Table 1. Antibodies used for immunoprecipitation (ChIP)

Protein Antibody Manufacturer References Western blot ChIP p65 sc-372 Santa Cruz (1, 2) (3-5) Biotechnology p50 sc-1190 Santa Cruz (6) (7) Biotechnology RelB sc-226 Santa Cruz (8) (5) Biotechnology c-Rel sc-71 Santa Cruz (8) (5, 9) Biotechnology p52 06-413 Upstate Biotechnology, (10) Lake Placid, NY sc-1082 Santa Cruz (11) (12) Biotechnology Pol II 8WG16 (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 (sc-1191). In test experiments, 90% of enriched promoters were shared between the two p50 antibodies (data not shown).

1. Rodriguez, M. S., Thompson, J., Hay, R. T. & Dargemont, C. (1999) J. Biol. Chem. 274, 9108-9115. 2. Huang, T. T., Kudo, N., Yoshida, M. & Miyamoto, S. (2000) Proc. Natl. Acad. Sci. USA 97, 1014-1019. 3. Nissen, R. M. & Yamamoto, K. R. (2000) Dev. 14, 2314-2329. 4. 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. 5. Saccani, S., Pantano, S. & Natoli, G. (2003) Mol. Cell 11, 1563-1574. 6. Yamazaki, T. & Kurosaki, T. (2003) Nat. Immunol 4, 780-786. 7. Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. & Rosenfeld, M. G. (2002) Cell 110, 55-67. 8. Wang, W., Tam, W. F., Hughes, C. C., Rath, S. & Sen, R. (1997) Immunity 6, 165-174. 9. Liu, J. & Beller, D. I. (2003) J. Immunol. 170, 4489-4496. 10. 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. 11. Takahashi, Y., Rayman, J. B. & Dynlacht, B. D. (2000) Genes Dev. 14, 804-816. 12. Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A. & Dynlacht, B. D. (2002) Genes Dev. 16, 245-256. 13. Hirakata, M., Okano, Y., Pati, U., Suwa, A., Medsger, T. A., Jr., Hardin, J. A. & Craft, J. (1993) J. Clin. Invest. 91, 2665-2672. 14. 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, 1378- 1381. Table 2. (GO) categories enriched in the set of genes bound by NF-κB

GO system GO category Number of Total number P value bound genes of genes in category Biological process Response to biotic stimulus 44 516 6.3E-07 Biological process Immune response 38 425 1.3E-06 Biological process 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 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 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 Antigen processing, endogenous 3 7 0.00193 antigen via MHC class I Biological process 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 Regulation of transcription from Pol II 13 143 0.0043 Biological process 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

