Chemogenetic Analysis of Human Protein Methyltransferases

Chem Biol Drug Des 2011; 78: 199–210 ª 2011 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2011.01135.x Research Article

Victoria M. Richon1,*, Danielle Johnston1, Abbreviations: DOT1L, disruptor of telomeric silencing-1-like; Christopher J. Sneeringer 1, Lei Jin1, HGNC, HUGO Gene Nomenclature Committee; IPTG, isopropyl b-D-1- thiogalactopyranoside; NSUN, NOP2 Sun domain family; PKMT, protein Christina R. Majer1, Keith Elliston2, ⁄ 2 1 lysine methyltransferase; PMT, protein methyltransferase; PRDM, (PRDI- L. Fred Jerva , Margaret Porter Scott and BF1 and RIZ homology) domain; PRMT, protein arginine methyltransfer- 1, Robert A. Copeland * ase; PSI-BLAST, position-specific iterated basic local alignment search tool; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SET- 1Epizyme, Inc., 840 Memorial Drive, Cambridge, MA 02139, USA domain, Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax 2Genstruct Inc., One Alewife Center, Cambridge, MA 02140, USA (trx) domain. *Corresponding authors: Victoria M. Richon, [email protected] or Robert A. Copeland, [email protected] Received 16 March 2011, revised 23 April 2011 and accepted for publi- cation 27 April 2011 A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their Posttranslational modifications of proteins provide powerful mecha- catalytic domains. We identified 51 protein lysine nisms for selective and stringent temporal control of many cellular methyltransferase proteins based on similarity to processes (1). For example, the timing and extent of protein matura- the canonical Drosophila Su(var)3-9, enhancer of tion and degradation are often controlled by proteolytic processing zeste (E(z)), and trithorax (trx) domain. Disruptor of precursor proteins and by a combination of covalent ubiquitin of telomeric silencing-1-like, a known protein modification and proteolysis. Also, cellular signaling pathways, lysine methyltransferase, did not fit within the which communicate binding events at cell surface receptors to protein lysine methyltransferase family, but did nuclear-localized transcription events, are tightly controlled by spe- group with the protein arginine methyltransferas- cific protein phosphorylation and dephosphorylation; these pro- es, along with 44 other proteins, including the METTL and NOP2 ⁄ Sun domain family proteins. We cesses are catalyzed respectively by protein kinases and protein show that a representative METTL, METTL11A, phosphatases (1). In a like manner, other posttranslational modifica- demonstrates catalytic activity as a histone meth- tions of proteins, catalyzed by specific enzymes, confer remarkable yltransferase. We also solved the co-crystal struc- levels of specificity to critical biological systems. Not surprisingly, tures of disruptor of telomeric silencing-1-like dysregulation of the enzymes that perform these critical functions with S-adenosylmethionine and S-adenosylhomo- often leads to pathologies such as cancer, inflammation, and other cysteine bound in its active site. The conformation serious human diseases. of both ligands is virtually identical to that found in known protein arginine methyltransferases, Regulation of gene transcription is yet another example of a critical METTL and NOP2 ⁄ Sun domain family proteins and biological process that is controlled in large part by enzyme-cata- is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax lyzed, covalent modification of proteins and nucleic acids (2). For (trx) domain protein lysine methyltransferases. We example, DNA methyltransferase-mediated modification of the 5- have developed biochemical assays for 11 mem- position of cytosine, within the CpG dinucleotide sites of genes, bers of the protein methyltransferase target class plays a direct role in silencing of gene transcription (3). The DNA of and have profiled the affinity of three ligands for chromosomes is found in complex with a number of proteins, most these enzymes: the common methyl-donating sub- notably the histone proteins; together, the DNA–histone complex strate S-adenosylmethionine; the common reac- constitutes chromatin, the main component of chromosomes (4). tion product S-adenosylhomocysteine; and the Conformational remodeling of chromatin regulates gene transcrip- natural product sinefungin. The affinity of each of tion and its silencing. This conformational remodeling has been these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein shown to result from specific posttranslational modifications of the arginine methyltransferases to reveal patterns of histone proteins of the chromatin complex (5). The types of post- ligand recognition by these enzymes. translational modification of histones known to contribute to the regulation of gene transcription include acetylation, methylation, Key words: chemical biology, cheminformatics, drug discovery, phosphorylation, and ubiquitinylation (6). Recently, our group has enzyme structure, molecular recognition reviewed the enzymes that methylate histones and other cellular

199 Richon et al. proteins, the protein methyltransferases (PMTs) (6), and has made basic local alignment search tool (PSI-BLAST) (http://blast.ncbi.nlm.- the case that the enzymes of this class represent particularly attrac- nih.gov). Multiple sequence alignments for each family group were tive targets for drug discovery efforts, because of the disease asso- generated using the CLUSTALW2 software (http://www.clustal.org). ciation and druggability of these proteins. The enzymes of the PMT Visualization and manual editing of alignments were performed class can be subdivided into two major families: the protein lysine using GeneDoc (http://www.nrbsc.org/gfx/genedoc/). Interfamily methyltransferases (PKMTs) and the protein arginine methyltransfe- sequence alignment was accomplished using a 'sparse alignment' rases (PRMTs). These two families are distinguished from one approach [S. Smith (2009), personal communication] that aligns rep- another not only on the basis of the methyl-accepting amino acid resentative members of each family using CLUSTALW2 to define side chain recognized but also on the basis of the primary sequence appropriate anchor points for the interfamily alignments. The final of their catalytic domains and their three-dimensional structures (6). alignments were generated by manually adding additional proteins All of the lysine methyltransferases, except one, contain a ca. 130- to the alignment, ensuring alignment of key anchor points identified amino acid domain – referred to as the Drosophila Su(var)3-9, in the sparse alignments. The final multiple sequence alignments enhancer of zeste (E(z)), and trithorax (trx) domain (SET-domain) – used for the phylogenetic analysis are illustrated in the supplemen- that constitutes the active site domain of these enzymes (7). The tal materials as Figures S1 and S2 for the PKMTs and the PRMTs one exception to this structural generality is the enzyme disruptor (plus DOT1L), respectively. of telomeric silencing-1-like (DOT1L), a PKMT that does not contain a SET-domain (8). The phylogenetic analysis of the aligned sequences was accomplished using the PHYLIP package developed at the University of To exploit most effectively the PMT target class for drug discovery Washington (http://evolution.genetics.washington.edu/phylip.html). and chemical biological studies, it is critical to understand the con- To facilitate bootstrap analysis of the data, the SEQBOOT program stituency of the class within an organism of interest (in the case of was used to create 250 randomized data representations of the drug discovery, this would usually mean humans) and the structural input alignment for analysis. The PROTDIST program was used to cal- relatedness of these proteins to one another. In terms of drug dis- culate pairwise distance measures between the sequences within covery and chemical biology, it is the structural relatedness of the the alignments using the BLOSUM45 matrix, and the program NEIGHBOR active site domains in particular, rather than the overall structures of was used to construct phylogenetic trees from the distance data the full-length proteins, that is most germane. While several groups using the neighbor joining method. Consensus trees were built have reported alignments of known members of the PKMT and using the CONSENSE utility and uprooted trees were drawn using the PRMT families, no systematic analysis of the constituency of PMTs DRAWTREE program. encoded by the human genome has yet been reported (7,9–13).

