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Table 1. Antibodies used for chromatin immunoprecipitation (ChIP) Protein Antibody Manufacturer p65 sc-372 p50 sc-1190 RelB sc-226 c-Rel sc-71 p52 06-413 E2F4 sc-1082 Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Upstate Biotechnology, Lake Placid, NY Santa Cruz Biotechnology Pol II 8WG16 References Western blot ChIP (1, 2) (3-5) (6) (7) (8) (5) (8) (5, 9) (10) (11) (12) (13) (14) The fact that the p65 dataset has no targets in unstimulated cells confirms the lack of background enrichment from the Dynal magnetic beads. The number of p50 targets in unstimulated cells is higher than for the other subunits. We were concerned that this might be due to the antibody used. Therefore, we tested another antibody raised against the N-terminus of the protein (sc-1191). In test experiments, 90% of enriched gene promoters were shared between the two p50 antibodies (data not shown). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Rodriguez, M. S., Thompson, J., Hay, R. T. & Dargemont, C. (1999) J. Biol. Chem. 274, 9108-9115. Huang, T. T., Kudo, N., Yoshida, M. & Miyamoto, S. (2000) Proc. Natl. Acad. Sci. USA 97, 1014-1019. Nissen, R. M. & Yamamoto, K. R. (2000) Genes Dev. 14, 2314-2329. Martone, R., Euskirchen, G., Bertone, P., Hartman, S., Royce, T. E., Luscombe, N. M., Rinn, J. L., Nelson, F. K., Miller, P., Gerstein, M., et al. (2003) Proc. Natl. Acad. Sci. USA 100, 12247-12252. Saccani, S., Pantano, S. & Natoli, G. (2003) Mol. Cell 11, 1563-1574. Yamazaki, T. & Kurosaki, T. (2003) Nat. Immunol 4, 780-786. Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. & Rosenfeld, M. G. (2002) Cell 110, 55-67. Wang, W., Tam, W. F., Hughes, C. C., Rath, S. & Sen, R. (1997) Immunity 6, 165-174. Liu, J. & Beller, D. I. (2003) J. Immunol. 170, 4489-4496. Senftleben, U., Cao, Y., Xiao, G., Greten, F. R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S. C. & Karin, M. (2001) Science 293, 1495-1499. Takahashi, Y., Rayman, J. B. & Dynlacht, B. D. (2000) Genes Dev. 14, 804-816. Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A. & Dynlacht, B. D. (2002) Genes Dev. 16, 245-256. Hirakata, M., Okano, Y., Pati, U., Suwa, A., Medsger, T. A., Jr., Hardin, J. A. & Craft, J. (1993) J. Clin. Invest. 91, 2665-2672. Odom, D. T., Zizlsperger, N., Gordon, D. B., Bell, G. W., Rinaldi, N. J., Murray, H. L., Volkert, T. L., Schreiber, J., Rolfe, P. A., Gifford, D. K., et al. (2004) Science 303, 13781381. Table 2. Gene ontology (GO) categories enriched in the set of genes bound by NF-κB GO system GO category Biological process Response to biotic stimulus Biological process Immune response Biological process Number of bound genes P value 44 Total number of genes in category 516 38 425 1.3E-06 Defense response 39 475 7.8E-06 Biological process Response to pest/pathogen/parasite 26 276 3E-05 Molecular function Cytokine activity 15 133 0.00011 Molecular function Transcription factor activity 32 468 0.00046 Biological process Response to stress 33 465 0.00068 Biological process Response to wounding 16 163 0.0007 Molecular function Tumor necrosis factor receptor binding 4 12 0.00072 Biological process Response to external stimulus 49 793 0.00083 Molecular function Transcription regulator activity 38 635 0.00172 Biological process 3 7 0.00193 Biological process Antigen processing, endogenous antigen via MHC class I Inflammatory response 12 116 0.00207 Molecular function Receptor binding 23 332 0.00255 Biological process Antigen processing 3 8 0.003 Biological process Response to stimulus 53 934 0.00345 Biological process Transcription 50 869 0.00347 Biological process Innate immune response 12 125 0.00387 Biological process 13 143 0.0043 Biological process Regulation of transcription from Pol II promoter Response to virus 5 28 0.00442 Molecular function GTP binding 12 139 0.00517 Molecular function Chemokine receptor binding 5 32 0.00592 Molecular function Chemokine activity 5 32 0.00592 Molecular function Guanyl nucleotide binding 12 143 0.00648 Molecular function G-protein-coupled receptor binding 5 33 0.00677 Biological process Transcription, DNA-dependent 47 833 0.00681 Biological process Cell motility 14 169 0.00707 Biological process Viral life cycle 4 20 0.00714 Molecular function DNA binding 48 930 0.00858 Biological process Transcription from Pol II promoter 20 285 0.00902 Molecular function Binding 149 3,561 0.0091 6.3E-07 Fisher exact scores were generated using the EASEonline tool (aaps1.niaid.nih.gov/david). A total of 5,707 genes present of the arrays were annotated by GO biological process information; 227 of these were bound by NF-κB. Table 3. Genes bound by NF-κB in U937 cells ABHD8 hypothetical protein FLJ11743 ACTR3 ARP3 actin-related protein 3 homolog AF093680 similar to mouse Glt3 AF15Q14 AF15q14 protein AIM2 absent in melanoma 2 ALAS1 aminolevulinate, delta-, synthase 1 AP1S2 adaptor-related protein complex 1 APBB1IP similar to proline-rich protein 48 