Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_wt_rep3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_wt_rep2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_wt_rep1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD048161/20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_Su-Hw_DLM3_4x3runs_Flybase_Step2_fixedLimma.pdResultprimaryOK200alexander.stark@imp.ac.atRichard ImreData-dependent acquisitionMass SpectrometryBottom-up proteomicsLc-msmsDna-affinity purificationDrosophila melanogasterDlm3-motifhttps://www.ebi.ac.uk/pride/archive/projects/PXD048161Schneider-2 CellBead-immobilized biotinylated DNA and S2 nuclear extracts were prepared similarly as described in Serebreni L. et al., EMBO J. 2023 15;42(10):e113519, modified as follows. A DNA array of either 4 DLM3 motif copies or 4 shuffled DLM3 motif copies was generated by self-annealing primer extension PCR with KAPA HiFi HotStart Ready Mix (Roche) of two oligonucleotides with 2 motif copies each (primer sequences: 2xDLM3_spacer_MS_fw: ccatcagacgtctggctcgagatctAGTTCGTTGCCTAAGTCTTTTGTTTAACCTCGTCGAGTTCGTTGCCTAAGTCTTTTGTTTGGCGGCGGCG, 2xDLM3_spacer_MS_rv: ccaacagtaccggaatgccaagcttAAACAAAAGACTTAGGCAACGAACTCGACGAGGTTAAACAAAAGACTTAGGCAACGAACTCGCCGCCGCC; 2xDLM3-sh_spacer_MS_fw: ccatcagacgtctggctcgagatctTCTTTGTACGTCTTTACTTGTGGTAAACCTCGTCGTCTTTGTACGTCTTTACTTGTGGTAGGCGGCGGCG, 2xDLM3-sh_spacer_MS_rv: ccaacagtaccggaatgccaagcttTACCACAAGTAAAGACGTACAAAGACGACGAGGTTTACCACAAGTAAAGACGTACAAAGACGCCGCCGCC). The PCR fragment was then digested with BglII and HindIII, gel-purified, phosphorylated with T4 polynucleotide kinase (NEB) and ligated into the BglII and HindIII digested pGL3 (Promega, E1751) with T4 DNA ligase (NEB). Amplification of linear biotinylated DNA from plasmid templates was done as described previously (primers are listed in Serebreni L. et al., EMBO J. 2023 15;42(10):e113519). PCR products were purified using AMPure XP beads (Agencourt) and eluted in water. 15µg of biotinylated DNA was used per reaction (=sample) and coupled to 50µL Dynabeads M-280 Streptavidin (Invitrogen) for 30min at room temperature, after which they were washed and placed in DBB Buffer (150 mM NaCl, 50 mM Tris/HCl pH, 8.0, 10 mM MgCl2) and immediately used for DNA affinity purification. For each sample the bead-coupled DNA was incubated with 400µg S2 cell nuclear extract and 1200ng sheard salmon sperm competitor DNA. Axygen 1.5mL low bind tubes were used for all subsequent steps. Reactions were incubated at room temperature for 40min. Beads were magnetically pelleted, resuspended in DBB buffer and proteins were crosslinked with 0.75% formaldehyde for 10min, followed by 6 wash steps in DBB (the first with addition of 0.5% NP-40). Samples were then processed and subjected to mass spectrometry analysis as described in Serebreni L. et al., EMBO J. 2023 15;42(10):e113519.PrideNot availableFor peptide identification, the RAW files were loaded into Proteome Discoverer (version 2.5.0.390, Thermo Scientific). All MS/MS spectra were searched using MSAmanda v2.0.0.16129 (Dorfer V. et al., J. Proteome Res. 2014 Aug 1;13(8):3679-84). The peptide mass tolerance was set to ±5 ppm and the fragment mass tolerance was set to ±15 ppm, and the maximal number of missed cleavages was set to 2, using tryptic enzymatic