In this study, we report the results of such a systematic survey of the Determination of PMT enzymatic activity human genome for PKMT- and PRMT-related enzymes on the basis of Recombinant protein production and activity assays were performed amino acid sequence alignments of the active site domains of these for the 11 PMTs as described elsewhere (15; S. R. Daigle, E. J. Olh- enzymes. We find that both families are composed of >40 putative ava, C. A. Therkelsen, C. R. Majer, C. J. Sneeringer, J. Song, D. enzymes and that each family can be further subdivided into discrete Johnston, M. Porter Scott, J. J. Smith, Y. Xiao, L. Jin, K. W. clusters of related proteins. Interestingly, the non-SET-domain PKMT, Kuntz, R. Chesworth, M. P. Moyer, K. M. Bernt, S. A. Armstrong, R. DOT1L, could not be rationally included within the PKMT family, but A. Copeland, V. M. Richon, R. M. Pollock, unpublished data). could be reasonably associated with the PRMT family. This seemingly anomalous association of DOT1L with the PRMTs, based on amino acid sequence, is consistent with crystallographic data comparing Determination of inhibitor IC50 values the conformation of the substrate and product ligands (S-adenosyl- IC50 values for enzymes in the protein methyltransferase panel were methionine, SAM, and S-adenosylhomocysteine, SAH) bound to SET- determined under balanced assay conditions with both SAM and domain PKMTs, PRMTs, and DOT1L. The implications of the data gen- protein ⁄ peptide substrate present at concentrations equal to their erated here for drug discovery and chemical biology studies are also respective KM values. Where a peptide was used as methyl-accept- discussed. ing substrate, the peptide is referred to here by the histone and amino acid residue numbers that it represents. For example, peptide H3:16–30 refers to a peptide representing histone H3 amino acid Methods residues 16 through 30. Flag and his-tagged CARM1 (2–585) purified from 293 cells was assayed at a final concentration of 0.25 nM Sequence alignment and family tree against a biotinylated peptide corresponding to histone H3:16–30 construction (R26-Me1). E. coli-expressed EHMT2 (913–1193) was assayed at a Amino acid sequence alignments and family tree construction were final concentration of 0.1 nM against a biotinylated peptide corre- performed by Genstruct, Inc. (Cambridge, MA, USA). Amino acid sponding to H3:1–15. Full-length EZH1 and EZH2 were expressed as sequences were obtained from annotated protein sequence files four-component complexes in insect cells and purified as described obtained from the NCBI reference proteins database, RefSeq elsewhere (15). EZH1 (4 nM) and EZH2 (4 nM) four-component com- (http://www.ncbi.nlm.nih.gov/RefSeq/) or from Swiss-Prot (http:// plexes were assayed against a biotinylated peptide corresponding www.expasy.ch/sprot/) (14). Rigorous identification of family mem- to histone H3:21–44. Insect expressed full-length PRMT1 was ber sequences was accomplished using position-specific iterated assayed at a final concentration of 0.75 nM against biotinylated

200 Chem Biol Drug Des 2011; 78: 199–210 Chemogenetic Analysis of Human Protein Methyltransferases peptide corresponding to H4:36–50. Flag-tagged full-length PRMT5 40 mg ⁄ mL for crystallization work. The His-DOT1L-1-416 was cloned purified from 293 cells was assayed at a final concentration of into pET30a vector with N-terminal His tag and expressed in BL21- 1.5 nM against a biotinylated peptide corresponding to H4:1–15. Gold (DE3) (Stratagene, Cedar Creek, TX, USA). The culture was

