IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation pjh3@po.cwru.edu Accepted 2007-APR-1 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extract fractions from DEAE sepharose column containing esc-ez and p55 were further analyzed by immuno-affinity chromatography. Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation The S-200 fractions containing the esc-ez complex were further immunoprecipitated and analysed. Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly ion exchange chrom ion exchange chromatography IEC IEC ion exchange chrom western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Fractions of S-200 that contained the esc-e(z)-p55 complex were further fractionated on a DEAE-Sepharose column. Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-APR-1 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation S-200 used for fractionation. Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extract from FLAG-esc expressing flies used for studies. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development (0950-1991) 2001 imex curation Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu 09-MAR-2007: Contacted by JYOTI. Only protein-protein interactions drome Drosophila melanogaster (Fruit fly) Fruit fly anti tag coip anti tag coimmunoprecipitation anti tag coip fingerprinting peptide massfingerprinting fingerprinting Tie F., Furuyama T., Prasad-Sinha J., Jane E., Harte PJ. Chromatin - Epigenetic interactions resulting in chromatin modulation Three chromatographic steps, including gel filtration - Superdex 200, ion exchange - DEAE-sepharose and antibody affinity - Anti FLAG M2 agarose affinity column chromatography used in the studies. Nuclear extact from flies constitutively expressing FLAG-esc was used in this study. pjh3@po.cwru.edu Accepted 2007-MAR-27 by DAVET. 09-MAR-2007: Contacted by JYOTI. Development (0950-1991) 2001 Only protein-protein interactions imex curation ez_drome Histone-lysine N-methyltransferase E(z) KMT6 E(z) CG6502 Protein enhancer of zeste Lysine N-methyltransferase 6 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MNSTKVPPEWKRRVKSEYIKIRQQKRYKRADEIKEAWIRNWDEHNHNVQDLYCESKVWQAKPYDPPHVDCVKRAEVTSYNGIPSGPQKVPICVINAVTPIPTMYTWAPTQQNFMVEDETVLHNIPYMGDEVLDKDGKFIEELIKNYDGKVHGDKDPSFMDDAIFVELVHALMRSYSKELEEAAPGTATAIKTETLAKSKQGEDDGVVDVDADGESPMKLEKTDSKGDLTEVEKKETEEPLETEDADVKPDVEEVKDKLPFPAPIIFQAISANFPDKGTAQELKEKYIELTEHQDPERPQECTPNIDGIKAESVSRERTMHSFHTLFCRRCFKYDCFLHRLQGHAGPNLQKRRYPELKPFAEPCSNSCYMLIDGMKEKLAADSKTPPIDSCNEASSEDSNDSNSQFSNKDFNHENSKDNGLTVNSAAVAEINSIMAGMMNITSTQCVWTGADQALYRVLHKVYLKNYCAIAHNMLTKTCRQVYEFAQKEDAEFSFEDLRQDFTPPRKKKKKQRLWSLHCRKIQLKKDSSSNHVYNYTPCDHPGHPCDMNCSCIQTQNFCEKFCNCSSDCQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCQACGADQFKLTKITCKNVCVQRGLHKHLLMAPSDIAGWGIFLKEGAQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSINPNCYAKVMMVTGDHRIGIFAKRAIQPGEELFFDYRYGPTEQLKFVGIEREMEIV 022360ED7772EB60 eed_human Polycomb protein EED EED WD protein associating with integrin cytoplasmic tails 1 Embryonic ectoderm development protein protein protein human Homo sapiens Human MSEREVSTAPAGTDMPAAKKQKLSSDENSNPDLSGDENDDAVSIESGTNTERPDTPTNTPNAPGRKSWGKGKWKSKKCKYSFKCVNSLKEDHNQPLFGVQFNWHSKEGDPLVFATVGSNRVTLYECHSQGEIRLLQSYVDADADENFYTCAWTYDSNTSHPLLAVAGSRGIIRIINPITMQCIKHYVGHGNAINELKFHPRDPNLLLSVSKDHALRLWNIQTDTLVAIFGGVEGHRDEVLSADYDLLGEKIMSCGMDHSLKLWRINSKRMMNAIKESYDYNPNKTNRPFISQKIHFPDFSTRDIHRNYVDCVRWLGDLILSKSCENAIVCWKPGKMEDDIDKIKPSESNVTILGRFDYSQCDIWYMRFSMDFWQKMLALGNQVGKLYVWDLEVEDPHKAKCTTLTHHKCGAAIRQTSFSRDSSILIAVCDDASIWRWDRLR