APG-1 heat shock protein (hsp110 family) AQP9 aquaporin 9 ARF3 ADP-ribosylation factor 3 ARHGAP4 Rho GTPase activating protein 4 ARHGEF2 rho/rac guanine nucleotide exchange factor ARL1 ADP-ribosylation factor-like 1 ARL6IP5 vitamin A responsive; cytoskeleton related ARTS-1 type 1 TNFR shedding aminopeptidase regulator ASGR2 asialoglycoprotein receptor 2 ASH1 hypothetical protein ASH1 ATF6 activating transcription factor 6 ATP5G1 ATP synthase, H+ transporting BAT3 HLA-B associated transcript 3 BAZ1B bromodomain adjacent to zinc finger domain, 1B BCL2A1 BCL2-related protein A1 BCL6 B-cell CLL/lymphoma 6 (zinc finger protein 51) BF B-factor, properdin BNIP1 BCL2/adenovirus E1B 19kD interacting protein 1 BPI bactericidal/permeability-increasing protein C10orf117 AD24 protein C11orf10 chromosome 11 open reading frame 10 C14orf111 CGI-35 protein C20orf28 chromosome 20 open reading frame 28 C21orf45 chromosome 21 open reading frame 45 C21orf59 chromosome 21 open reading frame 59 C3 complement component 3 C6orf109 DKFZP566C243 protein C6orf66 HSPC125 protein C7orf10 chromosome 7 open reading frame 10 C9orf100 hypothetical protein FLJ14642 CA11 carbonic anhydrase XI CAPZA1 capping protein (actin filament) muscle Z-line CARD15 caspase recruitment domain family, member 15 CAT catalase CCDC2 capillary morphogenesis protein 1 CCL1 small inducible cytokine A1, I-309 CCL3 small inducible cytokine A3, Mip-1-alpha CCL4 small inducible cytokine A4, Mip-1-beta CCL5 small inducible cytokine A5 (RANTES) CCNL1 cyclin L ania-6a CCR1 chemokine (C-C motif) receptor 1 CCRL2 chemokine (C-C motif) receptor-like 2 CD37 CD37 antigen CD58 CD58 antigen, (LFA 3) CD69 CD69 antigen (p60, early T-cell activation antigen) CDCA1 cell division cycle associated 1 CDH15 cadherin 15, M-cadherin (myotubule) CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) CHI3L2 chitinase 3-like 2 CLCN1 chloride channel 1, skeletal muscle (Thomsen disease) CLECSF14 macrophage lectin 2 (calcium dependent) COX4I1 cytochrome c oxidase subunit IV isoform 1 CREB1 cAMP responsive element binding protein 1 CRYBA1 crystallin, beta A1 CSF2 colony stimulating factor 2 (granulocyte-macrophage) CSF3 colony stimulating factor 3 (granulocyte) CST7 cystatin F (leukocystatin) CTSC cathepsin C CYB5 cytochrome b-5 CYP51A1 cytochrome P450, 51 (lanosterol 14-alpha-demethylase) DAF decay accelerating factor for complement DBI diazepam binding inhibitor (GABA receptor modulator) DBP D site of albumin promoter binding protein DDX21 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 21 DDX31 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 31 DDX47 hqp0256 protein DGCR6L DiGeorge syndrome critical region gene 6 like DGUOK deoxyguanosine kinase DJ434O14.3 hypothetical protein dJ434O14.3 DKFZp434K1210 hypothetical protein DKFZp434K1210 DKFZp762L0311 hypothetical protein DKFZp762L0311 - LPS + + + w + + + + + + + + + + w + + + + + + ++ +w w ++ ++ + ++ w ++ ++ +w ++ + +w + + ++ +w + + + ++ +w w ++ + + + + + w+ + + ++ + + + + + w++ + + + + + + ++ + + ++ + + ++ w w+ + + ++ +w + + ++ w w+ w+ ++ +w + + w+ w+ ++ + + ++ ++ ++ + + + + + + + + + + + w + X w w w + X X w + + + + ++ + w w + + + ++ + + + ++ + w + + w + + + + w ++ + + + w++ + + + + + X ++ + ++ + + +w + w + + + + + ++ + + ++ + + Known targets p65 c-Rel RelB p50 p52 + LPS p65 c-Rel RelB p50 p52 EntrezGene Description symbol + X X X X X X X w + + ++ ++ + w + + + + + ++ + X X DMAP1 DPP3 DR1 DXS9879E EFEMP2 EFNB1 EIF2S3 ELF3 ELK1 EMP3 ERBB2IP ERCC1 ET EXTL2 FBXW3 FCAR FEN1 FHL3 FIBP FKSG87 FLJ10420 FLJ10687 FLJ11082 FLJ11773 FLJ11838 FLJ13195 FLJ13639 FLJ20080 FLJ20257 FLJ20477 FLJ20825 FLJ22353 FLJ22555 FLJ22573 FLOT1 FUT4 GABPB2 GADD45B GDF5 GNA15 GNG5 GNL1 GPR132 GPR84 GPX1 GTF3C4 GTPBP4 GYPC HADHA HCST HIST1H4I HIST1H4K HLA-B HLA-F HLA-G HMGN4 HNMT HOXA7 HSPA1B HSPC003 HSPC052 HSPC073 HSPCB ICAM1 ICAM4 IER3 IFNGR1 IL1B IL1F9 IL2RG IL6ST IL8 ILK INHBE ITGA5 ITGAX ITSN2 IVNS1ABP JUN DNA methyltransferase 1-associated protein 1 dipeptidylpeptidase III down-regulator of transcription 1, TBP-binding DNA segment on chromosome X, expressed sequence EGF-containing fibulin-like extracellular matrix protein 2 ephrin-B1 eukaryotic translation initiation factor 2, subunit 3 E74-like factor 3 (ets domain transcription factor) ELK1, member of ETS oncogene family epithelial membrane protein 3 erbb2 interacting protein excision repair cross-complementing repair deficiency hypothetical protein ET exostoses (multiple)-like 2 F-box and WD-40 domain protein 3 Fc fragment of IgA, receptor for flap structure-specific endonuclease 1 four and a half LIM domains 3 fibroblast growth factor (acidic) intracellular binding protein FKSG87 protein hypothetical protein FLJ10420 hypothetical protein FLJ10687 hypothetical protein FLJ11082 hypothetical protein FLJ11773 hypothetical protein FLJ11838 hypothetical protein FLJ13195 similar to stromal antigen 3 hypothetical protein FLJ13639 hypothetical protein FLJ20080 hypothetical protein FLJ20257 