E. coli-expressed full-length PRMT8 was assayed at a final concen- incubated at 37 C until OD600 reached 0.3 and continued at 25 C tration of 1.5 nM against a biotinylated peptide corresponding to until OD600 reached 0.7. Expression was induced by adding 0.2 mM H4:31–45. Full-length SETD7 purified from E. coli was assayed at a IPTG, and cells were harvested after 4 h. Cell pellet was suspended final concentration of 1 nM against a biotinylated peptide corre- in buffer A (20 mM Tris–HCl, 500 mM NaCl, 10 mM imidazole, 10% sponding to H3:1–15. Flag and his-tagged full-length WHSC1 was glycerol, 5 mM b-mercaptoethanol, pH 7.8) with 1 mg ⁄ mL lysozyme purified from 293 cells and assayed at a final concentration of and incubated on ice for 30 min. The cells were sonicated and cen- 2.5 nM against avian oligonucleosomes. trifuged. The supernatant was loaded onto Ni-NTA column (Qiagen, Valencia, CA, USA) that preequilibrated with buffer A and washed with the same buffer. Protein was eluted with 200 mM imidazole in Determination of METTL11A enzymatic activity the buffer A and then dialyzed against 20 mM HEPES, 50 mM NaCl, Histone H3 and Histone H4 (NEB, Ipswich, MA, USA) were diluted 1mM EDTA, 1 mM DTT, pH 7.5. The sample was loaded on a SP separately in assay buffer (20 mM Tris, pH 8.0, 0.002% Tween20, Sepharose Fast Flow column (GE Healthcare) and eluted with a lin- 0.005% bovine skin gelatin, and 0.5 mM DTT) to a final concentra- ear gradient of 0–1 M NaCl. The target protein was concentrated tion of 200 nM in a 96-well plate (25 lL each well). To monitor the and further purified by Superdex200 column (GE Healthcare) with 3 reaction, 300 nM S-[methyl- H]-adenosyl-L-methionine (80 Ci ⁄ mmol, 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.8. Frac- from ARC) together with 200 nM cold SAM (total SAM concentrations of pure protein were pooled. tion = 500 nM) was used in each reaction. For initial determination of activity, METTL11A (Sino Biologics, Beijing, China) was added to SAH or SAM (Sigma-Aldrich) was dissolved in the final DOT1L pro- initiate the reaction (25 lL each well for final concentration of tein buffer to 100 mM. The DOT1L–SAH or DOT1L–SAM binary com- 50 nM). The reaction was incubated at room temperature and plex was prepared at a final concentration of DOT1L of 20 mg ⁄ mL quenched with 10 lL per well of 300 lM unlabeled S-adenosyl-L- (0.4 mM) and SAH or SAM of 2 mM. Crystals were obtained in methionine (Sigma, St. Louis, MO, USA) at various time-points. For 1.94 M ammonium sulfate, 0.1 M sodium acetate, pH 5.3, and 2 mM detection, 50 lL of reaction was transferred to a filter plate [Milli- TCEP by the hanging-drop method at 18 C. The crystals were cryo- pore (Billerica, MA, USA) Multiscreen HTS FB 1.0 ⁄ 0.65 lm], protected in 30% glucose and flash-frozen in liquid nitrogen. The dif- washed three times with 10% trichloroacetic acid, washed once fraction data set for DOT1L–SAH was collected at beamline 17 U at with 95% ethanol, and read on the Top Count (Perkin Elmer, Wal- Shanghai Synchrotron Radiation Facility and the data set for DOT1L– tham, MA, USA) after the addition of 30 lL scintillant. Having SAM was collected at beamline 21-ID-F at Advanced Photon Source established histone H4 as the preferred substrate, the enzyme con- in Argonne National Laboratory. All data were processed by HKL2000 centration was titrated from 5 to 40 nM, and the reaction progress (16). The SAH-bound crystal diffracted to 2.3 and the SAM-bound over time was determined at each enzyme concentration as crystal diffracted to 2.1 , in the space group of P65 with one protein described previously. molecule in the asymmetric unit. The structures were solved by molecular replacement (Molrep) (17) using the published DOT1L– SAM structure (PDB ID: 1NW3) as a search model (18). Refinement Structure determination of DOT1L with SAH or was carried out by Refmac5 (19), and the model building was carried SAM bound out by COOT (20). Detailed information on the diffraction data, refine- DOT1L-1-416 was produced in E. coli as either a GST or hexa-histi- ment, and structure statistics is provided in Table S1. dine N-terminal fusion protein. The GST-DOT1L-1-416 was cloned into the pGEX-KG vector and expressed in BL21 (DE3) cells (Nova- The enzyme thus purified was tested for enzymatic activity in a radio- 3 gen, Madison, WI, USA) with coexpression of chaperone plasmid metric assay of H-CH3 transfer from labeled SAM (Perkin-Elmer) to pGTf2 (Takara Bio Inc., Shiga, Japan) with 0.2 mM isopropyl b-D-1- purified nucleosomes from chicken erythrocytes according to the thiogalactopyranoside (IPTG), at 18 C for 16 h. Cell pellets were method of Fang et al. (21). The DOT1L used for crystallographic stud- suspended in buffer containing 50 mM Na2HPO4 ⁄ NaH2PO4, pH 7.5, ies was found to have reproducible activity as a histone methyltrans- 200 mM NaCl, 5% glycerol, and 5 mM b-mercaptoethanol. Cells ferase and displayed the following steady state kinetic parameters: SAM Nuc SAH were lysed by sonication. The supernatant was loaded onto a col- kcat = 0.3 per minute, KM = 0.67 lM,KM = 8.6 nM,Ki umn of glutathione-sepharose Fast Flow resin (GE Healthcare, Pitts- = 0.26 lM. A more complete description of the enzymatic mechanism burg, PA, USA). Protein was eluted with buffer containing 10 mM of DOT1L will be reported separately (A. Basavapathruni, C. R. Majer, reduced glutathione. The GST-tag was cleaved from DOT1L-1-416 R. A. Copeland and M. Porter Scott, manuscript in preparation). by thrombin and removed by a second run on a glutathione-sepharose column. The tag-free protein was purified further by SP Sepha- rose Fast Flow (GE Healthcare) chromatography and eluted with Results 25 mM Tris–HCl, pH 8.0, 675 mM NaCl, 5% glycerol, and 5 mM b- mercaptoethanol. The protein was further purified by Superdex75 Construction of PKMT and PRMT family tree (GE Healthcare) chromatography in buffer containing 20 mM Tris– diagrams HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. To facilitate the chemical biological and pharmacological exploration Fractions of pure protein were pooled and concentrated to of the human PMTs, it would be useful to know the relationships

Chem Biol Drug Des 2011; 78: 199–210 201 Richon et al. among members of this enzyme class and to illustrate these rela- putative PKMTs cluster into four major groups, illustrated as distinct tionships in a convenient format. This is commonly done by the branches on the family tree shown in Figure 1. Each of these clus- construction of family tree diagrams; such a diagram for the human ters includes at least one example of a protein for which biochemi- protein kinases (the human kinome) has proved an invaluable tool cal evidence of direct PKMT enzymatic activity has been reported in for scientists exploring the chemical biology of these enzymes (22). the literature. One large branch of the putative PKMT tree is com- Toward this end, we have systematically explored the protein posed of 16 PRDM proteins. The active site domain of these pro- sequences of human PKMTs and PRMTs with the goal of developing teins is the most divergent from that of the canonical SET-domain, a complete map of the relatedness among the active sites of being instead referred to as a PR (PRDI-BF1 and RIZ homology) enzymes in each of these protein families. Our initial intent was to domain. Despite the significant structural divergence of these pro- construct a single map that combined both the PKMT and PRMT teins from the other family members, there are literature reports families. However, we could find no clear rationale with which to that demonstrate the PKMT activity of PRDM2 (23), PRDM8 (24), define a point of intersection between the two families. For this and PRDM9 (Meisetz) (25). reason, we present here separate maps for each family within the overall PMT class. The tree diagram, dendogram, and alignments for the PRMT family are illustrated in Figure 2 and Figures S3 and S4, respectively. Prior Our interrogation of the human genome using PSI-BLAST and repre- to this report, 11 human proteins have been annotated as PRMTs in sentative PMT sequences revealed a total of 51 proteins with clear the literature, and most of these have been reported to have enzy- relatedness to the canonical SET-domain of enzymes with demon- matic activity as protein arginine methyltransferases (10). Early iter- strated biochemical activity as PKMTs. The relationship of these ations of our search and alignment process using PSI-BLAST resulted proteins to one another was determined and is illustrated by the in clustering of most, but not all, of the enzymatically active PRMTs. dendogram and alignments as shown in Figures S1 and S2. These However, PRMT5 and PRMT9 were not represented within the

Figure 1: Protein lysine methyltransferase (PKMT) family tree diagram. The relationship among the PKMT family members is shown in this unrooted tree, where the branch lengths are proportional to the distance calculated between the various PKMT family members. Sequence names are the HUGO Gene Nomenclature Committee standard gene names.