specificity without proline restriction. Peptide and protein identification was performed in two steps. For an initial search, the RAW files were searched against the database dmel‐all‐translation‐r6.34.fasta (Flybase.org, 22,226 sequences; 20,310,919 residues), supplemented with common contaminants, using the following search parameters: beta‐methylthiolation of cysteine was set as a fixed modification, oxidation of methionine as variable modification. The result was filtered to 1 % FDR on protein using the Percolator algorithm (Käll L. et al., Nat Methods. 2007 Nov;4(11):923-5.) integrated into Proteome Discoverer. A subdatabase of proteins identified in this search was generated for further processing. For the second search, the RAW files were searched against the created subdatabase using the same settings as above plus considering additional variable modifications in some data sets: Acetylation on lysine, phosphorylation on serine, threonine and tyrosine, methylation on lysine and arginine, di-methylation on lysine and arginine, tri-methylation on lysine, ubiquitinylation residue on lysine, biotinylation on lysine, formylation on lysine were set as additional variable modifications. The localization of the post‐translational modification sites within the peptides was performed with the tool ptmRS, based on the tool phosphoRS (Taus T. et al., J. Proteome Res. 2011, 10, 5354-62). Identifications were filtered again to 1 % FDR on protein and PSM level; additionally, an Amanda score cutoff of at least 150 was applied. Peptides were subjected to label‐free quantification using IMP‐apQuant (Doblmann J. et al., J. Proteome Res. 2019, 18(1):535-541). Proteins were quantified by summing unique and razor peptides and applying intensity‐based absolute quantification (iBAQ; Schwanhäusser B. et al., Nature. 2011 May 19;473(7347):337-42). Proteins were filtered to be identified by a minimum of 2 PSMs in at least one sample. Protein‐abundances‐normalization was done using sum normalization. Statistical significance of differential protein abundance was determined using limma (Smyth G.K. et al., Stat Appl Genet Mol Biol. 2004;3:Article3).ProteomicsAlexander StarkResearch Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, AustriaPARTIALDrosophila Melanogaster (fruit Fly)10.1016/J.MOLCEL.2024.10.041richard.imre@imba.oeaw.ac.atIMBA ViennaAustriaWater, nebb, Formol, Egos, CG10718, H(2)O, classic hairy cell leukaemia, Polynucleotide Hydroxylkinase, DNA Arrays, BOUND WATER, Hydrogenchlorid, NP-40, oxidane, Strepavidin, extracted material, WATER, HOH, Oncorhynchus nerka, SCHNEIDER S2 Cell, halite, hydrogen chloride, Magnesium chloride, Methanal, sl(2)ry, DGS, 5'-Hydroxyl-Kinase, 5'-Hydroxylkinase, Kinase, Polymerase Chain, Arrays, MIX, S2, X-Chromosome-Bearing Sperm, SL-2 cell, sperm cell, Polynucleotide 5'-Hydroxyl, thymus nucleic acid, X-Bearing, me75, Pink Salmon, DNA Kinase, cDNA Microarrays, Polynucleotide 5'-Hydroxyl Kinase, Chip, DmelCG13176, Formaldehyd, Microchips, klp38B, Oxomethane, Inverse Polymerase Chain Reaction, ORF19, Chinook, purification, DNA 5' Hydroxylkinase, Sockeye Salmon, D17Mit170, T1, ORF 3617., T4, cDNA Arrays, Ligase, cloruro sodico, isolation and purification, reaction, salt, Y Chromosome Bearing Sperm, Wasserstoffchlorid, Chips, Non-Idet