainserts into the Kac-binding pocket, whereas the side chain of K8ac is on the outer rim of the pocket and buttresses the insertion of K5ac into the pocket. (d ) Chemical formula of (+ )-JQ1. (e) Chemical formula of I-BET. ( f ) Chemical formula of I-BET151.( g ) Chemical formula of MS7972. (h) The 1.6- A structure of the rst bromodomain of BRD4 bound to the small-molecule inhibitorI-BET (PDB: 3P5O). I-BET inserts into the Kac-binding pocket and is anchored in place by shape complementarity and direct and water-mediated intermolecular contacts. ( i ) The 1.5- A structure of the rst bromodomain of BRD4 bound to the small-moleculeinhibitor I-BET151 (PDB: 3ZYU). I-BET151 inserts into the Kac-binding pocket and is anchored in place by shape complementarityand direct and water-mediated intermolecular contacts. ( j ) The NMR-based solution structure of insertion of the small-moleculeinhibitor MS7972 into the acetyl-lysine-binding pocket of the bromodomain of CBP. Abbreviation: N, N terminus.

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complex, a dimeric model has been proposed, whereby a pair of tandem PH domains withinthe Rtt106 dimer interacts with each of the twoH3K56ac sites in the H3-H4 tetramer. Muta-tional studies established that the interactionbetween H3K56ac and Rtt106 tandem PH do-mains contributes to gene silencing and to theDNA damage response.

BET-Family BromodomainsBromodomain and extraterminal (BET)family proteins play a role in transcriptionalelongation and cell-cycle progression. Onefamily member, mouse Brdt, contains twobromodomains separated by a long linker andtargets multiacetylated histones on H3 and

I-BET

Asn

Bromo1 (Brdt) (+)-JQ1 I-BET

Bromo1 (BRD4) + I-BET

G4 L10 H 4

K 5 a c H 4 K 8 a c

Asn

I-BET151

Bromo1 (BRD4) + I-BET151

I-BET151

H3K14ac

AspAspA1

L20

N

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R2

K4

K9

H 3 K 1 4 a c

T11CysCys

a b

c d e f

h i

Bromo (CBP) + MS7972

MS7972

MS

g

j

Tandem PHD (DPF3b) Tandem PHD (DPF3b)

NN

OO

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S

Cl

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NHN

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H4. The isolated bromodomain 1 of Brdt bound the H4K5acK8ac peptide with a Kdequal to 21.9 M and a 1:1 stoichiometry of bromodomain to peptide. The structure of the complex is shown in Figure 8 c , with bothacetyl-lysines pointing in the direction of anotherwise widened pocket (89). The side chain

of K5ac inserts deeply into the pocket whereit hydrogen bonds with an asparagine residueadopting the canonical recognition mode, andthe side chain of K8ac is positioned near theentrance of the pocket, stabilized throughhydrophobic contacts, where it buttresses theinserted K5ac side chain. The structure alsoexplains the requirement for a two-glycineresidue separation between K5ac and K8ac within the H4 sequence on the basis of stericprinciples, as well as why the K8 position has tobe acetylated rather than a charged unmodiedlysine on the basis of electrostatic principles.Functionally, the H4K5acK8ac ligand-bindingcapacity of bromodomain 1 of Brdt is requiredfor compaction of hyperacetylated chromatin. This system identies the Brdt bromodomain1 as a reader module that mediates positivecooperativity by engaging a pair of Kac marksin a common expanded binding pocket (89).

PHOSPHOSERINERECOGNITION Histone phosphorylationat serines, threonines,and tyrosines has been shown to link chromatinfunction with a diverse range of signal trans-duction pathways (reviewed in References 90and 91). Replacement of a hydroxyl by a phos-phate introduces a bulky andnegatively chargedmoiety, thereby enhancing the ion-pairing andhydrogen-bonding capabilities following phos-phorylation of serine, threonine, and tyrosineresidues. Functionally, histone phosphoryla-

tion impacts on diverse processes ranging fromtranscriptional regulation to DNA damage re-pair and cell-cycle control. Histone phosphorylation sites are located not only on canoni-cal histones but also on histone variants suchas H2AX, with phosphorylation at Ser139 and Tyr142 toward the C terminus.