202 Chem Biol Drug Des 2011; 78: 199–210 Chemogenetic Analysis of Human Protein Methyltransferases

Figure 2: Protein arginine methyltransferase (PRMT) family tree diagram. The relationship among the PRMT family members is shown in this unrooted tree, where the branch lengths are proportional to the distance calculated between the various PRMT family members. Sequence names are the HUGO Gene Nomenclature Committee standard gene names. clusters. To ensure inclusion of these latter PRMTs within the family and H4 as substrates. Both H3 and H4 were found to be methylat- tree, additional search and alignment iterations were performed. ed by the recombinant METTL11A, but H4 appeared to be utilized Through this method, the full complement of enzymatically active more efficiently by this enzyme (data not shown). We then studied PRMTs was incorporated into the family. This process also resulted the methyltransferase activity of METTL11A in more detail using H4 in the inclusion of multiple members of the METTL and NOP2 ⁄ Sun as a methyl-accepting substrate. As illustrated in Figure 3, linear domain family (NSUN) protein groups, both of which have previ- progress curves of methyl group incorporation into H4 were ously been described to be RNA methyltransferases (26,27). observed over a range of enzyme concentrations, suggesting that the reactions at all tested enzyme concentration conformed to steady state conditions. A replot of reaction velocity as a function Determination of methyltransferase activity of of enzyme concentration was linear over a range of 5–40 nM METTL11A enzyme, again suggesting well-behaved steady state kinetic behav- The structural relatedness of the METTL and NSUN protein groups ior (28,29). These data confirm the annotation of at least one to the bona fide PRMTs suggests that efforts to identify selective METTL group member as a PMT and more specifically as a histone ligands for PRMTs should consider carefully these other enzyme methyltransferase. groups for a full understanding of ligand selectivity. Beyond this, the inclusion of these protein groups within the PRMT tree raises the question of whether the members of these protein groups may Conformation of SAM and SAH in METTL, have heretofore unrecognized enzymatic activity as PMTs. To NSUN, and PRMT proteins address this question, we obtained a representative member of the To further understand the chemical biology relatedness of the METTL group of proteins, METTL11A. The recombinant METTL11A METTL and NSUN proteins to PRMTs, we compared the conforma- was initially tested for PMT activity using recombinant histones H3 tion of SAM or SAH in the crystal structures of representative

Chem Biol Drug Des 2011; 78: 199–210 203 Richon et al.

600 4.0 A B 3.5 500 3.0 400 2.5

300 2.0 CPM 1.5 200 1.0 Velocity (CPM/min) 100 0.5

0 0.0 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 40 Time (min) [E], nM

3 Figure 3: METTL11A is an active protein methyltransferase. (A) Reaction progress curves for the incorporation of H-CH3 from S-adenosylmethionine (SAM) into recombinant human histone H4 catalyzed by varying concentrations of METTL11A. Symbols: open circles, [E] = 5 nM; closed circles, [E] = 10 nM; open squares, [E] = 20 nM; closed squares, [E] = 40 nM. (B) Replot of the reaction velocity (measured as the slope of the linear least-squares best fit for each progress curve in panel A) as a function of METTL11A nominal enzyme concentration.

PRMTs, METTLs, and NSUNs that have been deposited at RCSB METTL, and PRMT enzymes although the overall structure for these PDB (http://www.pdb.org). Figure 4A illustrates the remarkable over- enzymes is quite different (Figure 4C,D). Only part of the SAM ⁄ SAH lap of SAM or SAH conformation in the active sites of NSUN, binding site is structurally conserved (the region immediately above

A D

B C

Figure 4: The superimposition of S-adenosylmethionine (SAM) ⁄ S-adenosylhomocysteine (SAH) in the crystal structures of NSUN5, MET- TL11A, and protein arginine methyltransferases (PRMTs). (A) Conformational similarity of SAM or SAH in NSUN5, METTL11A, and PRMTs. The SAM in NSUN5 (PDB ID: 2B9E) is presented as a ball-and-stick representation in yellow. The SAH in METTL11A (PDB ID: 2EX4) is presented as a ball-and-stick representation in green. The SAH in PRMT1 (PDB ID: 1OR8), PRMT3 (PDB ID: 2FYT), and CARM1 (PDB ID: 3B3F) is presented as a stick representation with carbon atom in gray, oxygen atom in red, nitrogen atom in blue, and sulfur atom in yellow. (B) The crystal structure of NSUN5 with SAM bound (PDB ID: 2B9E) in ribbon representation. The N- and C-termini are labeled. The b-sheets are colored in blue, a-helices in red, and loops in gray. The bound SAM molecule is in the same representation and color as in A. (C) The crystal structure of METTL11A with SAH bound (PDB ID: 2EX4). (D) The crystal structure of CARM1 with SAH bound (PDB ID: 3B3F). The general conformations of PRMT1 and PRMT3 (not shown) are similar to that of CARM1.

204 Chem Biol Drug Des 2011; 78: 199–210 Chemogenetic Analysis of Human Protein Methyltransferases the SAM ⁄ SAH ligand in the presented orientation). The structures A for the rest of the proteins vary significantly.

Inclusion of DOT1L in the PRMT family tree Despite its known activity as a PKMT, all attempts to include the non-SET-domain enzyme DOT1L in the PKMT family were unsuccessful. In contrast, DOT1L could be readily incorporated into the PRMT family tree (Figure 2). Again, we questioned whether the B amino acid alignment of the DOT1L active domain with that of the PRMTs translated into a structural relatedness that would be important from the perspective of ligand binding affinity and specificity. The crystal structure of DOT1L with the substrate SAM bound in the active site has been reported in the literature (PDB ID: 1NW3) (18). The crystal structures of several PRMTs have been reported with the product ligand, SAH, bound in the active site (PDB ID: 1ORH, 2FYT, 3B3F and 2V74). Preliminary comparisons of the ligand conformation of the DOT1L–SAM complex with that of PRMT–SAH complex suggested high similarity. However, some differences in specific torsion angles could be realized. Hence, we have solved the co-crystal structure of the DOT1L–SAH complex to 2.3 resolution (PDB ID 3QOX). The conformation of SAH within the DOT1L active site is compared with that in PRMT–SAH complexes in C Figure 5A. Here too we see remarkable overlap of the ligand conformation among DOT1L and PRMT enzymes, but clearly distinct from that of PKMTs (Figure 5B).