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

EntrezGene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52

ABHD8 hypothetical protein FLJ11743 + ACTR3 ARP3 actin-related protein 3 homolog + + AF093680 similar to mouse Glt3 + w AF15Q14 AF15q14 protein + w AIM2 absent in melanoma 2 + + + + + ALAS1 aminolevulinate, delta-, synthase 1 + + + + + AP1S2 adaptor-related protein complex 1 + w + + APBB1IP similar to proline-rich protein 48 + + + + + APG-1 heat shock protein (hsp110 family) w + AQP9 aquaporin 9 + + + + + ARF3 ADP-ribosylation factor 3 + + + + ARHGAP4 Rho GTPase activating protein 4 + w ARHGEF2 rho/rac guanine nucleotide exchange factor + + + + + + ARL1 ADP-ribosylation factor-like 1 + + + X ARL6IP5 vitamin A responsive; cytoskeleton related + + w + w ARTS-1 type 1 TNFR shedding aminopeptidase regulator + + ASGR2 asialoglycoprotein receptor 2 + ASH1 hypothetical protein ASH1 + + + + ATF6 activating transcription factor 6 + w ATP5G1 ATP synthase, H+ transporting + w BAT3 HLA-B associated transcript 3 w + BAZ1B bromodomain adjacent to domain, 1B + w BCL2A1 BCL2-related protein A1 + + + + w X BCL6 B-cell CLL/lymphoma 6 (zinc finger protein 51) + w BF B-factor, properdin + w X BNIP1 BCL2/adenovirus E1B 19kD interacting protein 1 + + + BPI bactericidal/permeability-increasing protein + C10orf117 AD24 protein + C11orf10 11 open reading frame 10 + w 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 w + + C3 complement component 3 + + + + w + X C6orf109 DKFZP566C243 protein + + C6orf66 HSPC125 protein + w + + C7orf10 chromosome 7 open reading frame 10 + + + C9orf100 hypothetical protein FLJ14642 + w + + CA11 carbonic anhydrase XI + + + + CAPZA1 capping protein (actin filament) muscle Z-line + CARD15 caspase recruitment domain family, member 15 + X CAT catalase + + CCDC2 capillary morphogenesis protein 1 + CCL1 small inducible cytokine A1, I-309 + + + + X CCL3 small inducible cytokine A3, Mip-1-alpha + + + + + + + + X CCL4 small inducible cytokine A4, Mip-1-beta + + + + w + X CCL5 small inducible cytokine A5 (RANTES) w w + + + X CCNL1 cyclin L ania-6a + + + + + + + + CCR1 chemokine (C-C motif) receptor 1 + w CCRL2 chemokine (C-C motif) receptor-like 2 + + + + w CD37 CD37 antigen w + CD58 CD58 antigen, (LFA 3) w + + CD69 CD69 antigen (p60, early T-cell activation antigen) w + + + + X CDCA1 cell division cycle associated 1 + w w CDH15 cadherin 15, M-cadherin (myotubule) + + CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) + X CHI3L2 chitinase 3-like 2 w + + + CLCN1 chloride channel 1, skeletal muscle (Thomsen disease) w + CLECSF14 macrophage lectin 2 (calcium dependent) + + w 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) + + + + + X CSF3 colony stimulating factor 3 (granulocyte) + + w X CST7 cystatin F (leukocystatin) + w CTSC cathepsin C + + CYB5 cytochrome b-5 + w 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 w + + + 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 + + + Gene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52