P-40, (1 (5-nitro-2-furanyl)methyleneamino)tetrahydropyrimidine-2-one methanol cyclohexanocarboxylate, Y-Bearing Sperm, DmelCG1106, X Chromosome Bearing Sperm, Reaction, sample, Y-Chromosome-Bearing Sperms, Oligonucleotide Microarray, Oligonucleotide Arrays, dihydridooxygen, DNA Array, T4 DNA Ligase, Double-Stranded DNA, Schneider, deoxyribonucleic acids, KLP 38B, DNAn, dihydrogen oxide, Sockeye, ejaculate, Y-Chromosome-Bearing, rock salt, Anchored Polymerase Chain Reaction, TX-144 surfactant, KIF14, Hydroxylkinase, [MgCl2], aqua, Ligases, Double-Stranded, classic hairy cell leukemia, Oligonucleotide, Tl3, tio, Tl2, (Deoxyribonucleotide)n+m, Oncorhynchus tshawytscha, Oligonucleotide Array, MgCl2, DNA Chips, H2O, common salt, mitotic nuclear envelope degradation, Chlorwasserstoff, 1110049F14Rik, WASH, desoxyribose nucleic acid, QPS1, Triton X-114, Hydrogen Oxide, PCR, MILD1, X-Chromosome-Bearing, cDNA Array, Nested Polymerase Chain Reaction, Hydroxykinase, 5' Hydroxylpolynucleotide Kinase, HCl, hydrogen hydroxide, SDH3, natrii chloridum, spermatozoid, X Bearing Sperm, polymerase chain reaction, CG1106, acqua, seminal fluid, NEB177D, Gene Expression Microarray Analysis, Schneider 2 cell, Sperms, ds DNA, Mot, Wasser, DNA, chlorane, S-2 cell, Schneider cell, SL2, Joinases, CYB560, Sequence Analysis, DNA Microchip, mitotic nuclear envelope catabolism, Sperm, DNS, 38B.12, (Deoxyribonucleotide)n, 38B.10, SCHNEIDER-2 cell, chlorure d'hydrogene, Polynucleotide Kinase, DmKlp38B, Gene, DNA Microchips, Synthetases, Polymerase Chain Reactions, Inverse, Y-Chromosome-Bearing Sperm, DmelCG10718, Deoxyribonucleic acids, KLP-38B, agua, Buffer, Inverse PCR, 5'-Hydroxylpolynucleotide Kinase, potassium salt, Deoxyribonucleic Acid, isolation, Oncorhynchus gorbuscha, DNA 5'-Hydroxylkinase, p63, p65, Klp38B, Spermatozoon, Gene Products, Low, DNA Chip, l(2)k00802, Y Bearing Sperm, cDNA Microarray, cloruro de hidrogeno, Microchip, Pro-Mega, KLP38B, 5'-Hydroxylpolynucleotide, Schneider 2, FAM39E, Oligonucleotide Microarrays, Self, Hydrogen chloride, S2 cell, gel, Chinook Salmon, chlorure de sodium, Double Stranded, Deoxyribonucleic acid, X-Bearing Sperms, NEM2, eau, CYBL, X-Chromosome-Bearing Sperms, Polynucleotide, Formalin, Salmo salmo, Neb, sperm, Kochsalz, T4 DNA, SPERM, (Deoxyribonucleotide)m, Anchored PCR, M-280, X-Bearing Sperm, Magnesiumchlorid, bead, cou, Microarray, Gene Chip, DNAn+1, mitotic nuclear envelope disassembly, Proteins, Igepal CA-630, chloridohydrogen, cDNA, motif, buffer, Magnesium, Polynucleotide Hydroxykinase, ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, spermatozoon, oligonucleotide primer, [HCl], RCB1153, l(2)03552, Wash2, Microarrays, Salmon, Wash1, Lr, Temperatures, Natriumchlorid, Gene Chips, water, Protein, Hydrochloride, table salt, Pink, FORMALIN, MIXL, 5'-Hydroxyl Kinase, ds-DNA, SL2 cell, NaCl, Y-Bearing, PGL3, Magnesium chloride anhydrous, DNA Microarray, CG13176, Anchored, l(2)k07614, Reactions, Nested, Polydeoxyribonucleotide, Polynucleotide 5' Hydroxyl Kinase, Chloride, DNA Joinases, Episome, sample population, Methylene oxide, Protein Gene Products, Episomes, Gene Proteins, Y-Bearing Sperms, DNA Microarrays, DNA Ligase, Klp38, Magnesium chloride (MgCl2), Oxomethylene, Dm0332, Nonidet