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/ Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP. PloS one 2010 imex curation Page SH., Wright EK., Gama L., Clements JE. jclements@jhmi.edu 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-u87 Human U-87 MG glioblastoma cell line u87 ch-ip chromatin immunoprecipitation assay ch-ip q-pcr quantitative pcr q-pcr Page SH., Wright EK., Gama L., Clements JE. -2578 A allele corresponds to the wt promoter, while -2578 G allele corresponds to mutant NC_000017.11:g.34252769A>G jclements@jhmi.edu Accepted 2020-FEB-19 AT 11:12 GMT AT 11:12 GMT by KALPANAP PloS one 2010 Only protein-protein interactions imex curation imex curation pbx2_human Pre-B-cell leukemia transcription factor 2 PBX2 G17 Homeobox protein PBX2 Protein G17 protein protein human Homo sapiens Human MDERLLGPPPPGGGRGGLGLVSGEPGGPGEPPGGGDPGGGSGGVPGGRGKQDIGDILQQIMTITDQSLDEAQAKKHALNCHRMKPALFSVLCEIKEKTGLSIRSSQEEEPVDPQLMRLDNMLLAEGVAGPEKGGGSAAAAAAAAASGGGVSPDNSIEHSDYRSKLAQIRHIYHSELEKYEQACNEFTTHVMNLLREQSRTRPVAPKEMERMVSIIHRKFSAIQMQLKQSTCEAVMILRSRFLDARRKRRNFSKQATEVLNEYFYSHLSNPYPSEEAKEELAKKCGITVSQVSNWFGNKRIRYKKNIGKFQEEANIYAVKTAVSVTQGGHSRTSSPTPPSSAGSGGSFNLSGSGDMFLGMPGLNGDSYSASQVESLRHSMGPGGYGDNLGGGQMYSPREMRANGSWQEAVTPSSVTSPTEGPGSVHSDTSN EF2FFA158C4DAF68 irf1_human Interferon regulatory factor 1 IRF1 protein protein human Homo sapiens Human MPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRSWAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRMLPPLTKNQRKERKSKSSRDAKSKAKRKSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGYMQDLEVEQALTPALSPCAVSSTLPDWHIPVEVVPDSTSDLYNFQVSPMPSTSEATTDEDEEGKLPEDIMKLLEQSEWQPTNVDGKGYLLNEPGVQPTSVYGDFSCKEEPEIDSPGGDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPSIQAIPCAP DIP protein P10914 original sequence version: 79 2E06245A212D1541 pknx1_human Homeobox protein PKNOX1 PBX/knotted homeobox 1 Homeobox protein PREP-1 PKNOX1 PREP1 protein protein human Homo sapiens Human MMATQTLSIDSYQDGQQMQVVTELKTEQDPNCSEPDAEGVSPPPVESQTPMDVDKQAIYRHPLFPLLALLFEKCEQSTQGSEGTTSASFDVDIENFVRKQEKEGKPFFCEDPETDNLMVKAIQVLRIHLLELEKVNELCKDFCSRYIACLKTKMNSETLLSGEPGSPYSPVQSQQIQSAITGTISPQGIVVPASALQQGNVAMATVAGGTVYQPVTVVTPQGQVVTQTLSPGTIRIQNSQLQLQLNQDLSILHQDDGSSKNKRGVLPKHATNVMRSWLFQHIGHPYPTEDEKKQIAAQTNLTLLQVNNWFINARRRILQPMLDSSCSETPKTKKKTAQNRPVQRFWPDSIASGVAQPPPSELTMSEGAVVTITTPVNMNVDSLQSLSSDGATLAVQQVMMAGQSEDESVDSTEEDAGALAPAHISGLVLENSDSLQ 381E901F435AC5F2 hxa9_human Homeobox protein Hox-A9 HOXA9 HOX1G Homeobox protein Hox-1G protein protein human Homo sapiens Human MATTGALGNYYVDSFLLGADAADELSVGRYAPGTLGQPPRQAATLAEHPDFSPCSFQSKATVFGASWNPVHAAGANAVPAAVYHHHHHHPYVHPQAPVAAAAPDGRYMRSWLEPTPGALSFAGLPSSRPYGIKPEPLSARRGDCPTLDTHTLSLTDYACGSPPVDREKQPSEGAFSENNAENESGGDKPPIDPNNPAANWLHARSTRKKRCPYTKHQTLELEKEFLFNMYLTRDRRYEVARLLNLTERQVKIWFQNRRMKMKKINKDRAKDE 823A1A22BB07A881 ccl2_human_gene C-C motif chemokine 2 CCL2 MCP1 SCYA2 HC11 Monocyte chemoattractant protein 1 Monocyte chemotactic and activating factor Monocyte chemotactic protein 1 Monocyte secretory protein JE Small-inducible cytokine A2 gene gene human Homo sapiens Human pknox1-ccl2-1 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation increasing strength mutation increasing interaction strength mutation increasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 4 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Table 1, Fig. 2B The TALE proteins bound to a greater extent to the -2578 G allele than to the -2578 A allele (Table 1). Mutation of the upstream TALE site reduced binding by both PREP1 and PBX2 for both the -2578 A and G alleles (Figure 2B, C, F and G). irf1-ccl2-1 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 5 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Table 1, Fig. 2D In addition, we found that there was significantly greater binding of HoxA9 protein and increased binding of IRF1 to the -2578 A allele (Table 1). Mutation of the upstream TALE site reduced binding by both PREP1 and PBX2 for both the -2578 A and G alleles (Figure 2B, C, F and G). pknox1-ccl2-2 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation increasing strength mutation increasing interaction strength mutation increasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 2 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Table 1, Fig. 2C The TALE proteins bound to a greater extent to the -2578 G allele than to the -2578 A allele (Table 1). Mutation of the upstream TALE site reduced binding by both PREP1 and PBX2 for both the -2578 A and G alleles (Figure 2B, C, F and G). irf1-ccl2-2 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 3 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Table 1, Fig. 2E While the A and G alleles bind IRF1 equally when the upstream site is intact, mutation of the upstream TALE site leads to a 15-fold (Figure 2E) decrease in binding of the A allele as compared to a 1.5-fold decrease for the G allele (Figure 2I). In addition, we found that there was significantly greater binding of HoxA9 protein and increased binding of IRF1 to the -2578 A allele (Table 1). irf1-ccl2-3 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation mutation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 3 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 2I While the A and G alleles bind IRF1 equally when the upstream site is intact, mutation of the upstream TALE site leads to a 15-fold (Figure 2E) decrease in binding of the A allele as compared to a 1.5-fold decrease for the G allele (Figure 2I). irf1-ccl2-4 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation mutation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 4 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 2F Mutation of the upstream TALE site reduced binding by both PREP1 and PBX2 for both the -2578 A and G alleles (Figure 2B, C, F and G). pknox1-ccl2-3 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation mutation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 2 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 2G Mutation of the upstream TALE site reduced binding by both PREP1 and PBX2 for both the -2578 A and G alleles (Figure 2B, C, F and G). pbx2-ccl2-1 1 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level promoter region 17:34252732-34252789 sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252732-34252789 NC_000017.11:g.34252769A>G 17:34252769-34252769 mutation mutation undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252769-34252769 NC_000017.11:g.34252742C>T mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 17:34252742-34252742 5 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 2H Since HOXA9 protein has been shown to form complexes with PBX2 is was not surprising that mutation of the upstream TALE site significantly reduced the amount of CCL2 promoter DNA immunoprecipitated by HoxA9 antibody for both the -2578 A and -2578 G alleles (Figure 2D and H)