DMAP1 DNA methyltransferase 1-associated protein 1 w + + DPP3 dipeptidylpeptidase III w + + DR1 down-regulator of transcription 1, TBP-binding + + DXS9879E DNA segment on chromosome X, expressed sequence + + EFEMP2 EGF-containing fibulin-like extracellular matrix protein 2 w + EFNB1 ephrin-B1 + + EIF2S3 eukaryotic translation initiation factor 2, subunit 3 w + + + + + ELF3 E74-like factor 3 (ets domain transcription factor) + X ELK1 ELK1, member of ETS oncogene family + EMP3 epithelial membrane protein 3 + + + + ERBB2IP erbb2 interacting protein + + + w ERCC1 excision repair cross-complementing repair deficiency + ET hypothetical protein ET w + EXTL2 exostoses (multiple)-like 2 + + FBXW3 F-box and WD-40 domain protein 3 + X FCAR Fc fragment of IgA, receptor for + + + + FEN1 flap structure-specific endonuclease 1 + + + + FHL3 four and a half LIM domains 3 + + w + w FIBP fibroblast growth factor (acidic) intracellular binding protein + + FKSG87 FKSG87 protein + + + FLJ10420 hypothetical protein FLJ10420 + + + + w FLJ10687 hypothetical protein FLJ10687 + FLJ11082 hypothetical protein FLJ11082 + FLJ11773 hypothetical protein FLJ11773 + + + FLJ11838 hypothetical protein FLJ11838 + + FLJ13195 hypothetical protein FLJ13195 similar to stromal antigen 3 + + FLJ13639 hypothetical protein FLJ13639 w + w FLJ20080 hypothetical protein FLJ20080 + + + + FLJ20257 hypothetical protein FLJ20257 + w FLJ20477 hypothetical protein FLJ20477 + + + + + + + FLJ20825 hypothetical protein FLJ20825 + FLJ22353 hypothetical protein FLJ22353 + w FLJ22555 hypothetical protein FLJ22555 + + + + w FLJ22573 hypothetical protein FLJ22573 w + + FLOT1 flotillin 1 + + + + + w + + FUT4 fucosyltransferase 4 (alpha (1,3) fucosyltransferase) + w GABPB2 GA binding protein transcription factor, beta subunit 2 + w + + w + GADD45B growth arrest and DNA-damage-inducible, beta + + + + + + + X GDF5 growth differentiation factor 5 + GNA15 guanine nucleotide binding protein (G protein), alpha 15 + GNG5 guanine nucleotide binding protein (G protein), gamma 5 w + GNL1 guanine nucleotide binding protein-like 1 w + GPR132 G protein-coupled receptor + + + w w GPR84 G protein-coupled receptor 84 + + w w GPX1 glutathione peroxidase 1 + + + + + w + GTF3C4 general transcription factor IIIC, polypeptide 4 (90kD) + + GTPBP4 G protein-binding protein CRFG + GYPC glycophorin C (Gerbich blood group) + + + + + + HADHA hydroxyacyl-Coenzyme A dehydrogenase + w HCST DNAX-activation protein 10 + w + w HIST1H4I H4 family, member M + + + HIST1H4K H4 histone family, member D + + w HLA-B major histocompatibility complex, class I, B w w + + + + X HLA-F major histocompatibility complex, class I, F + + X HLA-G HLA-G histocompatibility antigen, class I, G + HMGN4 high-mobility group protein 17-like 3 + w HNMT histamine N-methyltransferase w + HOXA7 homeo box A7 w w + HSPA1B heat shock 70kD protein 1B + HSPC003 HSPC003 protein + + HSPC052 HSPC052 protein + HSPC073 HSPC073 protein + + + w HSPCB heat shock 90kD protein 1, beta + + + + + ICAM1 intercellular adhesion molecule 1 (CD54) + + + + + w X ICAM4 intercellular adhesion molecule 4 + + IER3 immediate early response 3 + + w X IFNGR1 interferon gamma receptor 1 + + IL1B interleukin 1, beta + + X IL1F9 interleukin-1 homolog 1 + + + + IL2RG interleukin 2 receptor, gamma + + + + + + w + X IL6ST interleukin 6 signal transducer (gp130) + w IL8 interleukin 8 + + + w X ILK integrin-linked kinase w + INHBE hypothetical protein MGC4638 w + ITGA5 integrin, alpha 5 (fibronectin receptor) + + w + X ITGAX integrin, alpha X, alpha polypeptide) + + + w ITSN2 intersectin 2 + + IVNS1ABP NS1-binding protein + JUN v-jun sarcoma virus 17 oncogene homolog (avian) + w EntrezGene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52