To understand and confirm the torsional angle differences seen between SAM and SAH bound to DOT1L, we have also obtained a higher-resolution structure (2.1 ) of the DOT1L–SAM complex (PDB ID 3QOW; Figure 5C). To our surprise, the SAM conformation in this higher-resolution structure is identical to that of SAH in the enzyme–product complex. The difference in SAM conformation seen here and in the previously published structure may relate to the Figure 5: The conformations for S-adenosylmethionine (SAM) resolution difference. and S-adenosylhomocysteine (SAH) in disruptor of telomeric silencing-1-like (DOT1L). (A) The SAH conformation in DOT1L (ball-and- stick, in green) is similar to that in PRMT1, PRMT3, and CARM1 Ligand afﬁnity mapping (stick, colored as carbon atom in gray, oxygen atom in red, nitrogen One important application of family tree diagrams, such as those atom in blue, and sulfur atom in yellow). (B) The SAH conformation developed here for the PKMTs and PRMTs, is the assessment of in DOT1L (ball-and-stick, in green) is distinct from those in protein ligand affinity and selectivity from a target class perspective. To lysine methyltransferase (PKMTs) [stick, colored as protein arginine illustrate the power of this approach, we have evaluated three methyltransferases (PRMTs) in A]. The PKMTs include SETD7 (PDB ligands across a representative sampling of 11 PMTs comprising both ID: 1O9S), SETD8 (1ZKK), EHMT2 (2O8J), EHMT1 (2RFI), SUV39H2 PKMTs and PRMTs. We chose to profile SAM and SAH as the univer- (2R3A), SETMAR (3BO5), SETD2 (3H6L), and SMYD3 (3MEK). (C) The sal methyl donor substrate and reaction product of PMT catalysis, SAM and SAH conformations in DOT1L. SAH is presented in stick respectively, and the natural product analog sinefungin (Figure 6). representation, and SAM in our 2.1 DOT1L–SAM structure is presented in ball-and-stick representation with the same color scheme

The PMTs all carry out an SN2 group transfer reaction involving a as the PRMTs in A. The conformations for SAM and SAH in our methyl donor (SAM) and methyl acceptor [e.g., histone; (6)]. To date structures are identical and superimposed upon each other. SAM in every human PMT that has been studied has been found to conform the published structure (PDB ID: 1NW3) is presented in ball-and- to a ternary complex mechanism of catalysis, involving formation of stick representation in green, with the torsion angle differences a SAM–enzyme–acceptor complex and direct transfer of the methyl between the previously published structure and the current struc- group from SAM to the acceptor protein (6). This commonality not- ture evident. withstanding, bisubstrate-utilizing enzymes that conform to a ternary complex mechanism can vary in the preferred order (or lack thereof) strate saturation level for each substrate can have a profound effect of substrate binding and the degree to which individual substrate on the apparent affinity of other ligands, such as inhibitors and acti- binding affinity is influenced by the state of enzyme saturation with vators. This is a critical issue for chemical biology studies aimed at respect to the other substrate (28). Hence, the conditions of sub- identification of ligands through high-throughput screening and for

Chem Biol Drug Des 2011; 78: 199–210 205 Richon et al.

NH2 Table 1: Ligand affinity for S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and sinefungin for various protein meth- N yltransferases N A SAM SAM KM SAH Ki Sinefungin Ki 7 7 7 O CH3 Enzyme (M · 10 ) (M · 10 ) (M · 10 ) N N a S EZH2 12 (10–14) 75 (58–91) >250 HO EZH1 25 (10–30) 83 (60–100) >250 O WHSC1 3.7 (2.0–5.5) 35 (26–45) 150 (100–200) H H NH2 SETD7 0.4 (0.2–0.6) 230 (180–270) 1.7 (1.1–2.3) H H EHMT2 18 (16–20) 10 (8–13) 93 (80–110) OH OH SMYD2b £0.01 3.6 (2.3–4.8) 0.9 (0.6–1.2) DOT1L 4.5 (3.1–5.9) 2.7 (2.0–3.3) 37 (25–48) PRMT1 16 (14–18) 4.3 (3.6–4.9) 2.0 (1.8–2.2) NH2 CARM1 1.6 (1.3–1.9) 1.2 (1.0–1.3) 1.0 (0.7–1.1) PRMT5 1.5 (1.0–2.0) 1.6 (1.0–2.1) 5.1 (3.3–7.1) B SAH N N PRMT8 5.7 (3.1–8.3) 2.5 (2.0–3.0) 3.1 (2.8–3.4)

a O Values in table represent the best fit value of the parameter. Values in N N parentheses represent the 95% confidence interval for the parameter value. b S For SMYD2, an accurate SAM KM value could not be determined. Hence, HO the KM and Ki values reported here should be viewed as apparent values. O H H NH2

H H an apparent value; likewise, the SAH and sinefungin Ki values OH OH should also be considered apparent values.

NH 2 For the methyl-donating substrate SAM, the Michaelis constant, C Sinefungin K , was determined as a relative measure of ligand affinity, N M N despite the fact that the KM term is composed of kinetic rate con- stants representing steps in addition to initial substrate binding O NH 2 (28). Nevertheless, we felt that apparent KM values across various N N members of the PMT class would serve well as a measure of rela- HO tive substrate recognition and utilization. O H H NH2 Based on their close structural relatedness to SAM, we have H H assumed that both SAH and sinefungin are SAM-competitive inhibi- OH OH tors of PMTs, an assumption that is consistent with a significant body of crystallographic data. To support this assumption further, Figure 6: Chemical structures of (A) S-adenosylmethionine we have determined the modality of SAH inhibition for the 11 PMTs (SAM), (B) S-adenosylhomocysteine (SAH), and (C) sinefungin. studied here by mutual titration of substrate and inhibitor. In all cases, SAH was found to be competitive with SAM (data not the characterization of ligand selectivity among potential target shown). A similar systematic analysis of sinefungin modality was proteins. This issue has been quantitatively addressed in general precluded by the modest potency of this compound and its rela- by Copeland (28–30) and specifically for the case of bisubstrate tively limited solubility. Based on this preponderance of cumulative enzymes by Yang et al. (31). The results of these studies evidence, we felt confident in assuming that both SAH and sinefun- consistently demonstrate that quantitative comparisons of apparent gin were competitive inhibitors with respect to SAM and have ligand affinity across a spectrum of targets and ⁄ or ligands are best therefore used the Cheng–Prusoff equation for competitive inhibi- performed with assays under balanced conditions, in which all sub- tion (28,29,32) to calculate the apparent Ki values of SAH and sine- strate concentrations are poised at their apparent KM values (31). In fungin from the IC50 values obtained for each ligand under keeping with this widely accepted and common assay design tenet, balanced assay conditions (vide supra). we performed dual titrations of SAM and methyl-accepting substrate for all of the enzymes studied here to determine the apparent The results of this profiling are summarized in Table 1 and in Fig-

KM values for each substrate. With this information in hand, we ure 7. For illustrative purposes, we have converted the Ki and KM have adjusted the substrate concentrations to match their respective values to pKi and pKM ()log (X), where X is either Ki or KM) so that apparent KM values (31) and thus achieved balanced conditions for the parameter value increases with increasing ligand affinity and all subsequent assays in which ligands were evaluated. For SMYD2, have represented these values as spheres of varying diameter for the KM value for SAM was below the detection limits of our assay the enzymes studied in Figure 7; the diameter of the individual methods. Hence, the KM value for this enzyme should be viewed as spheres relates directly to the magnitude of the pKM ⁄ pKi value.

