Inhibition of Human Enhancer of Zeste Homolog 2 with Tambjamine Analogs
Martin KotevPilar Manuel-ManresaElsa HernandoVanessa Soto‐CerratoModesto OrozcoRoberto QuesadaRicardo Pérez‐TomásVı́ctor Guallar
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Combining computational modeling, de novo compound synthesis, and in vitro and cellular assays, we have performed an inhibition study against the enhancer of zeste homolog 2 (EZH2) histone-lysine N-methyltransferase. This enzyme is an important catalytic component of the PRC2 complex whose alterations have been associated with different cancers. We introduce here several tambjamine-inspired derivatives with low micromolar in vitro activity that produce a significant decrease in histone 3 trimethylation levels in cancer cells. We demonstrate binding at the methyl transfer active site, showing, in addition, that the EZH2 isolated crystal structure is capable of being used in molecular screening studies. Altogether, this work provides a successful molecular model that will help in the identification of new specific EZH2 inhibitors and identify a novel class of tambjamine-derived EZH2 inhibitors with promising activities for their use in cancer treatment.Keywords:
PRC2
vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy.
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Although central to regulating the access to genetic information, most lysine methyltransferases remain poorly characterised relative to other family of enzymes. Herein, I report new substrates for the lysine methyltransferase SETD6. Based on the SETD6-catalysed site on the histone variant H2AZ, I identified similar sequences in the canonical histones H2A, H3, and H4 that are modified by SETD6 in vitro, and putative non-histone substrates. I herein expend the repertoire of substrates for methylation by SETD6.
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PRC2
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The epigenetic control of gene expression could be affected by addition and/or removal of post-translational modifications such as phosphorylation, acetylation and methylation of histone proteins, as well as methylation of DNA (5-methylation on cytosines). Misregulation of these modifications is associated with altered gene expression, resulting in various disease conditions. G9a belongs to the protein lysine methyltransferases that specifically methylates the K9 residue of histone H3, leading to suppression of several tumor suppressor genes. In this review, G9a functions, role in various diseases, structural biology aspects for inhibitor design, structure–activity relationship among the reported inhibitors are discussed which could aid in the design and development of potent G9a inhibitors for cancer treatment in the future.
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Lysine methylation is one of the most important modification, which is regulated by histone lysine methyltransferases and histone lysine demethylases. Lysine-specific demethylase 1 (LSD1) specifically demethylates mono- and dimethyl-lysine on histone H3 (H3K4Me/Me2, H3K9Me/Me2) to control chromatin structure, resulting in transcriptional repression or activation of target genes. Furthermore, LSD1 is overexpressed in various cancers. Therefore, LSD1 inhibitors would be not only potential therapeutic agents for cancers but also chemical tools to research biological significance of LSD1 in physiological and pathological events. However, known assay methods to date have some inherent drawbacks. The development of simple method in detecting LSD1 activity has been indispensable to identify useful inhibitors. In this study, we designed and synthesized artificial substrates based on inhibitors of LSD1 to examine LSD1 activity by an absorption increment.
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Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using 15N metabolically labeled histones as the internal reference. Using this approach, we identified Tetrahymena trithorax related 1 (Txr1p) as a histone methyltransferase in Tetrahymena thermophila and characterized the relationships of the Txr1p and Ezl2p methyltransferases to histone H3 modification. We identified 32 PTMs in more than 60 tryptic peptides from histone H3 of the ciliate model organism Tetrahymena thermophila, and we quantified them (average coefficient of variation: 13%). We examined perturbations to histone modification patterns in two knockout strains of SET-domain-containing histone methyltransferases (HMT). Knockout of TXR1 led to