hypothetical protein FLJ20477 hypothetical protein FLJ20825 hypothetical protein FLJ22353 hypothetical protein FLJ22555 hypothetical protein FLJ22573 flotillin 1 fucosyltransferase 4 (alpha (1,3) fucosyltransferase) GA binding protein transcription factor, beta subunit 2 growth arrest and DNA-damage-inducible, beta growth differentiation factor 5 guanine nucleotide binding protein (G protein), alpha 15 guanine nucleotide binding protein (G protein), gamma 5 guanine nucleotide binding protein-like 1 G protein-coupled receptor G protein-coupled receptor 84 glutathione peroxidase 1 general transcription factor IIIC, polypeptide 4 (90kD) G protein-binding protein CRFG glycophorin C (Gerbich blood group) hydroxyacyl-Coenzyme A dehydrogenase DNAX-activation protein 10 H4 histone family, member M H4 histone family, member D major histocompatibility complex, class I, B major histocompatibility complex, class I, F HLA-G histocompatibility antigen, class I, G high-mobility group protein 17-like 3 histamine N-methyltransferase homeo box A7 heat shock 70kD protein 1B HSPC003 protein HSPC052 protein HSPC073 protein heat shock 90kD protein 1, beta intercellular adhesion molecule 1 (CD54) intercellular adhesion molecule 4 immediate early response 3 interferon gamma receptor 1 interleukin 1, beta interleukin-1 homolog 1 interleukin 2 receptor, gamma interleukin 6 signal transducer (gp130) interleukin 8 integrin-linked kinase hypothetical protein MGC4638 integrin, alpha 5 (fibronectin receptor) integrin, alpha X, alpha polypeptide) intersectin 2 NS1-binding protein v-jun sarcoma virus 17 oncogene homolog (avian) + LPS - LPS Known targets p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52 Entrez Gene Description symbol + w+ + + w + w+ ++ + + + + ++ + + +w + w + + + + + + ++ + + + + + + ++ + + + + + + w + ++ + +w + + ++ + + + + ++ w ++ + + ++ + w + w++ + + ++ + + + + + + + + + w w + + + w + w w + w w + + + + + +w + + ++ + + ++ + + + w + + + + + ++ + + ++ +w + + +w w w+ ++ + ++ ++ +w X + ++ + w + w + + + w + + ++ w w + w ++ w + + ++ X + + w + + +w + + ww + + ++ + + + + ++ + w + w+w + ++ + +w w w+ + X w + + w + + + + + ++ + + X X + + + + + + + w X X + w+ + w X + w + X X X KIAA0186 KIAA0218 KIAA0319 KIAA0409 KIAA0669 KIAA1008 KIAA1056 KLF15 LGALS1 LIM2 LIMK1 LIPT1 LMNA LOC113201 LOC116211 LOC51015 LOC51321 LOC55580 LOC92106 LRRC19 LRSAM1 M96 MAN2B1 MAPK6 MASTL MCCC2 MCP MDH1 MEF2B MGC10960 MGC10966 MGC14141 MGC4677 MGC4771 MGC5242 MID1IP1 MLLT10 MMAB MMP19 MMP8 MMP9 MRPL30 MRPL34 MRPS7 MX2 NAG73 NAP1L1 NAPA NCR1 NFATC2 NFKB2 NFKBIB NKG7 NME1 NOLC1 NOVA1 NR1D1 NRAS NRXN3 NUP107 OAS3 OAZ1 OAZ2 ORMDL1 P4HA1 PAFAH2 PAK4 PALM PDE6D PEX13 PHLDA1 PILRA PILRB PKM2 PLK1 PLXNB1 PMAIP1 PMS1 PMS2L8 KIAA0186 gene product KIAA0218 gene product KIAA0319 gene product KIAA0409 protein KIAA0669 gene product mitotic control protein dis3 homolog KIAA1056 protein Kruppel-like factor 15 lectin, galactoside-binding, soluble, 1 (galectin 1) lens intrinsic membrane protein 2 (19kD) LIM domain kinase 1 lipoyltransferase lamin A/C hypothetical protein BC012124 hypothetical protein BC013113 CGI-111 protein hypothetical protein hypothetical protein hypothetical protein BC008322 leucine rich repeat containing 19 leucine rich repeat and sterile alpha motif containing 1 putative DNA binding protein mannosidase, alpha, class 2B, member 1 mitogen-activated protein kinase 6 hypothetical protein FLJ14813 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) membrane cofactor protein (CD46) malate dehydrogenase 1, NAD (soluble) MADS box transcription factor 2, polypeptide B hypothetical protein MGC10960 hypothetical protein MGC10966 hypothetical protein MGC14141 hypothetical protein MGC4677 hypothetical protein MGC4771 hypothetical protein MGC5242 hypothetical protein STRAIT11499 myeloid/lymphoid leukemia translocated to 10 hypothetical protein MGC20496 matrix metalloproteinase 19 matrix metalloproteinase 8 (neutrophil collagenase) matrix metalloproteinase 9 (gelatinase B, 92kD gelatinase) mitochondrial ribosomal protein L30 mitochondrial ribosomal protein L34 mitochondrial ribosomal protein S7 myxovirus (influenza virus) resistance 2 (mouse) NPC-related protein NAG73 nucleosome assembly protein 1-like 1 N-ethylmaleimide-sensitive factor attachment protein, alpha lymphocyte antigen 94 homolog, NK-p46, (mouse) nuclear factor of activated T-cells, calcineurin-dependent 2 NF-κB p52/p100 NF-κB inhibitor, beta natural killer cell group 7 sequence non-metastatic cells 1, protein (NM23A) expressed in nucleolar and coiled-body phosphprotein 1 neuro-oncological ventral antigen 1 nuclear receptor subfamily 1, group D, member 1 neuroblastoma RAS viral (v-ras) oncogene homolog neurexin 3 nuclear pore complex protein 2'-5'-oligoadenylate synthetase 3 (100 kD) ornithine decarboxylase antizyme 1 ornithine decarboxylase antizyme 2 hypothetical protein procollagen-proline, 2-oxoglutarate 4-dioxygenase alpha platelet-activating factor acetylhydrolase 2 (40kD) p21(CDKN1A)-activated