206 Chem Biol Drug Des 2011; 78: 199–210 Chemogenetic Analysis of Human Protein Methyltransferases

A SMYD2 EZH1 EZH2 SETD7 PRMT5 PRMT1 DOT1L CARM1 PRMT8 EHMT2

WHSC1 LEGEND: Km, [M] Sized by pKm 10–9 10–8 10–7 10–6 10–5 >2.5 × 10–5

B SMYD2 EZH1 EZH2 SETD7 PRMT5 PRMT1 DOT1L CARM1 PRMT8 EHMT2

WHSC1 LEGEND: Ki, [M] Sized by pKi 10–9 10–8 10–7 10–6 10–5 >2.5 × 10–5

C SMYD2 EZH1 EZH2 SETD7 PRMT5 PRMT1 Figure 7: Ligand affinity maps DOT1L for S-adenosylmethionine (SAM) CARM1 (A), S-adenosylhomocysteine (SAH) (B), and sinefungin (C) binding to PRMT8 representative enzymes of the pro- EHMT2 tein lysine methyltransferase (PKMT) family (left) and the protein arginine methyltransferase (PRMT) family (right). The diameter of the WHSC1 LEGEND: spheres for each enzyme is directly Ki, [M] related to the magnitude of the Sized by pKi –9 –8 –7 –6 –5 >2.5 × 10–5 pKM (for SAM) or pKi (for SAH and 10 10 10 10 10 sinefungin) as described in the text.

Chem Biol Drug Des 2011; 78: 199–210 207 Richon et al.

Discussion have PRMT enzymatic activity and do not fall within the branch defined for the other enzymes. Inclusion of these two enzymes in Together, the human PRMTs and PKMTs (the PMTs) constitute an the alignments resulted in the additional incorporation of PRMT11, important class of enzymes that play critical roles in the methyla- a known PRMT, and two groups of proteins for which protein meth- tion of a number of cellular proteins. Most notably, from the per- ylation has not been previously suggested, the METTL and NSUN. spective of epigenetic regulation of gene transcription, a number of Both of these latter groups of proteins have been experimentally these enzymes have been shown to methylate arginine and lysine demonstrated to be RNA methyltransferases, and the crystal struc- residues of histones and to thus effect chromatin remodeling (5,6). tures of representative members of both groups display a well- The dysregulation of PMT enzyme activity – through gene amplifica- defined SAH binding pocket for each. We do not wish to imply that tion, gene rearrangement, point mutations, and other genetic the METTL and NSUN group members are necessarily PRMTs per changes – has been directly associated with various cancer types se. Rather, we include them within the PRMT tree on the basis of as well as with other human diseases [see Copeland et al. for their catalytic active site relatedness, which bears directly of chemi- review, (6)]. For example, overexpression of the SET-domain PKMT cal biology studies aimed at understanding small-molecule ligand EZH2 has been associated with prostate, breast, bladder, colon, affinity and cross-reactivity (i.e., selectivity) among structurally skin, liver, endometrial, lung, gastric, lymphoid, and myeloid cancers related proteins. This caveat notwithstanding, the data presented (33,34). More recently, a subset of patients with non-Hodgkin's lym- here suggest that at least one member of the METTL group, MET- phoma have been found to be heterozygous for point mutations TL11A, may be biochemically defined as a PMT and more specifi- within the catalytic active site of EZH2 (35). These mutations were cally as a histone methyltransferase (Figure 3). The results demonstrated to result in a change of function that, in concert with illustrated in Figure 3 clearly demonstrate that METTL11A has enzy- the wild-type enzyme, results in elevated levels of the tumorigenic matic activity as a PMT, but the current data do not define the sub- product histone H3K27me3 (15). Likewise, expression levels of a strate specificity of the enzyme with respect to methyl-accepting number of other PMTs have been linked to tumorigenesis and ⁄ or group. While this report was being prepared, Clarke's group (38) tumor invasiveness in human cancers (6). Based on this disease also reported PMT activity for METTL11A and specifically demon- association and the presumed druggability of the SAM binding strated that the protein acted as a peptide N-terminal methyltrans- pockets of these enzymes, we have made the case that the PMTs ferase. As illustrated in Figure 4A, the conformation of SAH within constitute an important, novel drug target class that should be the binding pockets of METTL and NSUN proteins shows remark- exploitable for small-molecule drug therapies against a number of able overlap with that seen in bona fide PRMTs. However, this serious human diseases (6). extended conformation of product ligand is also similar to that seen in the unrelated DNA methyltransferases. As stated in the introduction of this article, our aim here was to define the number of PMT-related proteins that exist in humans Conspicuously absent from the PKMT family tree is the non-SET- and to understand their relatedness with respect to catalytic active domain enzyme DOT1L. DOT1L has been well demonstrated to have domain structure. Toward this goal, we have performed a system- histone H3K79 methyltransferase activity when presented with atic survey of the human genome for proteins that display structural nucleosomes as the methyl-accepting substrate. In addition to lack- alignment with SET-domain PKMTs, and likewise with PRMTs, for ing a SET-domain, DOT1L is further differentiated from the other which biochemical evidence of enzymatic activity has been demon- PKMTs by its strict substrate specificity for nucleosomes; in con- strated. Through multiple, iterative database searches and multiple trast, many of the SET-domain enzymes have been shown to utilize sequence alignments, we have arrived at the family trees for recombinant histones or peptides as methyl-accepting substrates. human PKMTs and PRMTs that are displayed in Figures 1 and 2, DOT1L is additionally distinct from the SET-domain enzymes in how respectively. it binds ligands. As previously reported for SAM (18), and reported here for SAH, DOT1L binds substrate and product ligands in an A number of features of these family trees deserve comment. For extended conformation that is significantly different from the 'U- the SET-domain PKMTs, our analysis indicates that there are 51 shaped' conformation seen in SET-domain PKMTs and is in fact related proteins with sequences that suggest putative SAM binding highly overlapping with the ligand conformation seen for the PRMTs and MT activity. These cluster into four major branches or groupings (Figure 5A,B). of putative enzymes. One such branch is the PRDM cluster. The proteins within this branch display the greatest divergence from the All attempts to include DOT1L within the PKMT family tree using canonical SET-domain structure of the rest of the PKMTs. Whether PSI-BLAST were unsuccessful. However, we were able to rationally these proteins are indeed bona fide PKMTs is unclear based on the include DOT1L within the PRMT family tree. Inclusion of DOT1L also current literature. Three PRDM family members (PRDM2, PRDM8, resulted in the inclusion of additional METTL and NSUN group and PRDM9) have been reported to demonstrate methylation of a members, but these protein groups were already represented within protein or peptide substrate (23–25). This highlights an important the PRMT tree when PRMT5, PRMT9, and PRMT11 were brought in area for biochemical follow-up to the data reported here. (vide supra). Hence, the incorporation of additional METTL and NSUN proteins here seems reasonable. The biochemical literature For the PRMTs, the majority of enzymes with well-documented bio- leaves no question as to the ability of DOT1L to act as a highly chemical activity as arginine methyltransferases cluster together to specific protein lysine methyltransferase (8). The literature is silent, form a single branch of the family tree. However, the enzymes however, on whether or not this enzyme may have additional meth- PRMT5 (36) and PRMT9 (37) have been clearly demonstrated to yltransferase activities, including arginine methylation. To our