progressively decreased mono-, di-, and tri-methylation of H3K27 and apparent reduced monomethylation of H3K36 in vivo. In contrast, EZL2 knockout resulted in dramatic reductions in both di- and tri-methylation of H3K27 in vivo, whereas the levels of monomethylation of H3K27 increased significantly. This buildup of monomethyl H3K27 is consistent with its role as a substrate for Ezl2p. These results were validated via immunoblotting using modification site-specific antibodies. Taken together, our studies define Txr1p as an H3K27 monomethylation-specific HMT that facilitates the buildup of H3K27 di- and trimethylation by the canonical H3K27-specific HMT, Ezl2p. Our studies also delineate some of the interdependences between various H3 modifications, as compensatory increases in monomethylation at H3K4, H3K23, and H3K56 were also observed for both TXR1 and ELZ2 mutants. Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using 15N metabolically labeled histones as the internal reference. Using this approach, we identified Tetrahymena trithorax related 1 (Txr1p) as a histone methyltransferase in Tetrahymena thermophila and characterized the relationships of the Txr1p and Ezl2p methyltransferases to histone H3 modification. We identified 32 PTMs in more than 60 tryptic peptides from histone H3 of the ciliate model organism Tetrahymena thermophila, and we quantified them (average coefficient of variation: 13%). We examined perturbations to histone modification patterns in two knockout strains of SET-domain-containing histone methyltransferases (HMT). Knockout of TXR1 led to progressively decreased mono-, di-, and tri-methylation of H3K27 and apparent reduced monomethylation of H3K36 in vivo. In contrast, EZL2 knockout resulted in dramatic reductions in both di- and tri-methylation of H3K27 in vivo, whereas the levels of monomethylation of H3K27 increased significantly. This buildup of monomethyl H3K27 is consistent with its role as a substrate for Ezl2p. These results were validated via immunoblotting using modification site-specific antibodies. Taken together, our studies define Txr1p as an H3K27 monomethylation-specific HMT that facilitates the buildup of H3K27 di- and trimethylation by the canonical H3K27-specific HMT, Ezl2p. Our studies also delineate some of the interdependences between various H3 modifications, as compensatory increases in monomethylation at H3K4, H3K23, and H3K56 were also observed for both TXR1 and ELZ2 mutants. Histones, and especially their N-terminal tails, are subject to various covalent post-translational modifications (PTMs) 1The abbreviations used are:AcacetylationATXRArabidopsis trithorax relatedCVcoefficient of variationEZL2enhancer of zeste-like 2HMThistone methyltransferaseMe1monomethylationMe2dimethylationMe3trimethylationPCAprincipal component analysisPHDplant homeodomainPrpropionylationPTMpost-translational modificationSETsuppressor of variegation, enhancer of zeste, trithoraxTXR1Tetrahymena trithorax related 1. 1The abbreviations used are:AcacetylationATXRArabidopsis trithorax relatedCVcoefficient of variationEZL2enhancer of zeste-like 2HMThistone methyltransferaseMe1monomethylationMe2dimethylationMe3trimethylationPCAprincipal component analysisPHDplant homeodomainPrpropionylationPTMpost-translational modificationSETsuppressor of variegation, enhancer of zeste, trithoraxTXR1Tetrahymena trithorax related 1. including acetylation, methylation, phosphorylation, ubiquitination, and citrullination (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8036) Google Scholar, 2Campos E.I. Reinberg D. Histones: annotating chromatin.Annu. Rev. Genet. 2009; 43: 559-599Crossref PubMed Scopus (646) Google Scholar, 3Allis C.D. Jenuwein T. Reinberg D. Caparros M.L. Epigenetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2007: 23-62Google Scholar). A combinatorial set of PTMs on one or more histones, deposited by histone-modifying enzymes, effectively serves to modulate various DNA pathways, including gene expression and replication as postulated in the histone code hypothesis (4Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7633) Google Scholar, 5Strahl B.D. Allis C.D. The language of covalent histone modifications.Nature. 2000; 403: 41-45Crossref PubMed Scopus (6585) Google Scholar). Prominent among the PTMs is the reversible epigenetic mark, lysine methylation, present in mono-, di-, and trimethylation states. Methyl groups are added to the ε-amine of the lysyl residue by histone methyltransferases (HMTs) and removed by histone demethylases (6Klose R.J. Zhang Y. Regulation of histone methylation by demethylimination and demethylation.Nat. Rev. Mol. Cell. Biol. 2007; 8: 307-318Crossref PubMed Scopus (664) Google Scholar, 7Zhang Y. Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails.Genes Dev. 2001; 15: 2343-2360Crossref PubMed Scopus (1234) Google Scholar, 8Chi P. Allis C.D. Wang G.G. Covalent histone modifications—miswritten, misinterpreted and mis-erased in human cancers.Nat. Rev. Cancer. 2010; 10: 457-469Crossref PubMed Scopus (863) Google Scholar, 9Bannister A.J. Kouzarides T. Reversing histone methylation.Nature. 2005; 436: 1103-1106Crossref PubMed Scopus (391) Google Scholar). Different lysine methylation states are often associated with different—sometimes even opposite—biological functions. For example, the monomethylation of histone residues H3K9, H3K27, and H4K20 is linked to active transcription, whereas their trimethylation states are associated with transcriptional repression (6Klose R.J. Zhang Y. Regulation of histone methylation by demethylimination and demethylation.Nat. Rev. Mol. Cell. Biol. 2007; 8: 307-318Crossref PubMed Scopus (664) Google Scholar, 10Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome.Cell. 2007; 129: 823-837Abstract Full Text Full Text PDF PubMed Scopus (5056) Google Scholar). The functional distinction of the different methylation states is further underscored by the presence of divergent state-specific HMTs, such as SETDB1/SETDB2 (for H3K9Me1) and SUV39H1/SUV39H2 (for H3K9Me2 and H3K9Me3) (11Loyola A. Tagami H. Bonaldi T. Roche D. Quivy J.P. Imhof A. Nakatani Y. Dent S.Y. Almouzni G. The HP1alpha-CAF1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin.EMBO Rep. 2009; 10: 769-775Crossref PubMed Scopus (160) Google Scholar), SETD8 (for H4K20Me1) (12Beck D.B. Oda H. Shen S.S. Reinberg D. PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription.Genes Dev. 2012; 26: 325-337Crossref PubMed Scopus (218) Google Scholar), and SUV4–20 (for H4K20Me2 and H4K20Me3) (13Schotta G. Lachner M. Sarma K. Ebert A. Sengupta R. Reuter G. Reinberg D. Jenuwein T. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.Genes Dev. 2004; 18: 1251-1262Crossref PubMed Scopus (846) Google Scholar). It is a major challenge in the field of epigenetics to unravel the mechanism regulating histone lysine methylation events, which are dynamically affected by many factors and implicated in various biological processes. The modifications of histone lysines are dynamic, and mono-, di-, and trimethylated residues are generally considered to be progressively methylated in vivo (14Zee B.M. Levin R.S. Xu B. LeRoy G. Wingreen N.S. Garcia B.A. In vivo residue-specific histone methylation dynamics.J. Biol. Chem. 2010; 285: 3341-3350Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). acetylation Arabidopsis trithorax related coefficient of variation enhancer of zeste-like 2 histone methyltransferase monomethylation dimethylation trimethylation principal component analysis plant homeodomain propionylation post-translational modification suppressor of variegation, enhancer of zeste, trithorax Tetrahymena trithorax related 1. acetylation Arabidopsis trithorax related coefficient of variation enhancer of zeste-like 2 histone methyltransferase monomethylation dimethylation trimethylation principal component analysis plant homeodomain propionylation post-translational modification suppressor of variegation, enhancer of zeste, trithorax Tetrahymena trithorax related 1. Most HMTs for lysine methylation contain a conserved catalytic domain, the SET (suppressor of variegation, enhancer of zeste, trithorax) domain (15Albert M. Helin K. Histone methyltransferases in cancer.Semin. Cell. Dev. Biol. 2010; 21: 209-220Crossref PubMed Scopus (231) Google Scholar). HMTs, in particular lysine methyltransferases, have been implicated in human diseases, including cancers (16Zhang K. Dent S.Y. Histone modifying enzymes and cancer: going beyond histones.J. Cell. Biochem. 2005; 96: 1137-1148Crossref PubMed Scopus (129) Google Scholar). In the past decade, a large number of HMTs have been identified in a wide range of eukaryotic organisms, and they have been classified according to their sequence homology into subfamilies, whose members generally share the substrate specificity. Arabidopsis trithorax related 5 (ATXR5) and ATXR6, the founding members of a recently identified HMT subfamily, were first isolated as proliferating cell nuclear antigen interacting proteins in Arabidopsis thaliana (17Raynaud C. Sozzani R. Glab N. Domenichini S. Perennes C. Cella R. Kondorosi E. Bergounioux C. Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.Plant J. 2006; 47: 395-407Crossref PubMed Scopus (86) Google Scholar). Both ATXR5 and ATXR6 feature a divergent SET domain (17Raynaud C. Sozzani R. Glab N. Domenichini S. Perennes C. Cella R. Kondorosi E. Bergounioux C. Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.Plant J. 2006; 47: 395-407Crossref PubMed Scopus (86) Google Scholar), a plant homeodomain (PHD) finger that binds the modified histones (18Sanchez R. Zhou M.M. The PHD finger: a versatile epigenome reader.Trends Biochem. Sci. 2011; 36: 364-372Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 19Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF.Nature. 2006; 442: 91-95Crossref PubMed Scopus (183) Google Scholar, 20Peña P.V. Davrazou F. Shi X. Walter K.L. Verkhusha V.V. Gozani O. Zhao R. Kutateladze T.G. Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2.Nature. 2006; 442: 100-103Crossref PubMed Scopus (555) Google Scholar, 21Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Peña P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Côté J. Chua K.F. Gozani O. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression.Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar), and a proliferating cell nuclear antigen interacting protein box that binds proliferating cell nuclear antigen (22Maga G. Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners.J. Cell Sci. 2003; 116: 3051-3060Crossref PubMed Scopus (868) Google Scholar). Homologues of ATXR5 and ATXR6 are found in plants but not in animals. The atxr5 atxr6 double mutant exhibits reduced H3K27Me1 levels (23Jacob Y. Feng S. LeBlanc C.A. Bernatavichute Y.V. Stroud H. Cokus S. Johnson L.M. Pellegrini M. Jacobsen S.E. Michaels S.D. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.Nat. Struct. Mol. Biol. 2009; 16: 763-768Crossref PubMed Scopus (215) Google Scholar), supporting the notion that ATXR5 and ATXR6 function as the H3K27 monomethylation-specific HMTs. Tetrahymena trithorax related 1 (Txr1p) was recently identified as a putative HMT in the ciliate model organism Tetrahymena thermophila (encoded by the TXR1 gene) via homology to Arabidopsis ATXR5 and ATXR6. Txr1p carries two PHD domains (PHD1 and PHD2), one proliferating cell nuclear antigen interacting protein box (QKLIEDYF), and one C-terminal SET domain (Fig. 1), all of which are consistent with its being a bona fide member of the ATXR5/ATXR6 subfamily of HMTs. In Tetrahymena, there are also three homologues of the canonical H3K27-specific HMT enhancer of zeste, referred to as EZL1, EZL2, and EZL3, respectively (24Liu Y. Taverna S.D. Muratore T.L. Shabanowitz J. Hunt D.F. Allis C.D. RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena.Genes Dev. 2007; 21: 1530-1545Crossref PubMed Scopus (194) Google Scholar, 25Chung P.H. Yao M.C. Tetrahymena JMJD3 homolog regulates H3K27 methylation and nuclear differentiation.Eukaryot. Cell. 2012; 11: 601-614Crossref PubMed Scopus (16) Google Scholar). Only EZL2 is expressed at significant levels (24Liu Y. Taverna S.D. Muratore T.L. Shabanowitz J. Hunt D.F. Allis C.D. RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena.Genes Dev. 2007; 21: 1530-1545Crossref PubMed Scopus (194) Google Scholar, 25Chung P.H. Yao M.C. Tetrahymena JMJD3 homolog regulates H3K27 methylation and nuclear differentiation.Eukaryot. Cell. 2012; 11: 601-614Crossref PubMed Scopus (16) Google Scholar) and required for H3K27 di- and trimethylation in asexually dividing cells (see below). Mass spectrometry (MS) has played an important role in the study of histone PTMs for the following reasons: (1) MS is capable of simultaneously monitoring multiple PTMs; and (2) it can identify and quantify known and unknown PTMs in histones that cannot be easily determined via other analytical approaches such as micro-sequencing by Edman degradation or immunoblotting with modification site-specific antibodies (26Zhang K. Tang H. Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping.J. Chromatogr. 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The quantification of histone PTMs can be achieved via a label-free strategy based on the relative intensities of extracted ion chromatograms of precursors (31Beck H.C. Nielsen E.C. Matthiesen R. Jensen L.H. Sehested M. Finn P. Grauslund M. Hansen A.M. Jensen O.N. Quantitative proteomic analysis of post-translational modifications of human histones.Mol. Cell. Proteomics. 2006; 5: 1314-1325Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) or, more accurately through stable isotope labeling techniques such as SILAC or iTRAQ (32Plazas-Mayorca M.D. Bloom J.S. Zeissler U. Leroy G. Young N.L. DiMaggio P.A. Krugylak L. Schneider R. Garcia B.A. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown.Mol. Biosyst. 2010; 6: 1719-1729Crossref PubMed Scopus (29) Google Scholar, 33Jung H.R. Pasini D. Helin K. Jensen O.N. Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36.Mol. Cell. Proteomics. 2010; 9: 838-850Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 34Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar, 35Garcia B.A. Joshi S. Thomas C.E. Chitta R.K. Diaz R.L. Busby S.A. Andrews P.C. Ogorzalek Loo R.R. Shabanowitz J. Kelleher N.L. Mizzen C.A. Allis C.D. Hunt D.F. Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.Mol. Cell. Proteomics. 2006; 5: 1593-1609Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, the analysis of histone PTMs via LC/MS is particularly challenging because of the enormous number of isoforms generated by the combination of various densely deposited PTMs (29Ueberheide B. Mollah S. Deciphering the histone code using mass spectrometry.Int. J. Mass Spectrom. 2007; 259: 46-56Crossref Scopus (15) Google Scholar). The problem is further exacerbated by the basicity of histones, which, after trypsin digestion, generate peptides too small or hydrophilic to be effectively retained on reversed-phase HPLC columns and analyzed via MS. Chemical derivatization using propionic anhydride was introduced in order to overcome some of these challenges (36Syka J.E.P. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B.A. Busby S. Muratore T. Shabanowitz J. Hunt D.F. Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications.J. Proteome Res. 2004; 3: 621-626Crossref PubMed Scopus (333) Google Scholar, 37Garcia B.A. Mollah S. Ueberheide B.M. Busby S.A. Muratore T.L. Shabanowitz J. Hunt D.F. Chemical derivatization of histones for facilitated analysis by mass spectrometry.Nat. Protoc. 2007; 2: 933-938Crossref PubMed Scopus (279) Google Scholar). Briefly, the propionylated histones are only cleaved after arginyl residues when digested with trypsin and thus generate nicely sized, more hydrophobic peptides that can be more readily analyzed via LC/MS. When samples are chemically labeled with light and heavy isotopes (d0/d10-propionic anhydride), the relative levels of individual modifications from the two samples can be quantified by means of MS (32Plazas-Mayorca M.D. Bloom J.S. Zeissler U. Leroy G. Young N.L. DiMaggio P.A. Krugylak L. Schneider R. Garcia B.A. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown.Mol. Biosyst. 2010; 6: 1719-1729Crossref PubMed Scopus (29) Google Scholar). The deuterium isotope effect on chromatography and the variations in differential labeling from one peptide to another, however, place some limitations on the precise quantification of histone PTMs. If metabolic labeling is used for quantification, variations in propionylation can be minimized because different physiological samples are combined prior to reaction with propionic anhydride. The major aims of this study were to determine the roles of the Txr1p and Ezl2p methyltransferases in shaping histone modification patterns, with the focus on H3. This was achieved through quantifying the levels of various histone modifications in TXR1 and EZL2 knockout cells, as well as the wild-type cells. For this purpose, we developed a robust MS-based quantitative proteomics method for the study of histone PTMs using 15N metabolically labeled histones as internal standards spiked into histone preparations as references. The general strategy and experimental design for this uniform labeling technique are illustrated in Fig. 2. Similar studies using isotope-labeled tissue or cells as a global internal standard have been carried out in mammals (38Ishihama Y. Sato T. Tabata T. Miyamoto N. Sagane K. Nagasu T. Oda Y. Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.Nat. Biotechnol. 2005; 23: 617-621Crossref PubMed Scopus (195) Google Scholar, 39Wu C.C. MacCoss M.J. Howell K.E. Matthews D.E. Yates III, J.R. Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis.Anal. Chem. 2004; 76: 4951-4959Crossref PubMed Scopus (339) Google Scholar). Overall, more than 60 unique H3 tryptic peptides were successfully quantified using this technique with small statistical variation (average coefficient of variation (CV): ∼13%). Similar to Super-SILAC, which combines a mixture of several stable-isotope-labeled cell lines to serve as internal standards for MS-based analysis (40Geiger T. Cox J. Ostasiewicz P. Wisniewski J.R. Mann M. Super-SILAC mix for quantitative proteomics of human tumor tissue.Nat. Methods. 