14-3-3 Proteins Mammalian 14-3-3 proteins aphosphoserine-binding modules that regulatsignal transduction, chromosome condenstion, and apoptotic cell death. The 14-3-3isoform targets both H3S10ph (Kd = 78 Mand H3S28ph (Kd = 23 M) marks, bo

of which contain a common Ala-Arg-Lys-Ssequence motif. In the crystal structure 14-3-3 bound to the H3S10ph-containinpeptides (92), the partially extended bkinked H3S10ph-containing peptide wpositioned within a V-shaped binding channof the all -helical 14-3-3 protein scaffo(Figure 9 a shows dimeric arrangement this complex), with the phosphate of S10panchored through multiple intermoleculahydrogen bonds, involving the hydroxyl grou

of a tyrosine and the guanidinium grouof two Arg side chains, thereby facilitatcharge neutralization ( Figure 9 b). In additioH3R8 is also anchored in the complex throughydrogen bonding to S10ph and a Glu sidchain (Figure 9 b), thereby accounting for thsequence specicity of the recognition.

A fascinating example of phosphoserirecognition in plant biology emerged frostudies of 14-3-3 proteins that act as intracelular receptors for rice Hd3a origen, a protei

synthesized in the leaf and transported to thshoot apex, where it induces owering. Strutural studies were undertaken on the origeactivator complex, composed of the rice ogen analog Hd3a, the 14-3-3 protein GF14cand a phosphoserine peptide from Ory sativa transcription factor OsFD1, interactinpartners identied from yeast two-hybrstudies (93). The structure of one symmetrhalf of the dimeric topology is shown in Figur9c , with GF14c forming a stable compl

simultaneously with Hd3a and OsFD1, whimediating indirect binding between Hd3a anOsFD1. The S192ph-containing OsFD1 peptide is positioned within the GF14c 14-3-3 domain and anchored in place through hydrogebonding with arginine and tyrosine side chai(Figure 9 d ). This study highlights the role

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A7

R8

H3S10ph

K14A7

K14

H3S10ph

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14-3-3 (14-3-3)

A7 K14

R8

H3S10ph

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FOBISIN101FOBISIN101

FOBISIN101

14-3-3 (GF14c) + Hd3a

R189

S192phS192phF195

14-3-3 (GF14c) 14-3-3 (14-3-3)

b

d

e

f

N

C

N

C

R189F195F195

S192p hS 1 9 2 p h

Hd3a

a

cGF14c

GluTyr

N

O H

HOO P

O

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ONaN

N

OsFD1

Figure 9Structures of 14-3-3 proteins bound to phosphoserine marks and inhibitors. ( a) The 2.0- A crystal structure of 14-3-3 bound toH3(714)S10ph peptide, Protein Data Bank (PDB) reference 2C1N. The 14-3-3 protein in the complex adopts a symmetrical dalignment, with individual monomers shown in blue and green. The bound peptide can be traced from Ala7 to Lys14 (A7 to K14(b) Details of the alignment and hydrogen-bonding interactions involving the side chain of S10ph of the peptide in theH3(714)S10ph-14-3-3 complex. Also, note the pair of hydrogen bonds between the guanidinium group of Arg8 (R8) of the peand the carboxyl group of a Glu residue of the protein. (c ) The 2.4- A crystal structure of the ternary complex between Hd3a ( piGF14c (blue), and the bound OsFD1(187195)S192ph peptide ( yellow) (PDB: 3AXY). The side chain can be traced from Arg18Phe195 (R189 to F195). (d ) Details of the alignment and hydrogen-bonding interactions involving the side chain of phosphorylaSer192 (S192ph) and the peptide in the ternary complex. (e) The chemical formula of FOBISIN101. ( f ) Details of interactions ophosphate group of the cleaved pyridoxal-phosphate moiety of FOBISIN101 with 14-3-3 in the FOBISIN101-14-3-3 comple Abbreviations: C, C terminus; N, N terminus.

