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/ Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library 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 Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Nuclear extracts were prepared from 0 to 24 h HA-esc embryos and passed over a Superose 6 column. rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions drome-embryo drosophilla melanogaster embryo embryo Embryo Defined as the development stage prior to the initiation of tissue differentiation. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. Gene (0378-1119) 2002 Only protein-protein interactions imex curation Drosophila Enhancer of zeste protein interacts with dSAP18. Drosophila Enhancer of zeste protein interacts with dSAP18. Gene (0378-1119) 2002 imex curation Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Wang L., Ding L., Jones CA., Jones RS. Chromatin - Epigenetic interactions resulting in chromatin modulation Leucine prototrophy and expression of beta-galactosidase used to detect interaction. rjones@mail.smu.edu Accepted 2007-MAR-27 by DAVET. 12-MAR-2007: Contacted by JYOTI. 0�12 h embryo Drosophila cDNA library Gene (0378-1119) 2002 Only protein-protein interactions imex curation sap18_drome Histone deacetylase complex subunit SAP18 Bin1 SAP18 CG6046 18 kDa Sin3-associated polypeptide Bicoid-interacting protein 1 dSAP18 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MANVESMIVEEKTQVKQIDREKTCPMLLRVFCSTGRHHSVSEYMFGNVPTNELQIYTWQDATLHELTSLVRDVNPDTRKKGTYFDFAVVYPNFRSNHFQMREIGVTCTGQKGIDDNKTLAQAKFSIGDFLDISITPPNRLPPTARRQRPY 94947F206D8FA0AD 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 esc_drome Polycomb protein esc esc CG14941 Protein extra sex combs protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MSSDKVKNGNEPEESEESCGDESASYTTNSTTSRSKSPSSSTRSKRRGRRSTKSKPKSRAAYKYDTHVKENHGANIFGVAFNTLLGKDEPQVFATAGSNRVTVYECPRQGGMQLLHCYADPDPDEVFYTCAWSYDLKTSSPLLAAAGYRGVIRVIDVEQNEAVGNYIGHGQAINELKFHPHKLQLLLSGSKDHAIRLWNIQSHVCIAILGGVEGHRDEVLSIDFNMRGDRIVSSGMDHSLKLWCLNTPEFHHKIELSNTFSQEKSTLPFPTVTKHFPDFSTRDIHRNYVDCVQWFGNFVLSKSCENAIVCWKPGQLHQSFEQVKPSDSSCTIIAEFEYDECEIWFVRFGFNPWQKVIALGNQQGKVYVWELDPSDPEGAHMTTLHNSRSVATVRQIAFSRDASVLVYVCDDATVWRWNRRQTTSI 511C305E5DE86727 bin1-e_z_ 7 9 unspecified role unspecified role bait bait set domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Identification of E(Z)-dSAP18 interaction in a yeast two-hybrid system screen bin1-bin1 5 8 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Unlike the truncated dSAP18, full-length dSAP18 was found to self-interact 1B bin1-e_z_-1 5 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role bait bait set domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Additional yeast two-hybrid tests revealed that this truncated version of dSAP18 interacts not only with E(Z)512�760, but also with another E(Z) region located within E(Z)1�511 1A bin1-e_z_-2 1 9 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 8 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Following extensive washing, bound proteins were examined by SDS-PAGE and subsequent autoradiography. 35S-E(Z) bound to His6-dSAP18 2A bin1-e_z_-3 1 9 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 cxc region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 8 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain in vitro In vitro direct interaction direct interaction Thus, dSAP18 is capable of binding independently to two non-contiguous E(Z) regions: aa 377�446 and aa 512�605. 2B, C, D bin1-e_z_-4 6 9 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association The resulting western blot was probed with rabbit anti-dSAP18 antibodies, revealing co-immunoprecipitation of dSAP18 by anti-E(Z) antibodies 3A Interaction was detected in HA-esc embryos. bin1-e_z_-5 2 8 unspecified role unspecified role bait bait 9 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 In reciprocal experiments, anti-dSAP18 antibodies were found to co-immunoprecipitate E(Z). 3B bin1-esc 4 8 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association HA-ESC was immunoprecipitated from HA-esc extracts using the HA.11 MAb. An immunoblot of the precipitated proteins was probed with anti-dSAP18 antibodies, revealing co-immunoprecipitation of dSAP18 3C bin1-e_z_-6 3 9 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component physical association physical association dSAP18-containing complexes range in mass from not, vert, similar1100 to not, vert, similar450 kDa ( Fig. 4, bottom; fractions 22�30). These results are consistent with inclusion of dSAP18 in the ESC-E(Z) complex. 4 dSAP18-containing complexes range in mass from 1100 to 450 kDa.