Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation
2012; Elsevier BV; Volume: 11; Issue: 11 Linguagem: Inglês
10.1074/mcp.m112.017194
ISSN1535-9484
AutoresPavlos Fanis, Nynke Gillemans, Ali Aghajanirefah, Farzin Pourfarzad, Jeroen Demmers, Fatemehsadat Esteghamat, Ratna K. Vadlamudi, Frank Grosveld, Sjaak Philipsen, Thamar B. van Dijk,
Tópico(s)RNA modifications and cancer
ResumoChromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity. Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity. Transcription factor activity is often controlled by post-translational modifications such as acetylation, phosphorylation, methylation, and sumoylation. Some modifications are associated with both gene activation and repression, whereas others appear to be more exclusive: asymmetrical dimethylation of arginine residues is restricted to transcriptional activation, whereas modification by sumoylation correlates with inhibition of transcription (1Verger A. Perdomo J. Crossley M. Modification with SUMO. A role in transcriptional regulation.EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (375) Google Scholar). Arginine methylation occurs frequently within glycine-arginine-rich (GAR) 1The abbreviations used are:GARglycine-arginine-richaDMAasymmetrically dimethylated argininessDMAsymmetrically dimethylated argininesSUMOSmall ubiquitin-like modifierSIMsSUMO interacting motifsChtopChromatin target of Prmt1HGNCHUGO Gene Nomenclature CommitteeMELmouse erythroleukemia.1The abbreviations used are:GARglycine-arginine-richaDMAasymmetrically dimethylated argininessDMAsymmetrically dimethylated argininesSUMOSmall ubiquitin-like modifierSIMsSUMO interacting motifsChtopChromatin target of Prmt1HGNCHUGO Gene Nomenclature CommitteeMELmouse erythroleukemia. regions and is catalyzed by members of the protein arginine methyltransferase (Prmt) family. These enzymes are subdivided in two major classes: type I enzymes catalyze the formation of asymmetrically dimethylated arginines (aDMA), whereas type II enzymes form symmetrically dimethylated arginines (sDMA) (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar). Prmt1 and Prmt4/Carm1 (Coactivator-associated arginine methyltransferase 1) are the major type I enzymes and both are critical for mammalian development (3Pawlak M.R. Scherer C.A. Chen J. Roshon M.J. Ruley H.E. Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable.Mol. Cell. Biol. 2000; 20: 4859-4869Crossref PubMed Scopus (278) Google Scholar, 4O'Brien K.B. Alberich-Jordà M. Yadav N. Kocher O. Diruscio A. Ebralidze A. Levantini E. Sng N.J. Bhasin M. Caron T. Kim D. Steidl U. Huang G. Halmos B. Rodig S.J. Bedford M.T. Tenen D.G. Kobayashi S. CARM1 is required for proper control of proliferation and differentiation of pulmonary epithelial cells.Development. 2010; 137: 2147-2156Crossref PubMed Scopus (65) Google Scholar). 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Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). glycine-arginine-rich asymmetrically dimethylated arginines symmetrically dimethylated arginines Small ubiquitin-like modifier SUMO interacting motifs Chromatin target of Prmt1 HUGO Gene Nomenclature Committee mouse erythroleukemia. glycine-arginine-rich asymmetrically dimethylated arginines symmetrically dimethylated arginines Small ubiquitin-like modifier SUMO interacting motifs Chromatin target of Prmt1 HUGO Gene Nomenclature Committee mouse erythroleukemia. Small ubiquitin-like modifier (SUMO) has an important regulatory function in several cellular processes, including DNA repair, cell cycle progression, signal transduction, chromatin structure and transcriptional regulation (13Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). Mammalian cells express four SUMO paralogs (SUMO-1 to SUMO-4). SUMO-1 differs in sequence by about 50% from SUMO-2 and 3, whereas SUMO-2 and SUMO-3 are 97% identical to each other. Conjugation of SUMO to target proteins occurs by a series of reactions conducted