2010; 7: 383-385Crossref PubMed Scopus (432) Google Scholar), our method provides a cost-effective alternative for studying protein PTMs, especially in systems for which the SILAC medium is not available or cannot be easily formulated and for which iTRAQ-based quantitative proteomics techniques are not easily applicable. To generate the TXR1 and ELZ2 knockout constructs, the genomic regions flanking TXR1 or ELZ2 were PCR amplified from the wild-type Tetrahymena cells and fused with the neo4 cassette (41Loidl J. Mochizuki K. Tetrahymena meiotic nuclear reorganization is induced by a checkpoint kinase-dependent response to DNA damage.Mol. Biol. Cell. 2009; 20: 2428-2437Crossref PubMed Scopus (40) Google Scholar), which confers paromomycin resistance to Tetrahymena cells. The constructs were introduced into Tetrahymena cells via standard biolistic transformations (42Cassidy-Hanley D. Bowen J. Lee J.H. Cole E. VerPlank L.A. Gaertig J. Gorovsky M.A. Bruns P.J. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment.Genetics. 1997; 146: 135-147Crossref PubMed Google Scholar). Transformants were selected for paromomycin resistance and complete replacement was finally confirmed via quantitative PCR. Tetrahymena thermophila wild-type strain CU428 (Tetrahymena Stock Center) and TXR1 and ELZ2 knockout strains were grown in 1× SPP medium (2% protease peptone, 0.2% dextrose, 0.1% yeast extract, 0.003% sequestrine) at 30 °C with gentle shaking. Logarithmic-phase cells (2 × 105/ml) were collected for subsequent experiments. For 15N labeling of wild-type Tetrahymena cells, 15N-labeled Escherichia coli BL21 cells were grown in [15N] M9 minimal medium (30 mm Na2HPO4, 2 g/l KH2PO4, 0.5 g/l NaCl, 300 μm Na2SO4, 1 mm MgSO4, 0.3 mm CaCl2, 1 μg/ml biotin, 1 μg/ml thiamine, 10 g/l glucose, and 1 g/l (15NH4)2SO4 (Cambridge Isotope Laboratories, Andover, MA)) supplemented with 15N-substituted Bioexpress (Cambridge Isotope Laboratories). Briefly, E. coli BL21 cells were inoculated into a small LB starter culture and incubated at 37 °C with vigorous shaking until reaching the logarithmic phase (OD600: 0.5 to 1). 1.0 ml of the starter culture was inoculated into 500 ml of the [15N] M9 media and incubated overnight at 37 °C with vigorous shaking. Stationary-phase E. coli BL21 cells were collected via centrifugation, and the cell pellet was resuspended in 500 ml of 1× phosphate buffer (0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4). Added into this labeling medium were inoculated Tetrahymena cells from a logarithmic-phase small starter culture grown in 1× SPP medium. The culture was incubated at 30 °C with gentle shaking for 48 h, with 15N-labeled E. coli BL21 cells as the only nitrogen source. The labeled Tetrahymena cells were collected via centrifugation for subsequent nuclear preparations. The procedure for isolating the macronuclei from Tetrahymena cells was adapted from a previously reported protocol (43Gorovsky M.A. Studies on nuclear structure and function in Tetrahymena pyriformis. II. Isolation of macro- and micronuclei.J. Cell Biol. 1970; 47: 619-630Crossref PubMed Scopus (53) Google Scholar). Briefly, Tetrahymena cells were resuspended in 200 ml of medium A (0.1 m sucrose, 2 mm MgCl2, 4% gum arabic, 10 mm Tris, 5 mm EDTA, 10 mm butyric acid, 1 mm iodoacetamide, 1 mm PMSF, adjusted to pH 6.5). Cells were disrupted via vigorous blending in the presence of 1-octanol (0.7 ml). Tetrahymena macronuclei were pelleted by means of differential centrifugation. They were then acid extracted with 1 ml of 0.4 N sulfuric acid, as reported elsewhere (44Gorovsky M.A. Studies on nuclear structure and function in Tetrahymena pyriformis. 3. Comparison of the histones of macro- and micronuclei by quantitative polyacrylamide gel electrophoresis.J. Cell Biol. 1970; 47: 631-636Crossref PubMed Scopus (17) Google Scholar). The acid-extracted histones were precipitated by TCA (20% w/v). After being washed once with acidified actone (0.2% HCl) and once with acetone, the histone samples were air-dried and resuspended in 500 μl of water. The histone samples were further purified on a C8 reversed-phase HPLC column (Vydac Part No. 208TP54, 250 mm × 4.6 mm) on a Rainin Rabbit HPLC with 5 ml/min pump heads, with the HPLC run conditions as reported elsewhere (45Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). Briefly, the HPLC column was equilibrated with 100% solvent A (5% acetonitrile in 0.1% TFA) for 5 min, and then 35% solvent B was added (90% acetonitrile in 0.1% TFA) and the column was