DIP protein O75530 original sequence version: 70 DIP protein O75530-1 original sequence version: 78 DIP protein O75530 original sequence version: 78 DIP protein O75530 original sequence version: 73 DIP protein O75530-1 original sequence version: 89 DIP protein O75530-1 original sequence version: 87 DIP protein O75530 original sequence version: 77 D2E0A5BA27C0499A caf1_drome Chromatin assembly factor 1 p55 subunit Caf1-55 CAF-1 Caf1 Nucleosome-remodeling factor 55 kDa subunit CG4236 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MVDRSDNAAESFDDAVEERVINEEYKIWKKNTPFLYDLVMTHALEWPSLTAQWLPDVTKQDGKDYSVHRLILGTHTSDEQNHLLIASVQLPSEDAQFDGSHYDNEKGEFGGFGSVCGKIEIEIKINHEGEVNRARYMPQNACVIATKTPSSDVLVFDYTKHPSKPEPSGECQPDLRLRGHQKEGYGLSWNPNLNGYLLSASDDHTICLWDINATPKEHRVIDAKNIFTGHTAVVEDVAWHLLHESLFGSVADDQKLMIWDTRNNNTSKPSHTVDAHTAEVNCLSFNPYSEFILATGSADKTVALWDLRNLKLKLHSFESHKDEIFQVQWSPHNETILASSGTDRRLHVWDLSKIGEEQSTEDAEDGPPELLFIHGGHTAKISDFSWNPNEPWIICSVSEDNIMQVWQMAENVYNDEEPEIPASELETNTA DIP protein Q24572 original sequence version: 142 DIP protein Q24572 original sequence version: 124 90C5FB959F1660D5 hdac1_human Histone deacetylase 1 HDAC1 RPD3L1 protein protein human Homo sapiens Human MAQTQGTRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRFNVGEDCPVFDGLFEFCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVARCWTYETAVALDTEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTNEYLEKIKQRLFENLRMLPHAPGVQMQAIPEDAIPEESGDEDEDDPDKRISICSSDKRIACEEEFSDSEEEGEGGRKNSSNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTKEEKPEAKGVKEEVKLA DIP protein Q13547 original sequence version: 106 DIP protein Q13547 original sequence version: 173 DIP protein Q13547 original sequence version: 100 DIP protein Q13547 original sequence version: 166 DIP protein Q13547 original sequence version: 153 DIP protein Q13547 original sequence version: 164 DIP protein Q13547 original sequence version: 175 DIP protein Q13547 original sequence version: 191 DIP protein Q13547 original sequence version: 185 DIP protein Q13547 original sequence version: 165 DIP protein Q13547 original sequence version: 162 DIP protein Q13547 original sequence version: 189 DIP protein Q13547 original sequence version: 158 DIP protein Q13547 original sequence version: 134 DIP protein Q13547 original sequence version: 124 4D35B7C1ED7838D6 q9gss9_schma RBAP48 protein protein schma Schistosoma mansoni (Blood fluke) Blood fluke MMLHPSDSEDIVEERVINEEYKIWKRNTPFLYDMLMSHCLEWPSLTAQWLPSVERTGRDYSVHRLILGTHTSDEQNHLLIVTVHLPNDQAEFDASAYDSERGDFGGFYFPSGKLEISMKINHEGEVNRARFMPQNPDIIATKTPSGDVLIFNYPRHPPKTPSDRGCQPDLRLKGHQKEGYGLSWNVSLNGHLLSASDDQTICLWDVNAAPLDGCDLDAMAIFTGHHSVVEDVSWHLFHGHIFGSVADDNKLMVWDTRSSNRTKPQHQVDAHTAEVNCLAFNPFSEFIIATGSADKVIKYFTLVSFFYT 5F2CCA5480F0B0AD hdac1_drome Histone deacetylase HDAC1 HDAC1 Rpd3 CG7471 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MQSHSKKRVCYYYDSDIGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKATADEMTKFHSDEYVRFLRSIRPDNMSEYNKQMQRFNVGEDCPVFDGLYEFCQLSAGGSVAAAVKLNKQASEICINWGGGLHHAKKSEASGFCYVNDIVLGILELLKYHQRVLYIDIDVHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNIPLRDGMDDDAYESIFVPIISKVMETFQPAAVVLQCGADSLTGDRLGCFNLTVKGHGKCVEFVKKYNLPFLMVGGGGYTIRNVSRCWTYETSVALAVEIANELPYNDYFEYFGPDFKLHISPSNMTNQNTSEYLEKIKNRLFENLRMLPHAPGVQIQAIPEDAINDESDDEDKVDKDDRLPQSDKDKRIVPENEYSDSEDEGEGGRRDNRSYKGQRKRPRLDKDTNSNKASSETSSEIKDEKEKGDGADGEESTASNTNSNNNSNNKSDNDAGATANAGSGSGSGSGAGAKGAKENNI DIP protein Q94517 original