P-40, Polydeoxyribonucleotide Synthetases, Desoxyribonukleinsaeure, Array, Plasmid, Bra, Schneider's Line S2 cell, Nested PCR, sl(2)ry3, [OH2], Oligodeoxyribonucleotide Array Sequence Analysis, hydrochloric acid, Polydeoxyribonucleotide Ligasesnuclear chromatin, Delayed, cytoplasmic chromatin, False Memory, Memory, Repressed, Psychology Repression, False Memory Syndrome, Genomes, Delayed Memories, False, Repression, Delayed., Repressed Memory, chromosome scaffold, Syndrome, Delayed Memory, whole genome, Memory Syndrome, Chromatins, False Memory Syndromes, Memoriespara Tyrosine, Raw, Lysine Hydrochloride, AUTSX5, Irip, NOVH, NAALAdase, phosphorylation, 2-Amino-3-hydroxypropionic acid, 5730420M11Rik, Polypeptides, NAALADase I, FOLH, Lysine Acetate, Method, L-Proline, B1, l(2)SH1330, 2, C530001K22Rik, 2-amino-3-mercaptopropanoic acid, establishment and maintenance of substrate location, WMS, Threonin, L Lysine, somitogenic mesoderm, SET, C, (S)-2-amino-5-guanidinopentanoic acid, Membrane glutamate carboxypeptidase, K, M, DmSTAT, 2-amino-3-(4-hydroxyphenyl)propanoic acid, 3-(p-Hydroxyphenyl)alanine, P, R, Arginine, Monohydrate DL-Arginine Acetate, procedures, Y, Dstat 92E, (2S)-pyrrolidine-2-carboxylic acid, PROLINE, DmelCG4299, Biotinylations, set, LYS, Lysin, sample, s, Half Cystine, NOVh, SGS, NK/GPI, Gpi, D-stat, formylation, Zinc Cysteinate, unsegmented mesenchyme, DL-Proline, acetylation, arginine, pSTAT, D-STAT, para-Tyrosine, Procedure, 2-(acetylamino)-2-deoxy-, SH2-B PH domain-containing signaling mediator 1, Fsrg1, ACMICD, GIG27, RING3, l(2)br3, Algorithm, FSRG1, Methionin, NK|GPI, Hmet, (2R)-2-amino-3-sulfanylpropanoic acid, Stat92, l(2)SH2 1330., Plxn1, HLA-DR-associated protein II, DI-2, I-2Dm, FGCP, STAT92E, Methodological, alpha, (-)-proline, mKIAA4053, L isomer, I-2PP1, Pro-rich, DmelCG4257, TAF-IBETA, (5Z, TAF-Ibeta, PLXN1, L-Tyrosine, D-Stat, Liquimeth, L-pyrrolidine-2-carboxylic acid, Fsrg-1, Bglap-rs1, establishment and maintenance of cellular component location, NAT, Nat, 2-amino-4-(methylsulfanyl)butanoic acid, D17H6S113E, SD-stat, dstat, dSTAT, L-Zystein, 2-Amino-5-guanidinovaleric acid, alpha-L-N-acetyltalosaminuronic acid, protein-containing complex, FSH, DmelCG4482, Harg, pyrrolidine-2-carboxylic acid, Gene Products, Pgi, Lysine, Half-Cystine, dSTAT92E, Technique, Hpro, Gpi-1r, Gpi-1s, L Cysteine, E-920, Phi, Gpi-1t, 2pp2a, Hcys, IGFBP9, CG10574, Study, Kiaa4053, 2PP2A, 2-amino-4-(methylthio)butanoic acid, CYSTEINE, L Proline, dSET, dSet, L-Arg, FREE CYSTEINE, peptidos, Tyrosin, (2R)-2-amino-3-mercaptopropanoic acid, metionina, Methylations, l(3)j6C8, stat[92E], CG4482, prolina, DRODSRC, Proteins, Phosphorylations, KIAA1428, MF, L-Isomer, Amf, polypeptide, Serin, STAT 92E, native protein, I-2PP2A, tirosina, L-Isomer Arginine, Acetate, AW228947, Dm I-2, proline, MFS1, segmental plate, Mopsm, Sh2bpsm1, L-Lysine, NK, Met, dSTAT92E/marelle, Col4a-1, ensemble, (2S)-2-amino-5-guanidinopentanoic acid, Specificity, (R)-2-amino-3-mercaptopropanoic acid, 2-Amino-3-mercaptopropionic acid, Stat1alpha-like, Folate hydrolase 1, Gene Proteins, br3, localisation, tropinone reductase, EG:95B7.8, E920, 2600013D04Rik, NIP, Enisyl, alpha-L-2-N-acetylamino-2-desoxytaluronic acid, SH2-Bb, Bru, IPP2A2, 11alpha, E 920, TalANAc, DL-Arginine