Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.new_PeptideGroups.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant_PD_search_settings.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.new_Proteins.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Mock.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.mzid.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/Human_UniProtRev_Nov2017_LCORvariant.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD013390/20181222_Peder_Lewis_Proteome_E3_Kcv1.repeatLCORvariant.mgfprimaryOK200bgarci@pennmedicine.upenn.eduPeder LundAffinity purification coupled with mass spectrometry proteomicsMass Spectrometry2.2.0.388 2.2.0.388Cxorf67Prc2EpigeneticsH3k27meEzhiphttp://www.ebi.ac.uk/pride/archive/projects/PXD013390Cell CultureNuclei from control 293T cells or 293T cells expressing FLAG-tagged Cxorf67 (proposed to be re-named EZHIP) were isolated by resuspending cells in buffer A (15 mM HEPES pH 7.9, 4 mM MgCl2, 10 mM KCl, 1 mM EDTA, 4 mM PMSF) followed by centrifugation. The nuclear pellet was then re-suspended in buffer AC (15 mM HEPES pH 7.9) and nuclear extract was prepared by adding 1/10th volume of saturated ammonium sulfate followed by ultracentrifugation at 28,000 rpm for 90 mins. The supernatant (nuclear extract) was then dialyzed against FLAG-IP buffer (20 mM HEPES pH 7.9, 250 mM KCl, 1 mM EDTA, 2 mM β-mercaptoethanol, 0.4 mM PMSF, 0.1% Triton X-100) for 4 hrs. Nuclear extract was incubated with M2 anti-FLAG affinity gel (Sigma A2220) for 2 hrs. Beads were subsequently washed three times with wash buffer (20 mM HEPES pH 7.9, 500 mM KCl, 1mM EDTA, 2 mM β-mercaptoethanol, 0.4 mM PMSF, 0.1% Triton X-100). Captured proteins were eluted in 20 mM HEPES pH 7.9, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.4 mM PMSF, 500 ug/ml FLAG peptide. The eluates were reduced with 10 mM DTT for 30 mins at 56C, alkylated with 50 mM iodoacetamide for 40 mins in the dark at room temperature, and then diluted into 4 volumes of 50 mM Tris pH 8 with 2 mM CaCl2 for overnight digestion with trypsin (1 ug) at 37C. After desalting with C18 stage tips, the peptides were analyzed by LC-MS/MS with an EasyLC1000 nanoLC system (Thermo) and a Fusion Orbitrap mass spectrometer (Thermo). MS was performed over a 95 min LC gradient with a 2 second cycle of one full MS scan in the orbitrap followed by DDA MS/MS scans in the ion trap on the most abundant precursor ions fragmented by HCD at 27NCE. Dynamic exclusion was set to 40 sec.PrideNot availableCarbamidomethylAcetylMass spec raw files were processed by ProteomeDiscoverer software (Thermo) using a Sequest search against the human proteome (UniProt reviewed). An additional protein sequence, accession A0A1P0AZG4 corresponding to PALI1, which is related to LCOR, was also included. Precursor and fragment mass tolerances were set to 10 ppm and 0.5 Da with inclusion of an upstream node for mass recalibration (30 ppm precursor mass, 0.5 Da fragment mass). Carbamidomethylation of cysteine residues was set as a static modification and N-terminal acetylation as a variable modification. Trypsin was set as the protease. Protein abundance ratios were expressed as specific FLAG pulldown divided by mock pulldown. PD results were exported as text files to Excel. Proteins with at least 2 unique peptides and log2 ratios (FLAG/mock) greater than 4 were considered as hits.ProteomicsBenjamin A. GarciaOrbitrap FusionDept. of Biochemistry and Biophysics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104COMPLETEHomo Sapiens (human)pederjlund@gmail.comNot availableUniversity