kinase 4 paralemmin phosphodiesterase 6D, cGMP-specific, rod, delta peroxisome biogenesis factor 13 pleckstrin homology-like domain, family A, member 1 paired immunoglobulin-like receptor alpha paired immunoglobulin-like receptor beta pyruvate kinase, muscle polo-like kinase (Drosophila) plexin B1 phorbol-12-myristate-13-acetate-induced protein 1 PMS1 postmeiotic segregation increased 1 (S. cerevisiae) postmeiotic segregation increased 2-like 8 + LPS - LPS p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52 EntrezGene Description s ymbol + + + w + + + + ++ + w ++ w w + + + ++ + w+ + + ++ + + + + w + w w+ + + + + + + + + + ++ + + ++ + + w w+ w+ w w+ w++ + + + +w ++ ++ +w + + + ++ + + ++ + + + + ++ + + + + + + + + + ++ + w++ +w + +w Known targets w + + + w + + ++ + w w + + + + + + w w + + w + + + + + X + + + + ++ w + + w+ w+ + + w + + ++ + + + + w++ + w+ w+ + +w + w+ +w + +w w + w + w + + ++ + + + + + + + + + + ++ w + + + + X w + + + X POLB POLD4 POLR1C POLR2A POLR3E PPFIBP1 PPP1R15B PPP1R3B PPP5C PRDM1 PRDM11 PRDM8 PRKAB2 PTGS2 PTPN7 PTX3 PXMP3 QTRTD1 RAB7L1 RABL2A RABL2B RAC2 RB1 RB1CC1 RBBP4 RFP RFXANK RGC32 RGL1 RND1 RNF4 RPL27A RPL39 RPL39L RPS28 RPS8 RS1 S100A12 S100A3 S100A4 S100A9 S100Z SAS SCARB1 SDF2L1 SELPLG SEMA7A SEPW1 SERPINB2 SERPINE1 SFRS2 SIRT2 SLAMF8 SLC1A3 SLC25A12 SLC30A7 SLC39A6 SLC7A1 SMPD1 SOX8 SPATA2 SQSTM1 SREBF2 SRF SSSCA1 STARD10 STAT1 STAT5A STAT6 STATIP1 STXBP3 SVIL SYNPO TAGLN2 TAPBP TFAP2A TFDP3 polymerase (DNA directed), beta polymerase (DNA-directed), delta 4 RNA polymerase I subunit polymerase (RNA) II (DNA directed) polypeptide A hypothetical protein FLJ10509 PTPRF interacting protein, binding protein 1 protein phosphatase 1, regulatory subunit 15B protein phosphatase 1, regulatory subunit 3B protein phosphatase 5, catalytic subunit PR domain containing 1, with ZNF domain PR domain containing 11 PR domain containing 8 protein kinase, AMP-activated, beta 2 subunit prostaglandin-endoperoxide synthase 2, COX-2 protein tyrosine phosphatase, non-receptor type 7 pentaxin-related gene, rapidly induced by IL-1 beta peroxisomal membrane protein 3 (Zellweger syndrome) hypothetical protein FLJ12960 RAB7, member RAS oncogene family-like 1 RAB, member of RAS oncogene family-like 2A RAB, member of RAS oncogene family-like 2B ras-related C3 botulinum toxin substrate 2 (rho family) retinoblastoma 1 (including osteosarcoma) RB1-inducible coiled-coil 1 retinoblastoma binding protein 4 ret finger protein regulatory factor X-associated ankyrin-containing protein RGC32 protein RalGDS-like gene GTP-binding protein ring finger protein 4 ribosomal protein L27a ribosomal protein L39 ribosomal protein L39-like ribosomal protein S28 ribosomal protein S8 retinoschisis (X-linked, juvenile) 1 S100 calcium binding protein A12 (calgranulin C) S100 calcium binding protein A3 S100 calcium binding protein A4 (calvasculin) S100 calcium binding protein A9 (calgranulin B) S100Z protein sarcoma amplified sequence CD36 antigen (thrombospondin receptor)-like 1 stromal cell-derived factor 2-like 1 selectin P ligand sema domain, immunoglobulin domain, semaphorin 7A selenoprotein W, 1 serine (or cysteine) proteinase inhibitor, clade B serine (or cysteine) proteinase inhibitor, clade E splicing factor, arginine/serine-rich 2 sirtuin silent mating type information regulation 2 B lymphocyte activator macrophage expressed solute carrier family 1 (glutamate transporter), member 3 solute carrier family 25, member 12 zinc transporter like 2 LIV-1 protein, estrogen regulated solute carrier family 7, member 1 sphingomyelin phosphodiesterase 1, acid lysosomal SRY (sex determining region Y)-box 8 spermatogenesis associated 2 sequestosome 1 sterol regulatory element binding transcription factor 2 serum response factor Sjogren's syndrome/scleroderma autoantigen 1 serologically defined colon cancer antigen 28 signal transducer and activator of transcription 1 signal transducer and activator of transcription 5A signal transducer and activator of transcription 6 elongator protein 2 syntaxin binding protein 3 supervillin synaptopodin transgelin 2 TAP binding protein (tapasin) transcription factor AP-2 alpha E2F-like protein + LPS - LPS p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52 EntrezGene Description symbol + + + + + + + +w + + + ++ w + + + w+ + w w+ + ++ w + w w + + + + + + w + + + + + + + + +w + + + + + + + + + +w w+ +w + + + + +w + +w + + + + + w + + w + + + + + + w w + +w + w ++ + w + + ++ + +w +w + +ww w+ ++ + w + + + + + + + + + + + X X X X w w + + w X w + + + w w+ + + + +w ++ + w w+ w + + + ++ + w + Known targets + + + + X X ++ + ++ + ++ + + + + X w X + + + ++ TFE3 TIMP1 TJP2 TLN1 TMPIT TNF TNFRSF1A TNFSF13 TNFSF13B TNFSF14 TNIP3 TRA1 TRAF1 TRAPPC3 TRIM31 TRPM2 TTC17 UBE2H UBE2L6 USP2 USP25 USP52 VAV1 VSIG4 WBP4 YME1L1 YWHAB ZBTB11 ZDHHC6 ZNF217 ZNF221 ZNF581 ZNF76 ZNHIT1 transcription factor binding to IGHM enhancer 3 tissue inhibitor of metalloproteinase 1 tight junction protein 2 (zona occludens 2) talin 1 transmembrane protein induced by tumor TNF tumor necrosis factor (TNF superfamily, member 2) tumor necrosis factor receptor superfamily, member 1A tumor necrosis factor (ligand) superfamily, member 13 tumor necrosis factor (ligand) superfamily, member 13b tumor necrosis factor (ligand) superfamily, member 14 hypothetical protein FLJ21162 tumor rejection antigen (gp96) 1 TNF receptor-associated factor 1 similar to yeast BET3 (S. cerevisiae) tripartite motif-containing 31 transient receptor potential cation channel, subfamily M hypothetical protein FLJ10890 ubiquitin-conjugating enzyme E2H ubiquitin-conjugating enzyme E2L 6 ubiquitin specific protease 2 ubiquitin specific protease 25 KIAA0710 gene product vav 1 oncogene Ig superfamily protein WW domain binding protein 4 (formin binding protein 21) YME1-like 1 (S. cerevisiae) tyrosine 3-monooxygenase, beta polypeptide zinc finger protein hypothetical protein FLJ21952 zinc finger protein 217 zinc finger protein 221 HSPC189 protein zinc finger protein 76 (expressed in testis) putative cyclin G1 interacting protein +, Indicates high confidence binding (p< 0.002), at least one subunit had to match this criterion w, Indicates weaker binding events (p< 0.01) - LPS + ++ ++ ++ + + + +w + + w + +w + w + +w + + ++ ++ ++ + + + + w + + + +w + + w++ +w + ++ + + Known targets p65 c-Rel RelB p50 p52 + LPS p65 c-Rel RelB p50 p52 EntrezGene Description symbol + + ++ + + w + ++ + + + + + + + ++ w + + + ww + + ++ + + + + w + X + + + + ++ X Table 4. Genes bound by E2F4 in unstimulated U937 cells EntrezGene Description symbol EntrezGene Description symbol AK2 AP2S1 AP4B1 APG4C ARHGAP11A ASF1B ATF2 ATM ATP5G3 BAT4 BAZ1B BCAS1 BLM BRD8 BUB3 C10orf7 C11orf10 C12orf5 C14orf123 C20orf172 C20orf77 C21orf45 C6orf166 C6orf18 C6orf211 C6orf82 C9orf76 CASP2 CASP8AP2 CBX3 CBX5 CCNB1 CDC25A CDC25C CDC6 CDCA1 CDCA8 CENPF CKAP1 CLSPN CSTF3 DCLRE1B DDX10 DKFZP434B168 DLG7 EIF3S2 EXO1 FANCL FLJ10579 FLJ10922 FLJ11029 FLJ11222 FLJ13912 FLJ20354 FLJ20508 GHITM GPHN H2AFG H2AFP HAGHL HIST1H2AJ HIST1H2AL HIST1H2BJ HIST1H2BK HIST1H2BL HIST1H2BM HIST1H3B HIST1H4C HIST1H4K HMGB2 HNRPA1 HPCL2 HSPC052 HSPC150 HT007 IER5 IRF3 ITGA2 KIAA0042 KIAA0092 KIAA0101 KIAA0410 KIAA0747 KIAA0872 KIF20A KIF22 KIF2C KIF4A KNSL7 KNTC2 KPNB1 LENG5 LGALS1 LOC55924 LOC81691 LSM4 LUC7L2 M96 MAPK7 MASTL MCM3 MDH1 MDS009 MGC5469 MIS12 MRP63 MRPS11 MRPS15 MRPS18C MRPS27 MTAP MTRF1L MUT MYOHD1 NAP1L1 NBR2 NDUFA1 NDUFB9 NEK2 NFKBIB NFYA NFYC NUDT15 NUP98 NUSAP1 ORC3L ORMDL1 PAI-RBP1 PCNA PCNP PDZK11 PELO PEX12 PIGC PLCG2 PLK1 PLK4 PMS1 POLA POLD1 POLE3 POLR3K PRPS1 QPCTL RAD51 RAMP RBBP4 RBL1 RBM15 RFC5 RNASE4 RNPC2 RPGR RPL26 adenylate kinase 2 adaptor-related protein complex 2, sigma 1 subunit adaptor-related protein complex 4, beta 1 subunit APG4 autophagy 4 homolog C (S. cerevisiae) KIAA0013 gene product ASF1 anti-silencing function 1 homolog B activating transcription factor 2 ataxia telangiectasia mutated ATP synthase, H+ transporting, mitochondrial F0 complex HLA-B associated transcript 4 bromodomain adjacent to zinc finger domain, 1B breast carcinoma amplified sequence 1 Bloom syndrome bromodomain containing 8 BUB3 budding uninhibited by benzimidazoles 3 homolog chromosome 10 open reading frame 76 chromosome 11 open reading frame 10 chromosome 12 open reading frame 5 chromosome 14 open reading frame 123 chromosome 20 open reading frame 172 chromosome 20 open reading frame 77 chromosome 21 open reading frame 45 chromosome 6 open reading frame 166 chromosome 6 open reading frame 18 chromosome 6 open reading frame 211 chromosome 6 open reading frame 82 chromosome 9 open reading frame 76 caspase 2, apoptosis-related cysteine protease CASP8 associated protein 2 chromobox homolog 3 (HP1 gamma homolog, Drosophila) chromobox homolog 5 (HP1 alpha homolog, Drosophila) cyclin B1 cell division cycle 25A cell division cycle 25C CDC6 cell division cycle 6 homolog (S. cerevisiae) cell division cycle associated 1 cell division cycle associated 8 centromere protein F (350/400kD, mitosin) cytoskeleton-associated protein 1 claspin homolog (Xenopus laevis) cleavage stimulation factor, 3' pre-RNA, subunit 3 DNA cross-link repair 1B (PSO2 homolog, S. cerevisiae) DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10 DKFZP434B168 protein discs, large homolog 7 (Drosophila) eukaryotic translation initiation factor 3, subunit 2 exonuclease 1 Fanconi anemia, complementation group L hypothetical protein FLJ10579 hypothetical protein FLJ10922 hypothetical protein FLJ11029 hypothetical protein FLJ11222 hypothetical protein FLJ13912 hypothetical protein FLJ20354 hypothetical protein FLJ20508 growth hormone inducible transmembrane protein gephyrin H2A histone family, member G H2A histone family, member P hydroxyacylglutathione hydrolase-like histone 1, H2aj histone 1, H2al histone 1, H2bj histone 1, H2bk histone 1, H2bl histone 1, H2bm histone 1, H3b histone 1, H4c histone 1, H4k high-mobility group box 2 