sequence version: 67 B0F6503D42A1BCE9 esc_drome Polycomb protein esc esc CG14941 Protein extra sex combs protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MSSDKVKNGNEPEESEESCGDESASYTTNSTTSRSKSPSSSTRSKRRGRRSTKSKPKSRAAYKYDTHVKENHGANIFGVAFNTLLGKDEPQVFATAGSNRVTVYECPRQGGMQLLHCYADPDPDEVFYTCAWSYDLKTSSPLLAAAGYRGVIRVIDVEQNEAVGNYIGHGQAINELKFHPHKLQLLLSGSKDHAIRLWNIQSHVCIAILGGVEGHRDEVLSIDFNMRGDRIVSSGMDHSLKLWCLNTPEFHHKIELSNTFSQEKSTLPFPTVTKHFPDFSTRDIHRNYVDCVQWFGNFVLSKSCENAIVCWKPGQLHQSFEQVKPSDSSCTIIAEFEYDECEIWFVRFGFNPWQKVIALGNQQGKVYVWELDPSDPEGAHMTTLHNSRSVATVRQIAFSRDASVLVYVCDDATVWRWNRRQTTSI 511C305E5DE86727 hdac2_human Histone deacetylase 2 HDAC2 protein protein human Homo sapiens Human MAYSQGGGKKKVCYYYDGDIGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKATAEEMTKYHSDEYIKFLRSIRPDNMSEYSKQMQRFNVGEDCPVFDGLFEFCQLSTGGSVAGAVKLNRQQTDMAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNFPMRDGIDDESYGQIFKPIISKVMEMYQPSAVVLQCGADSLSGDRLGCFNLTVKGHAKCVEVVKTFNLPLLMLGGGGYTIRNVARCWTYETAVALDCEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTPEYMEKIKQRLFENLRMLPHAPGVQMQAIPEDAVHEDSGDEDGEDPDKRISIRASDKRIACDEEFSDSEDEGEGGRRNVADHKKGAKKARIEEDKKETEDKKTDVKEEDKSKDNSGEKTDTKGTKSEQLSNP DIP protein Q92769 original sequence version: 161 DIP protein Q92769 original sequence version: 154 DIP protein Q92769 original sequence version: 117 DIP protein Q92769 original sequence version: 113 DIP protein Q92769 original sequence version: 139 DIP protein Q92769 original sequence version: 163 DIP protein Q92769 original sequence version: 179 DIP protein Q92769 original sequence version: 153 DIP protein Q92769 original sequence version: 112 775419CCCDAE07FA esc-rpd3 4 15 unspecified role unspecified role bait bait 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 5A In addition, when anti-E(Z), anti-RPD3 and anti-p55 antibodies were used for immunoprecipitation, FLAG-ESC was detected in the immunoprecipitates esc-e_z 8 16 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component physical association physical association 1 FLAG-ESC and E(Z) co-fractionate on a Superose 6 gel filtration column with identical peaks at ~600 kDa Complex size is about 600 kDa esc-caf1 9 12 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 16 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 2 and materials and methods We confirmed the presence of ESC and E(Z) in the complex both by western blot analysis of the purified fraction and by peptide sequencing. One of the new proteins identified was the histone-binding protein p55 (also known as CAF-1 p55 and Nurf-55). esc-caf1-3 7 12 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3A A small proportion of p55 co-fractionates with FLAG-ESC and E(Z) in fraction 18-20. Complex was about 670 kda in size. esc-caf1-4 5 10 unspecified role unspecified role neutral component neutral component 12 unspecified role unspecified role neutral component neutral component 16 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3B On a DEAESepharose column, p55 elutes in multiple peaks and cofractionates with FLAG-ESC and E(Z) in fractions 18-25 esc-caf1-5 3 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 16 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 3C On an anti-FLAG M2 agarose affinity gel column, p55 is present in the bound fraction and is co-eluted by FLAG peptide along with FLAG-ESC and E(Z) esc-caf1-6 6 15 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 16 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 