KIAA0186 KIAA0186 gene product + KIAA0218 KIAA0218 gene product + w + KIAA0319 KIAA0319 gene product + w KIAA0409 KIAA0409 protein + + + KIAA0669 KIAA0669 gene product + w KIAA1008 mitotic control protein dis3 homolog + w KIAA1056 KIAA1056 protein + + KLF15 Kruppel-like factor 15 + LGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) + + LIM2 lens intrinsic membrane protein 2 (19kD) + LIMK1 LIM domain kinase 1 + + LIPT1 lipoyltransferase + + + + + LMNA lamin A/C w + + LOC113201 hypothetical protein BC012124 + LOC116211 hypothetical protein BC013113 + + w LOC51015 CGI-111 protein + w LOC51321 hypothetical protein w w + + LOC55580 hypothetical protein + LOC92106 hypothetical protein BC008322 + + LRRC19 leucine rich repeat containing 19 + + + + LRSAM1 leucine rich repeat and sterile alpha motif containing 1 + M96 putative DNA binding protein + MAN2B1 mannosidase, alpha, class 2B, member 1 + + + + w MAPK6 mitogen-activated protein kinase 6 + + MASTL hypothetical protein FLJ14813 + + + + + + MCCC2 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) + + MCP membrane cofactor protein (CD46) + MDH1 malate dehydrogenase 1, NAD (soluble) w w + w MEF2B MADS box transcription factor 2, polypeptide B w + w w MGC10960 hypothetical protein MGC10960 w + MGC10966 hypothetical protein MGC10966 w + + + MGC14141 hypothetical protein MGC14141 + + MGC4677 hypothetical protein MGC4677 + + + w MGC4771 hypothetical protein MGC4771 + + + + + MGC5242 hypothetical protein MGC5242 + + + MID1IP1 hypothetical protein STRAIT11499 + + w w w MLLT10 myeloid/lymphoid leukemia translocated to 10 + MMAB hypothetical protein MGC20496 + + MMP19 matrix metalloproteinase 19 + + w + MMP8 matrix metalloproteinase 8 (neutrophil collagenase) + MMP9 matrix metalloproteinase 9 (gelatinase B, 92kD gelatinase) + + + + + X MRPL30 mitochondrial ribosomal protein L30 + + + + MRPL34 mitochondrial ribosomal protein L34 + + MRPS7 mitochondrial ribosomal protein S7 + + + MX2 myxovirus (influenza virus) resistance 2 (mouse) + NAG73 NPC-related protein NAG73 + + + NAP1L1 assembly protein 1-like 1 + NAPA N-ethylmaleimide-sensitive factor attachment protein, alpha + + NCR1 lymphocyte antigen 94 homolog, NK-p46, (mouse) + NFATC2 nuclear factor of activated T-cells, calcineurin-dependent 2 + + NFKB2 NF-κB p52/p100 + + + + + + + + X NFKBIB NF-κB inhibitor, beta + w + + w + NKG7 natural killer cell group 7 sequence + w NME1 non-metastatic cells 1, protein (NM23A) expressed in + NOLC1 nucleolar and coiled-body phosphprotein 1 + w NOVA1 neuro-oncological ventral antigen 1 + + NR1D1 subfamily 1, group D, member 1 w + + NRAS neuroblastoma RAS viral (v-ras) oncogene homolog w + NRXN3 neurexin 3 + NUP107 nuclear pore complex protein + w + OAS3 2'-5'-oligoadenylate synthetase 3 (100 kD) + + + + + X OAZ1 ornithine decarboxylase antizyme 1 + + OAZ2 ornithine decarboxylase antizyme 2 + ORMDL1 hypothetical protein + + P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxygenase alpha + w + + + + + PAFAH2 platelet-activating factor acetylhydrolase 2 (40kD) w + w PAK4 p21(CDKN1A)-activated kinase 4 w + + PALM paralemmin + + PDE6D phosphodiesterase 6D, cGMP-specific, rod, delta + w + PEX13 peroxisome biogenesis factor 13 + w + PHLDA1 pleckstrin homology-like domain, family A, member 1 + w PILRA paired immunoglobulin-like receptor alpha + + w PILRB paired immunoglobulin-like receptor beta w + w w PKM2 pyruvate kinase, muscle + w + PLK1 polo-like kinase (Drosophila) + + PLXNB1 plexin B1 + + PMAIP1 phorbol-12-myristate-13-acetate-induced protein 1 + + + + PMS1 PMS1 postmeiotic segregation increased 1 (S. cerevisiae) + PMS2L8 postmeiotic segregation increased 2-like 8 + EntrezGene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52