heterogeneous nuclear ribonucleoprotein A1 2-hydroxyphytanoyl-CoA lyase HSPC052 protein HSPC150 protein similar to ubiquitin-conjugating enzyme uncharacterized hypothalamus protein HT007 immediate early response 5 interferon regulatory factor 3 integrin, alpha 2 (CD49B) KIAA0042 gene product KIAA0092 gene product KIAA0101 gene product KIAA0410 gene product KIAA0747 protein KIAA0872 protein kinesin family member 20A kinesin family member 22 kinesin family member 2C kinesin family member 4A kinesin-like 7 kinetochore associated 2 karyopherin (importin) beta 1 leukocyte receptor cluster (LRC) member 5 lectin, galactoside-binding, soluble, 1 hypothetical protein exonuclease NEF-sp U6 snRNA-associated Sm-like protein LUC7-like 2 (S. cerevisiae) putative DNA binding protein mitogen-activated protein kinase 7 microtubule associated serine/threonine kinase-like MCM3 minichromosome maintenance deficient 3 malate dehydrogenase 1, NAD (soluble) x 009 protein hypothetical protein MGC5469 MIS12 homolog (yeast) mitochondrial ribosomal protein 63 mitochondrial ribosomal protein S11 mitochondrial ribosomal protein S15 mitochondrial ribosomal protein S18C mitochondrial ribosomal protein S27 methylthioadenosine phosphorylase mitochondrial translational release factor 1-like methylmalonyl Coenzyme A mutase myosin head domain containing 1 nucleosome assembly protein 1-like 1 NBR2 NADH dehydrogenase 1 alpha subcomplex, 1 NADH dehydrogenase 1 beta subcomplex, 9 NIMA (never in mitosis gene a)-related kinase 2 NF-κB inhibitor, beta nuclear transcription factor Y, alpha nuclear transcription factor Y, gamma nudix-type motif 15 nucleoporin 98kD nucleolar and spindle associated protein 1 origin recognition complex, subunit 3-like ORM1-like 1 (S. cerevisiae) PAI-1 mRNA-binding protein proliferating cell nuclear antigen PEST-containing nuclear protein PDZ domain containing 11 pelota homolog (Drosophila) peroxisomal biogenesis factor 12 phosphatidylinositol glycan, class C phospholipase C, gamma 2 Polo like Kinase 1 Polo like Kinase 4 PMS1 postmeiotic segregation increased 1 polymerase (DNA directed), alpha polymerase (DNA directed), delta 1, catalytic subunit polymerase (DNA directed), epsilon 3 (p17 subunit) polymerase (RNA) III (DNA directed) polypeptide K phosphoribosyl pyrophosphate synthetase 1 glutaminyl-peptide cyclotransferase-like RAD51 homolog (RecA homolog, E. coli) RA-regulated nuclear matrix-associated protein retinoblastoma binding protein 4 retinoblastoma-like 1 (p107) RNA binding motif protein 15 replication factor C (activator 1) 5 (36.5kD) ribonuclease, RNase A family, 4 RNA-binding region (RNP1, RRM) containing 2 retinitis pigmentosa GTPase regulator ribosomal protein L26 EntrezGene Description symbol RPL37 RPL39L RQCD1 RRM1 RXRB SAMHD1 SF3A3 SFRS1 SFRS2 SLC2A4 SLC31A1 SLC35A5 SLC39A9 SLC8A3 SMC1L1 SMC2L1 SMC4L1 SMN2 SNRPD2 SNX13 SPOCK3 STOML2 SUCLG1 SULT1A3 SUMO1 TCF19 TDRD3 TIMELESS TMEM5 TOPK TTF2 TTRAP TU12B1-TY TUSC4 TYMS UCHL5 USP1 VILL VPS29 WDR19 XRCC5 YWHAB ZIC1 ZNF451 ribosomal protein L37 ribosomal protein L39-like RCD1 required for cell differentiation1 homolog ribonucleotide reductase M1 polypeptide retinoid X receptor, beta SAM domain and HD domain, 1 splicing factor 3a, subunit 3, 60kD splicing factor, arginine/serine-rich 1 splicing factor, arginine/serine-rich 2 solute carrier family 2, member 4 solute carrier family 31, member 1 solute carrier family 35, member A5 solute carrier family 39, member 9 solute carrier family 8, member 3 SMC1 structural maintenance of chromosomes 1-like 1 SMC2 structural maintenance of chromosomes 2-like 1 SMC4 structural maintenance of chromosomes 4-like 1 survival of motor neuron 2, centromeric small nuclear ribonucleoprotein D2 polypeptide (16.5kD) sorting nexin 13 sparc, cwcv and kazal-like domains proteoglycan 3 stomatin (EPB72)-like 2 succinate-CoA ligase, GDP-forming, alpha subunit sulfotransferase family, cytosolic, 1A, phenol-preferring 3 SMT3 suppressor of mif two 3 homolog 1 transcription factor 19 (SC1) tudor domain containing 3 timeless homolog (Drosophila) transmembrane protein 5 T-LAK cell-originated protein kinase transcription termination factor, RNA polymerase II TRAF and TNF receptor associated protein TU12B1-TY protein tumor suppressor candidate 4 thymidylate synthetase ubiquitin carboxyl-terminal hydrolase L5 ubiquitin specific protease 1 villin-like vacuolar protein sorting 29 (yeast) WD repeat domain 19 X-ray repair complementing defective repair in Chinese hamster cells 5 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Zic family member 1 (odd-paired homolog, Drosophila) zinc finger protein 451 Table 5. Genes bound by four or five NF-κB subunits EntrezGene Description symbol 4 subunits APBB1IP ASH1 BCL2A1 C3 CCL4 CCRL2 CD69 CREB1 DBI EMP3 FLJ10420 FLJ22555 HSPCB IL1F9 MAN2B1 OAS3 P4HA1 PPP1R15B TAPBP TIMP1 TNF WBP4 similar to proline-rich protein 48 hypothetical protein ASH1 BCL2-related protein A1 complement component 3 small inducible