Acetate, Dstat92E, Monohydrate, nip, L-Threonine, protein, Prolin, CCN3, QM, peptide, L-Isomer Methionine, Techniques, Methionine, DIP13A, peptido, Cystein, 3.4.17.21, Arg, protein aggregate, Gpi-1, fs(1)M104, Svc, SH2-B, appl, peptides, TAF-I, Nlk, cisteina, (S)-2-Amino-5-guanidinovaleric acid, CG4257, epsilon-diaminocaproic acid, Stat, 35Bb, NAALAD1, Folylpoly-gamma-glutamate carboxypeptidase, IBP-9, stat, IGAAD, 13E, Methodological Studies, 2-amino-3-hydroxypropanoic acid, DmelCG10574, Tyr, STAT, APPL, dip13alpha, GPHYSD2, Racemethionine, l(3)06346, L-Arginin, phapii, establishment and maintenance of substance location, 2-Amino-4-(methylthio)butyric acid, 2-Amino-3-(p-hydroxyphenyl)propionic acid, Arginine Hydrochloride, StF-IT-1, Arginin, Thermo Scientific, Cys, Tyrosine, stat92, single organism localization, l35Bb, mOC-X, 3-Hydroxyalanine, PSM, Psm, lysine, NOV, Prostate-specific membrane antigen, presumptive somite mesoderm, PlexA1, L-Arginine, Org, ORG, Pteroylpoly-gamma-glutamate carboxypeptidase, prolinum, MS/MS, L-isomer, Gpi1-r, nov, Gpi1-s, L-Cysteine, MASS, CG4299, 15S)-, (S)-pyrrolidine-2-carboxylic acid, Gpi1-t, Methodological Study, fs(1)Y[b], L-Cystein, Specificity and Sensitivity, C130088N23Rik, PSMA, L Serine, Polypeptide, BG:DS01219.1, DXS648E, L-Serine, i2pp2a, Proteomes, Gpi1s, SH2 domain-containing protein 1B, Cysteine Hydrochloride, alpha-L-Talopyranuronic acid, Procedures, YB, (-)-(S)-proline, AI461847, mol, L-alpha-pyrrolidinecarboxylic acid, unsegmented paraxial mesoderm, L Isomer, FBN, Gene, somitomeric mesoderm, GCPII, PHAPII, L-(+)-arginine, DSRC, Yb, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, ECTOL1, Studies, establishment and maintenance of position, L-Methionine, N-acetylated-alpha-linked acidic dipeptidase I, mKIAA1299, Del(8)44H, CG2706, Pedameth, l(2)br23, SH2B, mrL, MS2, ipp2a2, 2-Pyrrolidinecarboxylic acid, Acetylations, mGCP, Ring3, L Threonine, d-STAT, OCTD, (S)-2-pyrrolidinecarboxylic acid, mrl, L Tyrosine, taf-ibeta, L10, Dstat, Sensitivity, stat92e, (S)-2-carboxypyrrolidine, 2-amino-5-(carbamimidamido)pentanoic acid, DSTAT, F16P2.29, DL-Methionine, protein complex, DmelCG2706, igaad, l(2)35Bb, total expressed protein, DXS648, stat92E, 2-amino-5-guanidinopentanoic acid, DL Arginine Acetate, Peptide, group, GCP2, Glutamate carboxypeptidase II, Cell growth-inhibiting gene 27 protein, IGFBP-9, Protein, I2PP2A, Hydrochloride, establishment and maintenance of localization, DIP13alpha, mKIAA4005, L Arginine, techniques, methylation, mrl stat92E, tandem MS, Zystein, WMS2, Data Base, D6S113E, PH and SH2 domain-containing signaling mediator, (-)-2-pyrrolidinecarboxylic acid, L-Prolin, 2-amino-3-sulfanylpropanoic acid, RNF3, Rnf3, Frg-1, L-2-Amino-3-mercaptopropionic acid, single-organism localization, CG15268, sample population, D-stat/stat92E, Protein Gene Products, dSET/TAF-Ibeta, 2610030F17Rik, SSKS, AI425885, alpha-amino-gamma-methylmercaptobutyric acid, F16P2_29, Peptid, L-(-)-proline, AA407739, 6-diaminohexanoic acid, methodologyfruit fly, Materials, postnatal development, metazoans, growth and development, Nuclear Receptor, 2610511A05Rik, melanogaster, element, BB234005, ter, dmTAF[[II]]230, Readability, DSmurf, Tier, D17Ertd710e, animal, Chromatins, Animalia, thymus nucleic acid, TFIID