by the E1 activating enzyme, E2 conjugating enzyme, and an E3 SUMO ligase (14Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1384) Google Scholar). The reverse desumoylation process is mediated by the isopeptidase activity of SUMO-specific proteases (Senps). In mammals, six members of Senps have been reported, known as Senp1–3 and Senp5–7. Sumoylation of multiple transcription factors, including Sp3, Sox6, Zeb1, and Zbp-89, has a negative effect on their transactivation potential, as it promotes the recruitment of repressive complexes (15Stielow B. Sapetschnig A. Wink C. Krüger I. Suske G. SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing.EMBO Rep. 2008; 9: 899-906Crossref PubMed Scopus (69) Google Scholar, 16Fernández-Lloris R. Osses N. Jaffray E. Shen L.N. Vaughan O.A. Girwood D. Bartrons R. Rosa J.L. Hay R.T. Ventura F. Repression of SOX6 transcriptional activity by SUMO modification.FEBS Lett. 2006; 580: 1215-1221Crossref PubMed Scopus (21) Google Scholar, 17Wang J. Scully K. Zhu X. Cai L. Zhang J. Prefontaine G.G. Krones A. Ohgi K.A. Zhu P. Garcia-Bassets I. Liu F. Taylor H. Lozach J. Jayes F.L. Korach K.S. Glass C.K. Fu X.D. Rosenfeld M.G. Opposing LSD1 complexes function in developmental gene activation and repression programmes.Nature. 2007; 446: 882-887Crossref PubMed Scopus (431) Google Scholar). Many components of the repressor complexes CoREST1, NuRD, PRC1, Setdb1, and MEC themselves are also sumoylated, or have SUMO interacting motifs (SIMs). This suggests that sumoylation plays an important role in the formation and/or stabilization of these complexes (18Garcia-Dominguez M. Reyes J.C. SUMO association with repressor complexes, emerging routes for transcriptional control.Biochim. Biophys. Acta. 2009; 1789: 451-459Crossref PubMed Scopus (126) Google Scholar). We previously identified Friend of Prmt1 (Fop), also known as Small protein rich in arginine and glycine (SRAG), encoded by the mouse 2500003M10Rik and human C1orf77 genes, respectively (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar, 20Zullo A.J. Michaud M. Zhang W. Grusby M.J. Identification of the small protein rich in arginine and glycine (SRAG): a newly identified nucleolar protein that can regulate cell proliferation.J. Biol. Chem. 2009; 284: 12504-12511Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Recently, the name Chromatin Target of Prmt1 (Chtop) has been assigned to this gene/protein by the HUGO Gene Nomenclature Committee (HGNC). For reasons of clarity, we will also use the name Chtop for the murine homolog. Chtop is a chromatin-associated protein that plays an important role in the ligand-dependent activation of estrogen target genes such as TFF1 (pS2) in breast cancer cells (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar). In addition, it is a critical regulator of γ-globin gene expression (21van Dijk T.B. Gillemans N. Pourfarzad F. van Lom K. von Lindern M. Grosveld F. Philipsen S. Fetal globin expression is regulated by Friend of Prmt1.Blood. 2010; 116: 4349-4352Crossref PubMed Scopus (36) Google Scholar). However, little is known about the molecular mechanism of transcriptional control mediated by Chtop. Chtop contains a central GAR region that is recognized and methylated by Prmt1. Because arginine methylation controls protein-protein interactions, we used a biotinylation-proteomics approach to identify proteins that bind Chtop in the presence and absence of Prmt1. In this study we identified and characterized a protein complex that binds specifically to methylated Chtop. As this nuclear complex consists of five proteins—SUMO1/sentrin/SMT3 specific peptidase 3 (Senp3), proline-glutamate and leucine rich protein 1 (Pelp1), LAS1-like protein (Las1L), Testis expressed 10 protein (Tex10), and WD repeat domain 18 protein (Wdr18)—we call it Five Friends of Methylated Chtop (5FMC). We show that Pelp1 is critical for the integrity of 5FMC and that Chtop and 5FMC are recruited by Zinc finger binding