cytokine A4 chemokine (C-C motif) receptor-like 2 CD69 antigen (p60, early T-cell activation antigen) cAMP responsive element binding protein 1 diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein) epithelial membrane protein 3 hypothetical protein FLJ10420 hypothetical protein FLJ22555 heat shock 90kD protein 1, beta interleukin-1 homolog 1 mannosidase, alpha, class 2B, member 1 2'-5'-oligoadenylate synthetase 3 (100 kD) procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I protein phosphatase 1, regulatory (inhibitor) subunit 15B TAP binding protein (tapasin) tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor) tumor necrosis factor (TNF superfamily, member 2) WW domain binding protein 4 (formin binding protein 21) 5 subunits AQP9 ARHGEF2 CCL3 CCNL1 DAF FLJ20477 FLOT1 GADD45B GPX1 GYPC ICAM1 IL2RG NFKB2 SMPD1 TRAF1 aquaporin 9 rho/rac guanine nucleotide exchange factor (GEF) 2 small inducible cytokine A3, Mip-1-alpha cyclin L ania-6a decay accelerating factor for complement (CD55) hypothetical protein FLJ20477 flotillin 1 growth arrest and DNA-damage-inducible, beta glutathione peroxidase 1 glycophorin C (Gerbich blood group) intercellular adhesion molecule 1 (CD54) interleukin 2 receptor, gamma p52/p100 sphingomyelin phosphodiesterase 1, acid lysosomal TNF receptor-associated factor 1 Supporting Figure Legends Figure 6. Example scatter plots showing binding of p65 and p50 to gene promoters in living cells. Microarray data from p65 ChIP in unstimulated cells and p50 ChIP following LPS stimulation. The x-axis represents the hybridization signal intensity for input whole cell extract (WCE) DNA (log10 Cy3 background-subtracted signal). The yaxis shows the intensity for DNA enriched by ChIP using antibodies specific for p65 or p50 (log10 Cy5 background-subtracted signal). Microarray elements that were flagged as not found by the Axon software were not analyzed. The red lines either side of the scatter plots represent the ratio of IP signal to WCE signal necessary to achieve a significance score of 0.002. The position of the promoters for the known NF-κB target genes HLA-B, NFKB2, CCL1, PTX3, CSF2 and RAC2, the novel NF-κB target genes NFKBIB and P4HA1 and β-actin, a negative control, are marked in both scatter plots. No genes were enriched above the pvalue threshold of 0.002 by p65 ChIP in unstimulated cells. In contrast, HLA-B, NFKB2, CCL1, PTX3, CSF2, RAC2, NFKBIB and P4HA1 were among the 193 genes enriched above this threshold by p50 ChIP in stimulated cells. β-actin was not enriched in either p65 or p50 ChIP. Figure 7. Gene-specific PCRs of immunoprecipitated and whole cell extract DNA. A) Gene-specific PCRs of promoters at which binding was detected using the promoter arrays. All gene-specific PCRs were performed with DNA from LPS- stimulated cells. 1/30th of the immunoprecipitated (IP) DNA (equivalent to 1.5x106 cells) was amplified with the designated promoter specific primers and the results compared to the amplification of serial dilutions of whole cell extract DNA (1/18000th, 1/6000th and 1/2000th). Thus, a similar intensity DNA band in the IP amplification and the highest WCE dilution amplification indicates that the DNA sequence is present in the IP fraction at an amount ~600x lower than in the WCE. We amplified the β-actin promoter from the IP and WCE DNA as a negative control (β-actin was not bound by NF-κB in the array results, see Supporting Information Figure 6). The intensities of the bands were quantified using ImageQuant (Amersham). The enrichment ratio is the average foldenrichment of the promoter of interest in the IP fraction compared to WCE, normalized to the β-actin control (ie. a ratio greater than 1 indicates that the ratio of IP to WCE is greater for the NF-κB target gene than for β-actin). B) Gene-specific PCRs of promoters at which binding was not detected using the promoter arrays. C) Intersection between previously known directly bound NF-κB target genes and targets identified in this study. The complete list of previously known targets is compiled from all the cell types and conditions in the literature and is available through the Literature Targets link at web.wi.mit.edu/young/nfkb (based on Pahl, H.L. (1999). Oncogene 18, 6853-6866 and the Gilmore lab database at http://people.bu.edu/gilmore/nf-kb/target/index.html). Of the 224 literature targets, our array covered the promoters of 154 genes. Of these, 38 NF-κB targets were bound at a p-value of 0.002 (24%, p=4x10-19) and 49 at a p-value of 0.01 (32%, p=2x10-19). As a negative control, we compared the NF-κB literature targets to those identified for E2F4 using location analysis and found no significant overlap (p=0.96). A list of the E2F4 bound genes can be found in Supporting Information, Table 4. Figure 8. Immunoblotting of NF-κB proteins in the cytoplasm and nucleus of U937 cells before and after LPS stimulation. U937 cells were treated with LPS (2.5 µg/ml) and nuclear and cytosolic extracts