TAF250, Genomes, cel, FATE, Receptor Co-Repressors, methylase, Futurology, inhibition of homeostatic process, Nuclear Hormone Receptor Co-Repressor, Co Repressor Proteins, Smurf, Bsg75C, chromosome scaffold, Co Repressors, Protein G18, Double-Stranded DNA, deoxyribonucleic acids, DNAn, transcription from bacterial-type RNA polymerase promoter, CG4943, Receptor Corepressor, Nuclear Receptor Co Repressor, D-smurf, Predictions and Projections, nuclear chromatin, dTAF[[II]]230, animalia, TAF200, Double-Stranded, Lack, TAFII-250, TAF250/230, GAT8, Receptor Co-Repressor, (Deoxyribonucleotide)n+m, KMT1C, organism, TAFII250, Nuclear Receptor Corepressor, Term, Cis, CIS, G18, CT43, Genetic Materials, atomo, C6orf30, desoxyribose nucleic acid, atome, Genetic Material, CIS-1, Corepressors, Element, Factors, whole organism, DmelCG4943, Nuclear Hormone Receptor Co Repressor, atoms, 9030605P22Rik, INSDC_feature:gene, whole genome, CG17603, TAF[[II]], Nuclear Receptor Co Repressors, Co-Repressor Protein, Drosophila, Taf250, Material, SR3-5, bacterial transcription, ds DNA, Koerper, Cistron, DNA, Bat8, TAF230, BAT8, LOT1, cytoplasmic chromatin, d230, multi-cellular organism, DNS, (Deoxyribonucleotide)n, dSmurf1, Gene, dTAFII250, Sophophora melanogaster, DmelCG11247, animals, Transcription Factor, EfW1, Deoxyribonucleic acids, Deoxyribonucleic Acid, dmTAF1, Taf230, Drosophila melangaster, Nuclear Hormone Receptor Co Repressors, Projections and Predictions, Animal, Catalogs, TAF250, Genomes., Corepressor Protein, study, Transcription, Taf200, dTAF[[II]]250, Genetic, cell, ligand, Nuclear Receptor Co-Repressors, Double Stranded, Taf1p, Deoxyribonucleic acid, metazoa, Nuclear Receptor Corepressors, Nuclear Hormone Receptor Co-Repressors, dTAF250, DmelCG4216, d-smurf, (Deoxyribonucleotide)m, methyltransferase activity, elements, TAF, BACTS2, atom, Autoregulation, Co Repressor, ahl4, TAF[[II]]250, body, DNAn+1, l(3)84Ab, whole body, Factor, BG:DS00004.13, Dsmurf, Corepressor Proteins, Cistrons, D. melanogaster, ZAC1, Cell, dTAF230, development, dSmurf, p230, Smurf ubiquitin ligase, Nuclear Receptor Co-Repressor, Metazoa, Protein, G9A, TAF[[II]]250/230, TFIID, atomus, Nuclear, ds-DNA, INSDC_feature:regulatory, Taf[[II]]250, TAF[[II]]230, Corepressor, Co Repressor Protein, G9a, postnatal growth, CG4216, TAF[II]250, Ng36, Understanding, multicellular animals, activation of homeostatic process, DmelCG17603, Co-Repressor, SOCS, Drosophila melanogasters, Desoxyribonukleinsaeure, NG36, Suppressor of cytokine signaling, Co-Repressors, Future, growth, ZAC, TAF1falseA genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repressionDifferential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.2024-11-132023-12-28PXD048161NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:309807NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:4093NEWT:4096NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:8496NEWT:27448NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:231490NEWT:430615NEWT:563041NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:298315NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:45474NEWT:1590841NEWT:4513NEWT:1247NEWT:6938NEWT:376741NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:289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