208 Chem Biol Drug Des 2011; 78: 199–210 Chemogenetic Analysis of Human Protein Methyltransferases knowledge, no systematic evaluation of DOT1L activity beyond ality. In several instances, inhibitory ligands have been found to H3K79 methylation has been made. Again, on the basis of the cur- 'leap frog' over closely related enzymes to inhibit enzymes more rent data, we do not suggest that DOT1L is in fact an arginine distal to the desired target within the kinome. The same phenome- methyltransferase. Nevertheless, the inclusion of DOT1L within the non is observed for the PMTs with the ligands tested here. For PRMT family tree raises the question of what the full spectrum of example, EZH2 and EZH1 are very similar in active domain structure methyltransferase activity for DOT1L might be. Regardless of and are nearest neighbors on the PKMT tree. As expected from this whether or not DOT1L and ⁄ or the NSUN and METTL proteins are proximity, the two enzymes display identical KM values for the sub- bona fide arginine methyltransferases, the similarity of the active strate SAM and similar Ki values for the inhibitor sinefungin (Fig- sites of these enzymes to those of the PRMTs is nevertheless strik- ure 7 and Table 1). In contrast, however, these two enzymes show ing. Hence, any attempt to identify inhibitors of the PRMTs will more than an order of magnitude difference in product affinity. In require selectivity assessment against these other enzymes as this this latter respect, the affinity of EZH2 for SAH is more similar to may be a case where the structural determinants of pharmacologic that for SMYD2 and the PRMTs than to its nearest neighbor EZH1. selectivity and biological specificity are distinct. As additional inhibitors of PMTs are discovered, determining the pattern of selectivity among the PKMTs and PRMTs, as illustrated The data reported here provide a clear and simple mechanism for here, will be an important aspect of compound optimization, espe- understanding the full complement of human PMTs and their relat- cially for pharmacologic utility. edness to one another. The illustration of these relationships in the form of a family tree or dendogram has proved to be a very power- It is our hope that the information reported here will provide an ful tool for other enzyme families, most notably the protein kinases important starting point for the further exploration of the human (22); it is from examination of this type of family tree that the ki- PMTs as an enzyme class. The family trees that have been devel- nome concept was born. We believe that the data presented here oped in this report will be of value to the chemical biology and will be equally valuable as a research tool for investigators inter- drug discovery communities, as described earlier. The results also ested in studying the PMT enzymes. In particular, the information point to specific experiments to explore further the full repertoire of reported here will be of use in chemical biology and drug discovery PMT activities within humans. Whether or not DOT1L is a dual efforts aimed at identifying selective ligands for these enzymes. lysine ⁄ arginine methyltransferase and whether the NSUN proteins The PKMT and PRMT trees provide a cogent basis for investigating may methylate both nucleic acid and protein substrates are open ligand selectivity among these enzymes, as illustrated here for the questions that have been raised by the current analysis. The experi- ligands SAM, SAH, and sinefungin (Figure 7). mental resolution of these questions will be important tasks for the community to address in the continued efforts to understand protein Inspection of Figure 7 reveals some interesting patterns of ligand methylation and its impact on biology and disease. affinity among the PMTs, even with the limited ligand set considered here. For example, for all three ligands, one observes a significant range of affinity among the PKMTs, but a much narrower References affinity range among the PRMTs. Also, for the PKMTs, one generally observes greater relative affinity for the substrate SAM compared 1. Walsh C.T., Garneau-Tsodikova S., Gatto G.J. Jr (2005) Protein with the product SAH, which is typical of enzyme reactions; weaker posttranslational modifications: the chemistry of proteome diver- product affinity results in facile release of product, thus facilitating sifications. Angew Chem Int Ed Engl;44:7342–7372. subsequent rounds of substrate binding and turnover. In contrast, 2. Kouzarides T. (2007) Chromatin modifications and their function. the PRMTs display similar affinity for substrate and product. The Cell;128:693–705. more indiscriminate binding of substrate and inhibitors observed for 3. Feinberg A.P., Tycko B. (2004) The history of cancer epigenetics. the PRMTs may relate to the greater substrate promiscuity that is Nat Rev Cancer;4:143–153. inherent to the biological function of these enzymes. Thus, while 4. Kornberg R.D., Lorch Y. (1999) Twenty-five years of the nucleo- the substrate specificity among PKMTs is thought to be fairly some, fundamental particle of the eukaryote chromosome. restrictive, the PRMTs are known to methylate a broader range of Cell;98:285–294. protein substrates, including histones and cytosolic proteins. The 5. Strahl B.D., Allis C.D. (2000) The language of covalent histone data summarized in Figure 7 might lead one to infer that develop- modifications. Nature;403:41–45. ment of selective inhibitors for the PRMTs may prove to be a 6. Copeland R.A., Solomon M.E., Richon V.M. (2009) Protein meth- greater challenge than for the PKMTs. However, examples of selec- yltransferases as a target class for drug discovery. Nat Rev Drug tive inhibitors for enzymes in each of these families have already Discov;8:724–732. been reported [see (6) for a review]. 7. Schneider R., Bannister A.J., Kouzarides T. (2002) Unsafe SETs: histone lysine methyltransferases and cancer. Trends Biochem Thus, as just described, the selectivity of ligands for PKMTs and Sci;27:396–402. PRMTs can be rationally evaluated by reference to the trees pro- 8. Okada Y., Feng Q., Lin Y., Jiang Q., Li Y., Coffield V.M., Su L., Xu vided here. Drawing on the experience with the kinome, it will G., Zhang Y. (2005) hDOT1L links histone methylation to leuke- often be the case that enzymes in closest proximity to a desired mogenesis. Cell;121:167–178. target on a family tree will present the greatest challenge with 9. Dillon S.C., Zhang X., Trievel R.C., Cheng X. (2005) The SET- respect to ligand selectivity. However, the kinome experience also domain protein superfamily: protein lysine methyltransferases. teaches that there are important exceptions to the foregoing gener- Genome Biol;6:227.

Chem Biol Drug Des 2011; 78: 199–210 209 Richon et al.