protein-89 (Zbp-89), thereby regulating both (de)sumoylation of, and transactivation by, Zbp-89. The following plasmids have been described previously: GST-PELP1 deletions (22Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Potential role of a novel transcriptional coactivator PELP1 in histone H1 displacement in cancer cells.Cancer Res. 2004; 64: 6416-6423Crossref PubMed Scopus (85) Google Scholar), T7-Pelp1 (23Vadlamudi R.K. Wang R.A. Mazumdar A. Kim Y. Shin J. Sahin A. Kumar R. Molecular cloning and characterization of PELP1, a novel human coregulator of estrogen receptor alpha.J. Biol. Chem. 2001; 276: 38272-38279Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), pMT2_HA_Chtop, HA_mPRMT1 WT, and HA_mPRMT1 EQ (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar). The cDNA of human SUMO-2 (hSMT3b) was kindly provided by Dr. Guntram Suske (Philipps-Universität Marburg, Germany). The cDNA of full-length LAS1L was obtained from Open Biosystems (Clone ID 3140243; Huntsville, AL, USA), full-length cDNA of Wdr18 was obtained from RZPD/imaGenes (clone IRAVp968G04150D6; Berlin, Germany), and full-length cDNA of Senp3 was obtained from RZPD/imaGenes (clone IRAVp968B0184D6; Berlin, Germany). The cDNA of PELP1 was subcloned from T7-PELP1. After the introduction of the 23-amino acid (aa) biotinylation tag into the pMT2_HA (24Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar) and pMT2_HA_Chtop, LAS1L, Senp3, and PELP1 were cloned to pMT2_bio_HA using SalI and NotI, Wdr18 was cloned using SalI, and SUMO-2 was cloned using SalI and EcoRI. Bio_HA_LAS1L, Bio_HA_Senp3, Bio_HA_PELP1, Bio_HA_Wdr18, Bio_HA_SUMO-2, and Bio_HA_Chtop were subcloned into the erythroid expression vector pEV-neo (25Needham M. Gooding C. Hudson K. Antoniou M. Grosveld F. Hollis M. LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.Nucleic Acids Res. 1992; 20: 997-1003Crossref PubMed Scopus (65) Google Scholar) and electroporated into mouse erythroleukemia (MEL) cells expressing the BirA biotin ligase (26de Boer E. Rodriguez P. Bonte E. Krijgsveld J. Katsantoni E. Heck A. Grosveld F. Strouboulis J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7480-7485Crossref PubMed Scopus (346) Google Scholar). To make the Gateway pSG513_myc destination plasmid, the Attr1-CmR-ccdb-Attr2 fragment was subcloned from pDEST17 (Invitrogen) to a modified pSG5 (Stratagene, La Jolla, CA) using HindIII, downstream the myc-tag sequence that was introduced to the pSG513 plasmid using EcoRI and BamHI. cDNAs for Senp3, Wdr18, and LAS1L were cloned into pDONR221 (Invitrogen), from which they were cloned by Gateway LR reaction to pSG513_myc. Internal deletion mutants of LAS1L and Wdr18 were generated using the QuikChange site-directed mutagenesis kit (Stratagene). MEL and 293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS). Transient transfections in 293T cells, immunoprecipitations and Western blot analysis were performed as described previously (27van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Löwenberg B. von Lindern M. Stem cell factor induces phosphatidylinositol 3′-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells.Blood. 2000; 96: 3406-3413Crossref PubMed Google Scholar). For immunoprecipitations combined with Benzonase (Novagen) incubation, 250 units of Benzonase were used followed by 3 h incubation at 4 °C. Nitrocellulose membranes were blocked in 1% bovine serum albumin (BSA), probed with the appropriate primary antibodies and analyzed using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Cambridge, UK). GST pull-down assays were performed as described previously (22Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Potential role of a novel transcriptional coactivator PELP1 in histone H1 displacement in cancer cells.Cancer Res. 2004; 64: 6416-6423Crossref PubMed Scopus (85) Google Scholar). Western blots were probed with the following primary antibodies: Prmt1 (07-404), Asym24 (07-414), and Cbx4 (09-029) were from