equilibrated for another 5 min. A 60-min gradient to 65% solvent B was applied to elute core histones from the column. Finally, the column was washed with 100% solvent B for 10 min and then re-equilibrated with 100% solvent A. HPLC fractions were vacuum dried, resuspended in deionized water, and evaluated via 15% SDS-PAGE, and those containing individual histones were combined. Concentrations of the purified histones were determined via the Bradford method (Bio-Rad). For each biological replicate (n = 3), 5 μg of histone H3 from wild-type, ΔTXR1, or ΔEZL2 cells grown in 1× SPP medium was mixed with an equal amount of 15N-labeled H3 separated and purified from wild-type cells. The two-step chemical derivatization of histone H3 with propionic anhydride, adopted from the work of Garcia et al. (37Garcia B.A. Mollah S. Ueberheide B.M. Busby S.A. Muratore T.L. Shabanowitz J. Hunt D.F. Chemical derivatization of histones for facilitated analysis by mass spectrometry.Nat. Protoc. 2007; 2: 933-938Crossref PubMed Scopus (279) Google Scholar), was performed before and after trypsin digestion to increase the hydrophobicity of tryptic peptides, as histones are very basic proteins with relatively short retention times on a reversed-phase column. Briefly, dried histone H3 samples were resuspended in 5 μl 100 mm ammonium bicarbonate. Samples were then treated with 20 μl propionylation reagent made with 3:1 (v/v) anhydrous methanol (Alfa Aesar, Ward Hill, MA):propionic anhydride (Sigma Aldrich), and this was immediately followed by the addition of ∼15 μl ammonium hydroxide (Sigma Aldrich) to raise the pH to 8.0. The reaction mixtures were incubated for 15 min at 50 °C and then concentrated to ∼5 μl in a SpeedVac concentrator. The propionylation reaction was performed twice to ensure the maximum conversion of primary amines to propionyl amides. Generally, more than 95% propionylation efficiency was achieved after two rounds of chemical derivatization. No significant evidence of Asn or Gln deamidation (a potential side reaction of propionylation) for H3 peptides was found through database searches. Propionylated samples were again brought up in 100 mm ammonium bicarbonate buffer and in-solution digested with sequencing-grade trypsin (Promega, Madison, WI) at a ratio of 1:20 (enzyme:substrate) at 37 °C for 6 h. The reactions were quenched by TFA (10% w/v). A second round of propionylation was performed as described above to convert the newly generated N termini to propionyl amides. Finally, the reaction mixture was vacuum dried and reconstituted in 0.1% formic acid. After filtering (Millipore Ultracel YM-10), the sample was stored in a −20° Celsius freezer until the MS analysis. The histone-derived peptides were resolved with a C18 capillary column (3 μm, 300 Å, 150 mm × 100 μm; CVC Technologies,
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The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z′>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.
PRC2
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Post translational modification of histone proteins including lysine methylation is an important epigenetic mark, essential for gene regulation and development. Recently, several examples of lysine methylation of non-histone proteins have been discovered suggesting that this is a common post-translational modification for regulation of protein activity. Here, we review assays for the detection of protein methylation based on mass spectrometry, radiolabel and immunological approaches using protein and peptide substrates including application of SPOT peptide arrays. Candidates for new methylation targets of protein methyltransferases can be predicted using the specificity of the enzyme and protein interaction data.
Protein methylation
Histone Methylation
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ABSTRACT Although central to regulating the access to genetic information, most lysine methyltransferases remain poorly characterised relative to other family of enzymes. Herein, we report new substrates for the lysine methyltransferase SETD6. Based on the SETD6- catalysed site on the histone variant H2AZ, we identified similar sequences in the canonical histones H2A, H3, and H4 that are modified by SETD6 in vitro , and putative non-histone substrates. We herein expend the repertoire of substrates for methylation by SETD6.
Histone Methylation
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The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.
Identification
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