of PennsylvaniaUnited States10.6019/PXD013390Sfrs5, liquid chromatography tandem mass spectroscopy, Hac-1/Dark, ZFYVE8, IPP2A2, muriate of potash, ion, Sulfuric acid diammonium salt, DmelCG6829, DmelCG2903, fond, Ethylenediaminetetraacetic acid, l(2)23AB5, HEPES Monosodium Salt, KCl, CycEI, Apaf-1, extracted material, (ethylenedinitrilo)tetraacetic acid, 5730420M11Rik, ammonium sulfate (2:1), peptide, Polypeptides, sulphate of ammonia, Ethanol, B37, Glycerol, peptido, Ccne, Magnesium chloride, Klotrix, Vps27, B1, SEC, l(2)23Ad(Hrs), 1, 2, Min, 2', Apaf1, sylvite, present in fewer numbers in organism, ARK, DmelCG42273, RCB2202, Edetic acid, HEK293T, 3-Propanetriol, HEPES Monosodium, 2-diyldinitrilo)tetraacetate, SET, Oelsuess, LCMSMS, reference sample, ion(4-), hac-1, peptides, TAF-I, vps27, DmelCG13176, N, phosphatase 2A inhibitor I2PP2A, sec, Salt, hypoplasia, 2 Mercaptoethanol, SFRS5, arc, min, ORF19, mAPC, proteins, CaCl2, ark, SCAN, T1, AI047805, DmelCG4299, Hek 293T, set, IGAAD, decreased, 1-Piperazineethanesulfonic acid, DmelCG10574, D1, diammonium salt, s, stage, dApaf1, (ethane-1, acidum edeticum, Calcium chloride anhydrous, LC-MS2, [KCl], Glyceritol, phapii, ethylenediaminetetraacetate, Klor-con, HRS, Hrs, D-Apaf-1, [MgCl2], anon-53Fa, LC-MS/MS, 2'', Dmel_CG7826, l(2)SH0173, StF-IT-1, {[-(BIS-CARBOXYMETHYL-AMINO)-ETHYL]-CARBOXYMETHYL-AMINO}-ACETIC ACID, SRp40, diazanium sulfate, Cyc E, br37, ug/ml, fragmented, NOD, MgCl2, hrs, BG:DS07108.3, HCD, Dm1, Ionen, 1-L-aspartic acid-2-L-tyrosine-3-L-lysine-, glycerol, Kaon-Cl 10, SRP40, 1110049F14Rik, WASH, l(2)05206, CG2903, CG6829, Dmel_CG7835, dark/hac-1/dapaf-1, Mnb, MNB, parent ion, calcium chloride, Gro, HLA-DR-associated protein II, glycerine, DI-2, I-2Dm, cracked, hac1, CG4299, inhibitor of granzyme A-activated DNase, N'-1, AW124434, 293 T, beta Trypsin, magnesium dichloride, organ system, I-2PP1, dapaf-1S, apaf-1, dapaf-1L, l35Dd, TAF-IBETA, precursor ion, Acetamide, liquid chromatography-tandem mass spectroscopy, 2-Mercaptoethanol, TAF-Ibeta, Polypeptide, HEPES, Monopotassium chloride, ammonium sulphate, i2pp2a, DYKDDDDK peptide, Ammonium, 2-ME, GLYCEROL, Selb., cycline, 293tsA1609neo, Dapaf-1/HAC-1, fractured, DmcyclinE, developmental stage, Gene, cycE, ethylenediaminetetraacetic acid, precursor, body system, l(2)br37, EDTA tetraanion, (NH4)2SO4, CG7826, PHAPII, 293T, LC-MS-MS, CYCLE, Buffer, Dark/Hac-1/dApaf1, cdi7, Glycerine, glycyl alcohol, Hac1, Propanetriol, Dark/Hac-1/dApaf-1, cyclinE, reduced, template-activating factor I, Trypsinogen (human pancreas isoenzyme 2 activation peptide), subnumerary, IT, p63, Acide ethylenediaminetetracetique, p65, Trihydroxypropane, LC-MSMS, Gene Products, CG7835, Cdi7, CDI7, CG42273, Hgr, system, tiny, acide edetique, calcium dichloride, T6K12_14, D12S755E, anatomical systems, 2'''-(ethane-1, FAM39E, CYCE, 293-T, 2-Ethane diylbis-(N-(carboxymethyl)glycine), propane-1, ethylenediamine tetraacetic acid tetraanion, sulfuric acid, DmelCG3938, iones, CyclE, Glycerin, ipp2a2, Monosodium Salt, dapaf-1, beta-Trypsin, 3938, 2pp2a, dapaf, dark, decreased number, DmcycE, ions, CG10574, calcium chloride anhydrous, l(2)k05007, edta, 2-diyldinitrilo)tetraacetic acid, dm-cycE, glycerolum, 2PP2A, HEK-293T, DARK, taf-ibeta, EDTA, dSET, dSet, peptidos, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, dArk, Dapaf-1, 4-(2-hydroxyethyl)-, 2-iodo-, PTHB1, Controlled, small, Human Embryonic Kidney 293T, dark/dapaf-1/hac-1, Controlling, DYRK1, Magnesiumchlorid, potassium chloride, USH3B, Glyzerin, dApaf-1, Proteins, 3-triol, igaad, DRPLA, split, HEK 