Upstate, Charlottesville, VA; Actin (clone I-19; sc-1616), Taf1β (clone H-120; sc-25564), Lamin B (sc-6216), HA (monoclonal F7; sc-7392), HA (polyclonal Y11; sc-805), Myc (monoclonal 9E10; sc-40), Myc (polyclonal A-14; sc-789), and Pol II (polyclonal N-20; sc-899) were from Santa Cruz Biotechnology, Santa Cruz, CA; Tex10 (17372-1-AP), Las1L (16010-1-AP), Senp3 (17659-1-AP) and Wdr18 (15165-1-AP) were from Proteintech Group; Zbp-89 (ab69933) was from Abcam, Cambridge, MA, SUMO 2/3 (clone 1E7; M114-3) was from MBL, Woburn, MA; Pelp1 (A300-876A) was from Bethyl Laboratories, Montgomery, TX; T7 (69522-3) was from Novagen, Madison, WI; Taf1α (B100-56353) was from Novus Biologicals, Littleton, CO and Chtop (KT64) was from Absea Biotechnology. Ring1B antibody was kindly provided from Dr. Miguel Vidal (Madrid, Spain). Preparation of nuclear and whole cell extracts from MEL and 293T cells were carried out as described previously (27van Dijk T.B. van Den Akker E. Amelsvoort M.P. Mano H. Löwenberg B. von Lindern M. Stem cell factor induces phosphatidylinositol 3′-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells.Blood. 2000; 96: 3406-3413Crossref PubMed Google Scholar). Purification of biotinylated proteins, digestion with trypsin (Promega, sequencing grade) on paramagnetic streptavidin beads and liquid chromatography-tandem MS (LC-MS/MS) were performed as described previously (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar, 26de Boer E. Rodriguez P. Bonte E. Krijgsveld J. Katsantoni E. Heck A. Grosveld F. Strouboulis J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7480-7485Crossref PubMed Scopus (346) Google Scholar). Nanoflow LC-MS/MS was performed on an 1100 series capillary LC system (Agilent Technologies, Santa Clara, CA) coupled to an LTQ-Orbitrap mass spectrometer (Thermo), operating in positive mode and equipped with a nanospray source. Peptide mixtures were trapped on a ReproSil C18 reversed phase column (Dr Maisch GmbH; column dimensions 1.5 cm × 100 μm, packed in-house) at a flow rate of 8 μl/min. Peptide separation was performed on ReproSil C18 reversed phase column (Dr Maisch GmbH; column dimensions 15 cm × 50 μm, packed in-house) using a linear gradient from 0 to 80% B (A = 0.1% formic acid; B = 80% (v/v) acetonitrile, 0.1% formic acid) in 70 min and at a constant flow rate of 200 nl/min using a splitter. The column eluent was directly sprayed into the electrospray ionization (ESI) source of the mass spectrometer. Mass spectra were acquired in continuum mode; fragmentation of the peptides was performed in data-dependent mode. Peak lists were automatically created from raw data files using the Mascot Distiller software (version 2.3; MatrixScience). The Mascot search algorithm (version 2.2, MatrixScience) was used for searching against the IPI_mouse database (version 3.83, containing 60,010 sequences and 27,475,843 residues). The peptide tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.8 Da. A maximum number of two missed cleavages by trypsin were allowed and carbamidomethylated cysteine and oxidized methionine were set as fixed and variable modifications, respectively. Search results were parsed into a home-built database system for further analysis. Entries were parsed if they had a minimum peptide Mascot score of 25, and a significance threshold of p < 0.05; the option "require red bold" was also selected. Using these parameters yields an estimated peptide false discovery rate (FDR) of 3–5% against a target decoy database. The Mascot data have been uploaded to the PRIDE database (28Vizcaino J.A. Côté R. Reisinger F. Barsnes H. Foster J.M. Rameseder J. Hermjakob H. Martens L. The Proteomics Identifications database: 2010 update.Nucleic Acids Res. 2010; 38: D736-742Crossref PubMed Scopus (211) Google Scholar) (www.ebi.ac.uk/pride) under accession numbers 21750–21767. The data was converted using PRIDE Converter (29Barsnes H. Vizcaino J.A. Eidhammer I. Martens L. PRIDE Converter: making proteomics data-sharing easy.Nat. Biotechnol. 