14-3-3 proteins as intracellular receptors fororigen in shoot apical cells, with new oppor-tunities to manipulate the owering process.

Tandem BRCT Domains Tandem breast cancer susceptibility (BRCT)domains are readers of phosphorylation marksin response to DNA double-strand breaksas was rst demonstrated for recognition of Ser139ph of the histone variant H2A.X by

MDC1 (mediator of DNA damage checkpoint protein 1) (94), and more recently by recog-nition of both Ser139ph along with Ser139ph, Tyr142ph dual mark of H2A.X by MCPH1(microcephalin 1) (95). Given that the structureof the MDC1 complex was reviewed previously (6), the present review focuses on the structureof the MCPH1 complex.

Under basal cellular conditions, the histone variant H2A.X is phosphorylated at C-terminal Tyr142 by the Williams-Beuren syndrome


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transcription factor (WSTF), a component of the WSTF-ISWI chromatin remodeling com-plex (96). Under DNA double-strand break conditions, H2A.X is very rapidly phospho-rylated at Ser139 by the ataxia telangiectasiamutated kinase (97), while progressively dephosphorylated at Tyr142 by Eya tyrosine

phosphatase (98). This results in a temporalswitch from a diphosphorylated (Ser139phand Tyr142ph; designated di- H2A.X) to amonophosphorylated (Ser139ph; designated H2A.X) state of this histone variant. Struc-tural, biochemical, and cellular studies haveestablished that the tandem BRCT domainsof MCPH1, an early DNA damage responseand maintenance of genome integrity protein,can read both states of H2A.X, thereby servingas a versatile sensor of both H2A.X anddi- H2A.X phosphorylation marks (95).

The tandem BRCT domains of MCPH1bound to both Ser139ph-modied H2A.X(Kd = 0.7 M) and Ser139ph, Tyr142phmodied di- H2A.X (Kd = 4.4 M) peptides,but not to the Tyr142ph-modied H2A.Xpeptide. Structures have been determined forthe tandem BRCT domains of MCPH1 boundto a Ser139ph-modied H2A.X peptide andto a Ser139ph,Tyr142ph-modied di- H2A.Xpeptide (Figure 10 a), thereby dening theintermolecular contacts accounting for thesequence specicity of recognition (95).Specically, the / folds of individual BRCTdomains are connected through a helical linkersegment, with both H2A.X and di- H2A.X(Figure 10 b) peptides bound within a cleft at the interface of the tandem BRCT domains and with recognition specicity associated with a ty-rosine at position 142 of the bound peptide that is positioned in a hydrophobic pocket. In bothcomplexes, the phosphate group of Ser139phis recognized by the rst BRCT domain(Figure 10 b) and hydrogen bonded to accep-tor atoms through direct and water-mediatedhydrogen bonds, with mutations of phosphate-pocket lining residues resulting in reducedbinding afnity. In the di- H2A.X peptide- MCPH1 complex, the Ser139ph phosphate isrecognized by the rst BRCT domain, whereas

the Tyr142ph phosphate (in two alternatalignments) is recognized by the second BRCdomain (Figure 10 b). The phosphate group Tyr142ph is also hydrogen bonded to acceptoatoms through direct and water-mediatehydrogen bonds, with mutations of phosphatepocket lining residues resulting in reduc

binding afnity. In the structures of bo MCPH1 complexes, the C-terminal carboxylate of Tyr142is anchored bya pairof hydrogebonds to the side chain of an evolutionarconserved arginine residue (Figure 10 whose mutation results in loss of binding.