4 In contrast, the FLAG-ESC and E(Z) present in the transgenic extract (lane 4) were recovered by anti-FLAG affinity gel (lane 6). Although most of the p55 was present in the unbound fraction from the anti-FLAG affinity gel, a substantial amount of p55 was detected in the bound fraction eluted with FLAG peptide esc-ez-1 4 10 unspecified role unspecified role bait bait 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 5A In addition, when anti-E(Z), anti-RPD3 and anti-p55 antibodies were used for immunoprecipitation, FLAG-ESC was detected in the immunoprecipitates esc-caf1-7 4 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 12 unspecified role unspecified role bait bait physical association physical association 5A In addition, when anti-E(Z), anti-RPD3 and anti-p55 antibodies were used for immunoprecipitation, FLAG-ESC was detected in the immunoprecipitates esc-rpd3-1 2 15 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 5B As shown in Fig. 5B, GST-E(Z)1-73 pulls down RPD3, p55 and ESC Drosophila nuclear extract used. esc-rpd3-2 2 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 5C pull-down assay indicates that GST-RPD3 can pull down E(Z), p55 and ESC from nuclear extract Drosophila nuclear extract used. esc-esc 1 16 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 16 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction 6A GST-ESC1-60 fusion protein encoding just the N-terminal 60 residues of ESC is sufficient to pull down full-length in vitro translated ESC eed-eed 1 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction 6A GST-EED1-81, which contains the corresponding N-terminal region of EED (Schumacher et al., 1996), binds directly to in vitro translated EED esc-rpd3-3 2 15 unspecified role unspecified role prey prey drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. 16 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role prey prey drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. 16 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. association association 6B GST-ESC1- 60 specifically pulls down the ESC complex. eed-hdac1 2 17 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 14 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 13 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 11 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) association association 6C GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48 from HeLa cell nuclear extract esc-rpd3-4 1 15 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 16 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction 7B GST-ESC and GST-E(Z) directly interacts with RPD3 ez-rpd3 1 15 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction 7B GST-ESC and GST-E(Z) directly interacts with RPD3 ez-rpd3-1 1 15 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 7B GST-ESC and GST-E(Z) directly interacts with RPD3 esc-rpd3-5 1 15 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 16 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 7B GST-ESC and GST-E(Z) directly interacts with RPD3 caf1-rpd3 1 12 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 15 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 7A GST-p55 was used to pull down in vitro translated full-length ESC, E(Z) and RPD3. caf1-ez 1 12 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 7A GST-p55 was used to pull down in vitro translated full-length ESC, E(Z) and RPD3.