293 T, l(2)23Ad, apaf1, buffer, Cell, Peptide, acido edetico, Kaliumchlorid, polypeptide, Wash2, APAF1, Wash1, LC/MS/MS, secret agent, l(2)k02514, Temperatures, DmCycE, Hac-1, I-2PP2A, C18, Protein, Dm I-2, I2PP2A, hemorrhaged, diammonium sulfate, Dyrk1, Dark, CyeE, connected anatomical system, Dark/Apaf-I, Magnesium chloride anhydrous, mascagnite, CG13176, T6K12.14, AU020952, torn, 3-Trihydroxypropane, l(2)k02602, underdeveloped, CC1, H4edta, l(2)35Dd, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, Protein pp110, Dark/Dapaf-1/HAC1, sulfuric acid ammonium salt (1:2), dApaf-1/DARK/HAC-1, ME-IV, Protein Gene Products, Gene Proteins, Trypure, D-CycE, dSET/TAF-Ibeta, 2610030F17Rik, Ion, [CaCl2], 2-mercapto-, liquid chromatography tandem mass spectrometry, HRS-2, Peptid, edetic acid, AA407739, CG3938, Sulfate, dAPAF-1, HEK-293-T, ethylenediaminetetraacetate tetraanionBru, IPP2A2, Raw, primitive node, alpha-Spe, protein, clefted, alpha-Spc, 5730420M11Rik, peptide, Polypeptides, l(3)04276, protein polypeptide chains, peptido, dorsal marginal zone, node, Software Engineering, protein aggregate, WMS, Computer Program, alpha-spec, Svc, SET, kus, peptides, TAF-I, Open, phosphatase 2A inhibitor I2PP2A, Computer Programs and Programming, proteins, alpha-spectrin, DmelCG4299, set, IGAAD, DmelCG10574, ppm, Hensen node, Hensen's node, mKIAA1795, Half Cystine, GPHYSD2, 3110023F06Rik, embryo organizer, SGS, phapii, subdivided, Zinc Cysteinate, acetylation, StF-IT-1, L-ornithine carboxy-lyase activity, Source Softwares, results, Software Tools, ACMICD, Programs, Program, Computer Applications, alphaSpec, l(1)G0108, Computer Applications Software, Computer Applications Softwares, Henson's node, Softwares, spectrin, forked, parent ion, Software Applications, divided, HLA-DR-associated protein II, DI-2, Source Software, I-2Dm, L-Cysteine, MASS, alpha, CG4299, inhibitor of granzyme A-activated DNase, septate, beta Trypsin, human, I-2PP1, Applications, TAF-IBETA, precursor ion, i6, DMZ, TAF-Ibeta, Polypeptide, i2pp2a, Proteomes, Specbeta, Computer Software Applications, anon-EST:fe2E2, human being, Cysteine Hydrochloride, l(3)alpha-Spec, alpha-Spect, FBN, Gene, Computer, precursor, protein-containing complex, Mblk1-related protein 2, text, PHAPII, B-spec, polypeptide chain, template-activating factor I, ECTOL1, betaspec, klo, Gene Products, alpha-Sp, Del(8)44H, Half-Cystine, CG1977, Application, shield, Open Source Softwares, nodus primitivus, Software Application, L Cysteine, ipp2a2, beta-Trypsin, dre3, Open Source Software, Acetylations, 2pp2a, Spemann's organizer, LCoR, man, CG10574, Computer Software Application, OCTD, anon-EST:fe1B3, 2PP2A, Tools, 10^[-6], SPEC8, taf-ibeta, stem node, A630025C20Rik, dSET, dSet, peptidos, CG5870, organizer, l(3)dre3, Applications Software, Spec-beta, Open Source, alpha-Spectrin, Computer Software, 3A9, protein complex, DmelCG1977, Proteins, igaad, total expressed protein, i168, embryonic organizer, SpeC, L-ornithine carboxy-lyase (putrescine-forming), Tool, polypeptide, Software Tool, native protein, natural protein, I-2PP2A, Protein, Dm I-2, I2PP2A, Spemann Mangold organizer, sequence, betaSpec, MFS1, alphaSp, b-Spec, Software, WMS2, DmelCG5870, l(1)G0074, l(1)G0198, Col4a-1, nodal stem, Engineering, FCP-B, Tripcellim, b spectrin, MLR2, Mlr2, primary structure of sequence macromolecule, beta, Protein Gene Products, Computer Programs, Trypure, Gene Proteins, dSET/TAF-Ibeta, 2610030F17Rik, Applications Softwares, l(3)62Bd, beta-spec, SSKS, alfa-Spec, embryonic shield, Peptid, AA407739, Peptide., Specextent, cytoplasmic