Functionally, MDC1 (and MCPH1) werfound to colocalize with H2AX (or d H2A.X) and 53BP1 to DNAdamageinducenuclear foci in a manner dependent on H2A(or di- H2A.X). Disruption of the MDC1 H2A.X (or MCPH1-di- H2A.X) interactiothrough incorporation of appropriate mutations resulted in a failure to recruit MDC1 ( MCPH1) to DNA damage foci and also pr vented the recruitment of downstream DNdamage repair proteins (94, 95).

A recent study determined that the struture of the tandem BRCT repeats of Rtt107a protein that restores stalled replication forkfollowing DNA damage, bound to phosphorylatedH2A( H2A) (99), thereby reinforcing threcognition principles outlined above for th MDC1 (94) and MCPH1 (95) complexes.

BIR DomainsSurvivin is an inhibitor of apoptosis family prteins implicated in mitosis and apoptosis. such, Survivin is a component of the chromsomal passenger complex, a key player durimitosis. It also recognizes the Smac/DIABLprotein that plays a role in apoptosis. The baulovirus IAP repeat (BIR) domain of Surviv which adopts an scaffold stabilized bybound Zn ion (100), recognizes the H3T3pmark; this interaction is necessary for accumlation of the chromosomal passenger complat centromeres, an event required for accuracell division (101103). Structural studies wundertaken on H3T3ph-containing (104, 105

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C

A1R2

H3T3phH3T3ph

K4

Q5

N

C

A1A1

Q5

H3T3phH3T3ph

S139ph(H2A.X)S139ph(H2A.X)

Y142ph(H2A.X)Y142ph(H2A.X)

Arg

A1

I4

BIR (Survivin) BIR (Survivin) BIR (Survivin)

Tandem BRCT (MCPH1)

Tandem BRCT (MCPH1)

a b

c d e

Y142ph(H2A.X)Y142ph(H2A.X)

Lys

His

S139ph(H2A.X)S139ph(H2A.X)

Figure 10Structures of BRCT (MCPH1) and BIR (Survivin) domains bound to phosphoserine/threonine/ tyrosine-containing peptides. ( a) The 1.5- A crystal structure of the complex of the tandem BRCT domainsof MCPH1 bound to di- H2A.X(139142)S139phY142ph peptide, Protein Data Bank (PDB) reference3U3Z. (b) Details of the alignment and interactions involving the side chain of phosphorylated Ser139(S139ph) and phosphorylated Tyr142 (Y142ph) (arrows ) of the di- H2A.X(139142)S139phY142ph peptide with the rst (blue) and second ( green) BRCT domains of MCPH1. Note that Y142ph adopts twoconformations in the structure of the complex. ( c ) The 2.4- A crystal structure of the complex of the BIR domain of Survivin bound to the H3(115)T3ph peptide (PDB: 3UIG). The peptide chain can be tracedfrom Ala1 to Gln5 (A1 to Q5). (d ) Details of the alignment and interactions between the boundH3(115)T3ph peptide and the BIR domain of Survivin. Interactions involving the side chains of Arg2 (R2)and Lys4 (K4) are also shown in the panel. (e) Details of the alignment and interactions between the boundSmac/DIABLO(115) peptide and the BIR domain of Survivin (PDB: 3UIH). The peptide chain can betraced from Ala1 to Ile4 (A1 to I4). Abbreviations: C, C terminus; MCPH1, microcephalin 1; N, N terminus.

and Smac/DIABLO N-terminal (104) pep-tides bound to the BIR domain of Survivin(Figure 10 c ). The peptides bind in an extendedconformation, with the N-terminal Ala residuerecognized in both complexes (Figure 10 d ,e).Inaddition,theNterminusandArg2arehydro-gen bonded to acidic side chains, the H3T3phmark is positioned in a basic pocket, and recog-nition is associated with intermolecular hy-drogen bonding with lysine and histidine side

chains (Figure 10 d ). Survivin binds tighter tothe H3T3ph-containing peptide relative to theN-terminal Smac/DIABLO peptide, and thispreference can be reversed through structure-guided mutations that increase the hydropho-bicity of the phosphate-binding pocket (104).Structural analysis also permitted identicationof putative Survivin-binding epitopes in othermitotic proteins, including human Shugoshin 1(105).