2009; 27: 598-599Crossref PubMed Scopus (149) Google Scholar) (http://pride-converter.googlecode.com). The Chtop and Prmt1 shRNA lentiviral vectors were described previously (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar, 21van Dijk T.B. Gillemans N. Pourfarzad F. van Lom K. von Lindern M. Grosveld F. Philipsen S. Fetal globin expression is regulated by Friend of Prmt1.Blood. 2010; 116: 4349-4352Crossref PubMed Scopus (36) Google Scholar). For mouse Pelp1, Senp3, and human PELP1, SENP3, WDR18, LAS1L, clones from the TRC Mission shRNA library ((30); Sigma Aldrich, St. Louis, MO, USA) were used for knockdown experiments in MEL and 293T cells respectively, including a non-targeting shRNA control virus (SHC002). Lentivirus was produced by transient transfection of 293T cells as described before (31Zufferey R. Nagy D. Mandel R.J. Naldini L. Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat. Biotechnol. 1997; 15: 871-875Crossref PubMed Scopus (1563) Google Scholar). The following clones were used from the TRC shRNA library: TRCN0000177043 (shPelp1 #1), TRCN0000178252 (shPelp1 #2), TRCN0000031014 (shSenp3 #1), TRCN0000031017 (shSenp3 #2), TRCN0000159617 (shPELP1 #2), TRCN0000159673 (shPELP1 #3), TRCN0000004106 (shSENP3 #1), TRCN0000004107 (shSENP3 #2), TRCN0000078088 (shWDR18 #1), TRCN0000078089 (shWDR18 #2), TRCN0000121835 (shLAS1L #2), TRCN0000142144 (shLAS1L #4), TRCN0000035931 (sh hPRMT1). Nuclear extracts from MEL cells expressing BirA biotin ligase enzyme, were chromatographed over a Superose 6 column (Amersham Biosciences) using an AKTA fast-performance liquid chromatography apparatus. Fractions were collected and precipitated with trichloroacetic acid and analyzed by Western blotting. Subcellular fractionation was performed as described previously (19van Dijk T.B. Gillemans N. Stein C. Fanis P. Demmers J. van de Corput M. Essers J. Grosveld F. Bauer U.M. Philipsen S. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.Mol. Cell. Biol. 2010; 30: 260-272Crossref PubMed Scopus (36) Google Scholar). Reverse transcription (RT), RT-quantitative PCR (RT-QPCR), and ChIP were performed as described previously (32Esteghamat F. van Dijk T.B. Braun H. Dekker S. van der Linden R. Hou J. Fanis P. Demmers J. van Ijcken W. Ozgur Z. Horos R. Pourfarzad F. von Lindern M. Philipsen S. The DNA binding factor Hmg20b is a repressor of erythroid differentiation.Haematologica. 2011; 96: 1252-1260Crossref PubMed Scopus (13) Google Scholar). Primers used for RT-QPCR and ChIP-QPCR are summarized in supplemental Table S1. ANOVA statistical analysis was performed by GraphPad Prism 5.02. To identify interaction partners of Chtop, an N-terminal double-tagged version of Chtop protein (Bio_HA_Chtop) was expressed in MEL cells. These cells also stably expressed BirA, a bacterial biotin ligase which efficiently biotinylates the Bio-tag (26de Boer E. Rodriguez P. Bonte E. Krijgsveld J. Katsantoni E. Heck A. Grosveld F. Strouboulis J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7480-7485Crossref PubMed Scopus (346) Google Scholar). Note that tagged Chtop was not overexpressed, as endogenous levels were reduced in Bio_HA-Chtop transfected cells (Fig. 1C). Protein complexes from nuclear lysates were recovered by streptavidin pull down followed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) and were compared with samples from cells expressing BirA alone (Fig. 1A, supplemental Table S2). This confirmed the association of Chtop with Prmt1 and Prmt5, factors that we have previously identified as Chtop binding proteins (19van Dijk T.B. Gillemans N. Stein C. Fanis P. 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When compared with cells transduced with a control lentivirus, no major differences were observed in the binding of Chtop to Prmt5, Pc2, Ring1B, Phc2, 40-42-3, and Set (Fig. 1B, supplemental Table S2). Interestin
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