chromatin, 2-amino-4-(methylsulfanyl)butanoic acid, Activity, 293tsA1609neo, Lysine Hydrochloride, L Isomer, A4, Gene, protein, protein-containing complex, DIPG, supernumerary, TYPE, regulation of gene product expression, FGARAT, 293T, Mutations, DAGA4, L-Isomer Methionine, encircling, protein polypeptide chains, Lysine Acetate, Methionine, posterior, polypeptide chain, concavities, Gene Products, L-Methionine, 2, Lysine, Low, MAM, protein aggregate, SCG3, Chromatins, Pedameth, L Lysine, RCB2202, HEK293T, increased, me75, 293-T, reference sample, fossa, K, M, scattered, epsilon-diaminocaproic acid, proteins, concavity, D17Mit170, T1, Hek 293T, LYS, 2-amino-4-(methylthio)butanoic acid, HEK-293T, Lysin, chromosome scaffold, associated, metionina, Racemethionine, Methylations, posterior end of organism, Controlled, nuclear chromatin, Human Embryonic Kidney 293T, Controlling, cou, diffuse, DL-Methionine, enclosing, completeness, protein complex, PFAS, Proteins, 2-Amino-4-(methylthio)butyric acid, HEK 293 T, L-Isomer, Tl3, Tl2, Cell, LGMD2C, Lr, native protein, natural protein, Drives., Acetate, wrapping, fossae, Protein, lysine, sequence, Methionin, gene regulation, methionine, methylation, Hmet, L-Lysine, Met, FGAMS, infiltrative brainstem glioma, groove, DMDA1, increased number, diffuse intrinsic pontine glioma, depressions, alpha, PURL, 293 T, primary structure of sequence macromolecule, regulation of protein expression, Protein Gene Products, Gene Proteins, present in greater numbers in organism, diffuse midline glioma, DMDA, SCARMD2, alpha-amino-gamma-methylmercaptobutyric acid, Bra, FGAR-AT, 6-diaminohexanoic acid, Liquimeth, Enisyl, General activity, depression, accessory, HEK-293-T, IntrinsicCo Immunoprecipitation, HEK293T, Hek 293T, Co-Immunoprecipitations, Human Embryonic Kidney 293T, Immune, 293-T, HEK-293T, 293tsA1609neo, determination, chemical analysis, Co-Immunoprecipitation, Immune Precipitations, Precipitation, Cell., HEK 293 T, assay, Precipitations, 293 T, Immune Precipitation, HEK-293-T, RCB2202, 293TtrueCxorf67-FLAG co-immunoprecipitation analysis from 293T cellsThe regulation of gene expression is controlled in part by post-translational modifications to histone proteins. Methylation at histone H3, lysine 27 (H3K27), which is catalyzed by Polycomb repressive complex 2 (PRC2), is associated with silenced chromatin. Previous studies have identified dysregulation of H3K27 methylation in pediatric diffuse intrinsic pontine gliomas (DIPGs), the majority of which feature mutation of lysine 27 to methionine. This “oncohistone” potently inhibits PRC2 activity and leads to a global reduction in H3K27 methylation. Similar to DIPG, posterior fossa type A (PFA) ependymomas also show low levels of H3K27 methylation. Although PFAs do not possess the H3K27M oncohistone mutation, they do show increased expression of Cxorf67. Interestingly, Cxorf67 contains a C-terminal sequence that resembles the sequence surrounding H3K27, and we find that this portion of Cxorf67 inhibits PRC2 activity to an even greater extent than the H3K27M oncohistone. Thus, we suggest re-naming Cxorf67 as EZHIP (Enhancer of Zeste Homologs Inhibitory Protein). Furthermore, when expressed in 293T cells, Cxorf67 interacts with several members of PRC2 and induces changes in H3K27 methylation patterns that mirror the changes in H3K27 methylation induced by expression of H3K27M. We propose that PFAs have dysregulated H3K27 methylation by a mechanism that involves inhibition of PRC2 by Cxorf67, which could drive 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