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MULTIVALENT READOUT BY LINKED BINDING MODULES The PHD-bromo cassette involving adjacently positioned PHD nger and bromodomains isthe most frequently observed combination of a distinct pair of readers positioned in tan-dem that impact on epigenetic regulation (re-

viewed in References 5 and 106). In principle,the PHD-bromo cassette has the potential forreadout of different combinations of methyl-lysine (PHD nger) and acetyl-lysine (bromo-domain) marks, and potentially doing so in acombinatorial manner.

BPTF PHD-Bromo Cassette The rst insights into structure-function as-pects of PHD-bromodomain (PHD-bromo)cassettes emerged from studies of this cassettein BPTF, where structural studies establishedthat the two domains were separated by an -helical linker, resulting in a dened separa-tion and orientation of the binding pocket do-mains (Figure 11 a) (20). Furthermore, struc-tural and binding studies established that theBPTF PHD nger exhibited selectivity for theH3K4me3 mark (20, 21), whereas the BPTFbromodomain targeted a range of Kac marks,demonstrating a preference for H4K16ac, withcooperative binding observed for simultaneousreadout of these two marks on different histonetails (107).

These studies on dual readout by the BPTFPHD-bromo cassette at the histone tail level(20, 21) have been extended to the nucle-osomal level (107). Experimentally, despitethe BPTF bromodomain displaying limiteddiscrimination among different acetyl-lysineson H4 (K12ac, K16ac, and K20ac) at thehistone peptide level, a marked selectivity wasobserved solely for H4K16ac, in combination with H3K4me3, by the PHD-bromo cassetteat the nucleosomal level (Figure 11 b) (107).Indeed, a signicant pool of doubly modiedH3K4me3/H4K16ac nucleosomes was iden-tied in vivo, and additional validation forthe multivalent readout emerged on demon-stration that the BPTF PHD-bromo cassette

colocalizes with the H3K4me3 and H4K16amarks in the genome (107). Furthermorstructure-based model building suggested ththe BPTF PHD-bromo cassette can be dockeonto a nucleosome core particle such that thPHD nger has the potential to target thH3K4me3 mark and the bromodomain t

target the H4K16ac mark, while positioninthe linker in the groove of the DNA (5).

MLL1 PHD-Bromo Cassette The MLL1 gene is essential for embryonic d velopment and hematopoiesis, and this genealso a frequent target for recurrent chromosomal translocations, resulting in hematopoietprecursor transformation into leukemia cell MLL1 is required for maintenance of HOXAexpression in stem and progenitor cells, as was for switching expression off during blood cematuration, with the induction of this epigenetic switch dependent on the PHD nger of MLL1 (reviewed in Reference 108). Strutural studies of the MLL1 PHD-bromo cassette have been undertaken in the free anH3K4me3-bound states (109). The individudomains adopt characteristic PHD nger anbromodomain folds with the connecting linkeadopting a turn segment that leads into the extended -helix Z of the bromodomain, suthat the two domains interact with each othe(Figure 11 c ). This interaction is manifested an increase in the binding afnity of the PHnger for the H3K4me3 mark in the PHDbromo cassette relative to the isolated PHnger. Furthermore, MLL1 and H3K4me3marks colocalize on HOX genes, with theChIP-based results implying that binding othe H3K4me3 mark by the MLL1 PHD3 contributes to MLL1 localization at target gene(109). By contrast, the MLL1 bromodomaifor as yet to be determined reasons, has lost ability to bind Kac-containing histone peptide

Cyclophilin CyP33, a peptidyl-prolyl ismerase that contains an attached RNArecognition motif (RRM) domain, binds to the PHDbromo cassette of MLL1, wherein it regulat MLL1 function through HDAC recruitmen

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H 3 K 4

m e 3

K14V21

H 4 K 1 6 a c

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PHD-bromo (MLL1)