10. Cheng X., Collins R.E., Zhang X. (2005) Structural and sequence 27. Sakita-Suto S., Kanda A., Suzuki F., Sato S., Takata T., Tatsuka motifs of protein (histone) methylation enzymes. Annu Rev Bio- M. (2007) Aurora-B regulates RNA methyltransferase NSUN2. phys Biomol Struct;34:267–294. Mol Biol Cell;18:1107–1117. 11. Bachand F. (2007) Protein arginine methyltransferases: from uni- 28. Copeland R.A. (2000) Enzymes: A Practical Introduction to Struc- cellular eukaryotes to humans. Eukaryot Cell;6:889–898. ture, Mechanism and Data Analysis, 2nd edn. Hoboken: Wiley. 12. Krause C.D., Yang Z.H., Kim Y.S., Lee J.H., Cook J.R., Pestka S. 29. Copeland R.A. (2005) Evaluation of enzyme inhibitors in drug dis- (2007) Protein arginine methyltransferases: evolution and assess- covery. A guide for medicinal chemists and pharmacologists. ment of their pharmacological and therapeutic potential. Phar- Methods Biochem Anal;46:1–265. macol Ther;113:50–87. 30. Copeland R.A. (2003) Mechanistic considerations in high- 13. Wu H., Min J., Lunin V.V., Antoshenko T., Dombrovski L., Zeng throughput screening. Anal Biochem;320:1–12. H., Allali-Hassani A., Campagna-Slater V., Vedadi M., Arrow- 31. Yang J., Copeland R.A., Lai Z. (2009) Defining balanced condi- smith C.H., Plotnikov A.N., Schapira M. (2010) Structural biology tions for inhibitor screening assays that target bisubstrate of human H3K9 methyltransferases. PLoS ONE;5:e8570. enzymes. J Biomol Screen;14:111–120. 14. Boeckmann B., Bairoch A., Apweiler R., Blatter M.C., Estreicher 32. Cheng Y., Prusoff W.H. (1973) Relationship between the inhibi- A., Gasteiger E., Martin M.J., Michoud K., O'Donovan C., Phan tion constant (K1) and the concentration of inhibitor which I., Pilbout S., Schneider M. (2003) The SWISS-PROT protein causes 50 per cent inhibition (I50) of an enzymatic reaction. Bio- knowledgebase and its supplement TrEMBL in 2003. Nucleic chem Pharmacol;22:3099–3108. Acids Res;31:365–370. 33. Simon J.A., Lange C.A. (2008) Roles of the EZH2 histone methyl- 15. Sneeringer C.J., Scott M.P., Kuntz K.W., Knutson S.K., Pollock transferase in cancer epigenetics. Mutat Res;647:21–29. R.M., Richon V.M., Copeland R.A. (2010) Coordinated activities 34. Martinez-Garcia E., Licht J.D. (2010) Deregulation of H3K27 of wild-type plus mutant EZH2 drive tumor-associated hypertrim- methylation in cancer. Nat Genet;42:100–101. ethylation of lysine 27 on histone H3 (H3K27) in human B-cell 35. Morin R.D., Johnson N.A., Severson T.M., Mungall A.J., An J., lymphomas. Proc Natl Acad Sci U S A;107:20980–20985. Goya R., Paul J.E. et al. (2010) Somatic mutations altering EZH2 16. Otwinowski Z., Minor W. (1997) Processing of X-ray diffraction (Tyr641) in follicular and diffuse large B-cell lymphomas of ger- data collected in oscillation mode. Methods Enzymol;276:307– minal-center origin. Nat Genet;42:181–185. 326. 36. Rho J., Choi S., Seong Y.R., Cho W.K., Kim S.H., Im D.S. (2001) 17. Vagin A., Teplyakov A. (1997) MOLREP: an automated program Prmt5, which forms distinct homo-oligomers, is a member of the for molecular replacement. J Appl Cryst;30:1022–1025. protein-arginine methyltransferase family. J Biol Chem;276: 18. Min J., Feng Q., Li Z., Zhang Y., Xu R.M. (2003) Structure of the 11393–11401. catalytic domain of human DOT1L, a non-SET domain nucleoso- 37. Cook J.R., Lee J.H., Yang Z.H., Krause C.D., Herth N., Hoffmann mal histone methyltransferase. Cell;112:711–723. R., Pestka S. (2006) FBXO11 ⁄ PRMT9, a new protein arginine 19. Vagin A.A., Steiner R.A., Lebedev A.A., Potterton L., McNicholas methyltransferase, symmetrically dimethylates arginine residues. S., Long F., Murshudov G.N. (2004) REFMAC5 dictionary: organi- Biochem Biophys Res Commun;342:472–481. zation of prior chemical knowledge and guidelines for its use. 38. Webb K.J., Lipson R.S., Al-Hadid Q., Whitelegge J.P., Clarke Acta Crystallogr D Biol Crystallogr;60:2184–2195. S.G. (2010) Identification of protein N-terminal methyltransferas- 20. Emsley P., Cowtan K. (2004) Coot: model-building tools for es in yeast and humans. Biochemistry;49:5225–5235. molecular graphics. Acta Crystallogr D Biol Crystallogr;60:2126– 2132. Supporting Information 21. Fang J., Wang H., Zhang Y. (2004) Purification of histone methyltransferases from HeLa cells. Methods Enzymol;377:213–226. Additional Supporting Information may be found in the online ver- 22. Manning G., Whyte D.B., Martinez R., Hunter T., Sudarsanam S. sion of this article: (2002) The protein kinase complement of the human genome. Science;298:1912–1934. Figure S1. PKMT dendogram. 23. Kim K.C., Geng L., Huang S. (2003) Inactivation of a histone methyltransferase by mutations in human cancers. Cancer Figure S2. PKMT alignment. Res;63:7619–7623. 24. Eom G.H., Kim K., Kim S.M., Kee H.J., Kim J.Y., Jin H.M., Kim J.R., Figure S3. PRMT dendogram. Kim J.H., Choe N., Kim K.B., Lee J., Kook H., Kim N., Seo S.B. (2009) Histone methyltransferase PRDM8 regulates mouse testis Figure S4. PRMT alignment. steroidogenesis. Biochem Biophys Res Commun;388:131–136. 25. Hayashi K., Yoshida K., Matsui Y. (2005) A histone H3 methyl- Table S1. Data collection and refinement statistics. transferase controls epigenetic events required for meiotic pro- phase. Nature;438:374–378. Please note: Wiley-Blackwell is not responsible for the content or 26. Alexandrov A., Martzen M.R., Phizicky E.M. (2002) Two proteins functionality of any supporting materials supplied by the authors. that form a complex are required for 7-methylguanosine modifi- Any queries (other than missing material) should be directed to the cation of yeast tRNA. RNA;8:1253–1266. corresponding author for the article.

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