were harvested at 0, 30, 60 and 180 minutes following stimulation. p50 and p52 were both present in the nuclei of unstimulated cells and increased in nuclei after LPS stimulation, consistent with the results of location analysis. The p52 antibody (Upstate, 06-413) detects an additional band in the nucleus corresponding to a protein of approximately 58 kD. This protein is unlikely to be an NF-κB subunit because it is not present in the cytoplasm and does not increase upon LPS stimulation. This unspecific band is also apparent in western blots performed using this antibody by other investigators (Senftleben et al. 2001. Science 293,1495-1499). We do not believe that this unspecific binding significantly affects our location analysis results because the vast majority of the p52 targets we identify were also detected by antibodies to other NF-κB subunits, either before or after LPS stimulation. Small amounts of RelB and c-Rel are apparent in the nuclei of unstimulated U937 cells after long exposures to film. We also detect a relatively small number of targets for these proteins in unstimulated cells (Figure 1). Location analysis can be more sensitive than western blotting for small amounts of DNA binding proteins because the technique uses immunoprecipitation to concentrate protein from ~5x107 cells and then LM-PCR to amplify co-precipitating DNA. Materials and methods: Extracts were prepared using a protocol modified from Dignam and colleagues (Dignam et al. (1983). Nucleic Acids Res. 11, 14751489). Cells were washed with PBS and lysed on ice in hypertonic buffer with NP-40 (10mM Hepes, pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 0.1% NP40 with Complete protease inhibitor (Roche)) for 5 minutes. The cytosolic fraction was harvested by centrifuging the lysate at 4500g for 3 min and collecting the supernatant. The nuclear pellet was washed twice in hypertonic buffer without NP-40. Nuclear proteins were then extracted on ice in high-salt buffer (20mM Hepes, pH 7.9, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM DTT, 10% glycerol with Complete protease inhibitor (Roche)) for 30 minutes. The nuclear extract was harvested by centrifuging the lysate at 7000g for 3 min and collecting the supernatant. Protein concentrations were determined using the BCA kit (Pierce) and 8µg of protein extracts were loaded per lane and separated by SDSpolyacrylamide gel electrophoresis. Proteins were transferred onto Hybond-P membrane (Amersham) and the membrane blocked with 5% milk in PBS containing 0.1% Tween-20 (Sigma). The primary antibodies used were identical to those used for location analysis. The blots were developed with ECL (Amersham). Figure 9. Recruitment of activatory NF-κB subunits to activated genes upon LPS stimulation. A. NF-κB binding at genes to which RNA polymerase II is recruited and to genes where it is lost (top 5% of genes for both categories). The change in RNA polymerase binding is represented as a ratio, with red indicating an increase and green a decrease, according to the scale on the left. Only genes bound by RNA polymerase II in either unstimulated or activated cells were used. NF-κB subunits that were already bound (p<0.002) before LPS stimulation are marked in blue and the recruitment of new subunits is marked in red. B. NF-κB binding at genes that are upregulated or downregulated over time in response to LPS in U937 cells. Genes were either upregulated by 2-fold or downregulated by 2-fold at two consecutive time points. Red indicates an increase in expression and green a decrease according to the scale on the left. Figure 10. Model of the transcriptional regulatory network coordinated by NF-κB family members. Genes (boxes) bound by NF-κB subunits (circles) in U937 cells before and after stimulation with LPS. Among the NF-κB target genes identified here are many transcription factors with roles in regulating the immune response. Therefore we suggest a model in which NF-κB controls the response to pathogens, not only by binding to its direct targets, but also through the activation of other transcription factors. Supporting Information Figure 6 log(IP) log(IP) 0 targets 4 p65 -LPS HLA-B NFKB2 NFKBIB CCL1 P4HA1 PTX3 β-actin CSF2 RAC2 3 2 3 2 1 0 193 targets 4 p50 +LPS HLA-B NFKB2 NFKBIB P4HA1 CCL1 CSF2 RAC2 PTX3 β-actin 1 0 1 2 3 44 log(WCE) 0 0 1 2 3 4 log(WCE) Supporting Information Figure 8 Cytoplasm 0 30 60 180 p65 p105 p50 RelB c-Rel p100 p52 Nucleus 0 30 60 180 NF-κB binding B 2 5/3 3/2 2/3 3/5 1/2 >8 4 2 1 1/2 1/4 <1/8 Change in gene expression NF-κB binding p50 p52 p65 RelB c-Rel Change in RNA Pol II p50 p52 p65 RelB c-Rel A 0 hrs 1 hr 3 hrs 9 hrs 27 hrs Supporting Information Figure 9 Supporting Information Figure 10 RB1 BCL6 p50 PRDM1 TAPBP IL8 JUN p52 STAT1 NFKBIB TFAP2A p65 VAV1 RB1 ICAM1 STAT6 CDKNA1 RelB CREB1 CCL3 CCL4 + LPS c-Rel SRF NFKB2 BCL6 p50 PRDM1 TAPBP IL8 JUN p52 STAT1 NFKBIB TFAP2A p65 VAV1 ICAM1 STAT6 CDKNA1 RelB CREB1 CCL3 CCL4 c-Rel SRF NFKB2