PHD-bromo (MLL1) + RRM (CyP33)

c i s - p r o

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t r a n s

- p r o

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MODEL

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A1

H 3 K 4

H 3 K 9 m

e 3

H3K

18ac

H 3 K 1 8 a c

L20

PHD-bromo (TRIM33)

a b

c d

e

ZZ

2.5% input GST-PHD-bromo

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8

40

Rel.CPM

U n m o

d

U n m o

d - C

H 3 K 4

m e 3 (

M e )

H 4 K 1

2 a c +

M e

H 4 K 1

6 a c +

M e

H 4 K 2

0 a c +

M e

U n m o

d

U n m o

d - C

H 3 K 4

m e 3 (

M e )

H 4 K 1

2 a c +

M e

H 4 K 1

6 a c +

M e

H 4 K 2

0 a c +

M e

RRM(CyP33)

PHD Bromo

PHD

Bromo

PHD Bromo

PHD

Bromo

Figure 11Structures and in vivo functional studies of PHD-bromo cassettes bound to methyl-lysine- and acetyl-lysine-containing histonepeptides. (a) The 2.0- A crystal structure of the complex of the PHD-bromo cassette of BPTF bound to the H3(115)K4me3 pepProtein Data Bank (PDB) reference 2F6Z. A separate 1.8- A crystal structure of the bromodomain of BPTF bound to theH4(1221)K16ac peptide was also solved (PDB: 3QZS), and that information was superimposed on the structure shown in this p The bound H3(115)K4me3-containing peptide can be traced from Ala1 to Thr6 (A1 to T6), and the bound H4(1221)K16ac-containing peptide can be traced from Lys14 to Val21 (K14 to V21) in the complexes. (b) A glutathione S -transferase (GST) pul

of modied nucleosomes containing semisynthetic histones produced by expressed protein ligation. Nucleosomes containing duamarks involving H4K12ac, H4K16ac, or H4K20ac in combination with H3K4me3 are pulled down with a resin-bound GST-BPTPHD-bromo cassette and detected by autoradiography after native gel electrophoresis. ( c ) The 1.72- A crystal structure of the MPHD-bromo cassette exhibits a cis linker proline in the free state (PDB: 3LQH). The PHD nger and bromodomains are blue angreen, respectively. (d ) Model of the MLL1 PHD-bromo cassette with a trans linker proline, with this alignment stabilized by thbound RRM domain of CyP33. ( e) The 2.7- A crystal structure of the complex of the PHD-bromo cassette of TRIM33 bound to H3(122)K9me3K18ac peptide (PDB: 3U5O). The bound H3(122)K9me3K18ac peptide can be traced from Ala1 to Leu20 (AL20) in the complex. Abbreviations: C, C terminal; CyP33, cyclophilin 33; MLL1, mixed-lineage leukemia 1 protein; N, N termRRM, RNA recognition motif; Rel CPM, relative counts per minute.


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(110, 111). Examination of the structure of the MLL1 PHD-bromo cassette identied acis proline in the linker segment between do-mains (Figure 11 c ), which in the presence of CyP33 undergoes cis-trans proline isomeriza-tion. The trans proline isomer, where the twodomains no longer interact with each other,

is stabilized by binding to the RRM moduleof CyP33 (model in Figure 11 d is based onthe structure of CyP33 RRM bound to a frag-ment of MLL1 PHD3) (109). Thus, disrup-tion of the PHD-bromo interface, as a result of the cis-trans proline transition, facilitates ac-cess to an otherwise occluded PHD nger inthe cassette to the CyP33 RRM domain. Fur-thermore, the H3K4me3 and the RRM do-main targetdifferentsurfaces of PHD3, thereby potentially integrating distinct regulatory in-puts by coexisting as a ternary complex. Func-tional experiments demonstrate that the inter-action between MLL1 and CyP33 is requiredfor HDAC-mediated repression of HOX tar-get genes in vivo (109). These results collec-tively establish the role of the MLL1 PHD-bromo cassette both as

epigenetic modification

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