POLB polymerase (DNA directed), beta + + POLD4 polymerase (DNA-directed), delta 4 w + POLR1C RNA polymerase I subunit + + + POLR2A polymerase (RNA) II (DNA directed) polypeptide A + + POLR3E hypothetical protein FLJ10509 + + + w PPFIBP1 PTPRF interacting protein, binding protein 1 + w PPP1R15B protein phosphatase 1, regulatory subunit 15B + + + + w w PPP1R3B protein phosphatase 1, regulatory subunit 3B + + PPP5C protein phosphatase 5, catalytic subunit + + w PRDM1 PR domain containing 1, with ZNF domain + + PRDM11 PR domain containing 11 w + w PRDM8 PR domain containing 8 + w + + PRKAB2 protein kinase, AMP-activated, beta 2 subunit w + + + PTGS2 prostaglandin-endoperoxide synthase 2, COX-2 + + w X PTPN7 protein tyrosine phosphatase, non-receptor type 7 w + w + PTX3 pentaxin-related gene, rapidly induced by IL-1 beta w + X PXMP3 peroxisomal membrane protein 3 (Zellweger syndrome) + QTRTD1 hypothetical protein FLJ12960 + RAB7L1 RAB7, member RAS oncogene family-like 1 + RABL2A RAB, member of RAS oncogene family-like 2A + RABL2B RAB, member of RAS oncogene family-like 2B + X RAC2 ras-related C3 botulinum toxin substrate 2 (rho family) w + X RB1 retinoblastoma 1 (including osteosarcoma) + + RB1CC1 RB1-inducible coiled-coil 1 + w RBBP4 retinoblastoma binding protein 4 + RFP ret finger protein + RFXANK regulatory factor X-associated ankyrin-containing protein + RGC32 RGC32 protein + RGL1 RalGDS-like gene + RND1 GTP-binding protein + w RNF4 ring finger protein 4 + w RPL27A ribosomal protein L27a + + RPL39 ribosomal protein L39 + RPL39L ribosomal protein L39-like + + RPS28 ribosomal protein S28 + RPS8 ribosomal protein S8 + RS1 retinoschisis (X-linked, juvenile) 1 + S100A12 S100 calcium binding protein A12 (calgranulin C) + + + w S100A3 S100 calcium binding protein A3 w + + w S100A4 S100 calcium binding protein A4 (calvasculin) + + + w S100A9 S100 calcium binding protein A9 (calgranulin B) + + w S100Z S100Z protein + SAS sarcoma amplified sequence + w SCARB1 CD36 antigen (thrombospondin receptor)-like 1 + X SDF2L1 stromal cell-derived factor 2-like 1 + w SELPLG selectin P ligand + + + X SEMA7A sema domain, immunoglobulin domain, semaphorin 7A + SEPW1 selenoprotein W, 1 + SERPINB2 serine (or cysteine) proteinase inhibitor, clade B + SERPINE1 serine (or cysteine) proteinase inhibitor, clade E w + X SFRS2 splicing factor, arginine/serine-rich 2 + + SIRT2 sirtuin silent mating type information regulation 2 + + SLAMF8 B lymphocyte activator macrophage expressed + + w + SLC1A3 solute carrier family 1 (glutamate transporter), member 3 w + + + + SLC25A12 solute carrier family 25, member 12 + + + w SLC30A7 zinc transporter like 2 + + + SLC39A6 LIV-1 protein, estrogen regulated + w w + w + + SLC7A1 solute carrier family 7, member 1 + + SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal + + + + + + + + SOX8 SRY (sex determining region Y)-box 8 w + SPATA2 spermatogenesis associated 2 + SQSTM1 sequestosome 1 + SREBF2 sterol regulatory element binding transcription factor 2 + + SRF + + X SSSCA1 Sjogren's syndrome/scleroderma autoantigen 1 + w STARD10 serologically defined colon cancer antigen 28 + w STAT1 signal transducer and activator of transcription 1 + + w w + STAT5A signal transducer and activator of transcription 5A w + + + w X STAT6 signal transducer and activator of transcription 6 + w STATIP1 elongator protein 2 + + + STXBP3 syntaxin binding protein 3 + + SVIL supervillin + SYNPO synaptopodin + TAGLN2 transgelin 2 + TAPBP TAP binding protein (tapasin) + + + + + + + TFAP2A transcription factor AP-2 alpha + TFDP3 -like protein EntrezGene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52

TFE3 transcription factor binding to IGHM enhancer 3 + TIMP1 tissue inhibitor of metalloproteinase 1 + + + + TJP2 tight junction protein 2 (zona occludens 2) + + TLN1 talin 1 + + + TMPIT transmembrane protein induced by tumor TNF + + + + TNF tumor necrosis factor (TNF superfamily, member 2) + + + w + X TNFRSF1A tumor necrosis factor receptor superfamily, member 1A + + w w TNFSF13 tumor necrosis factor (ligand) superfamily, member 13 + + + + TNFSF13B tumor necrosis factor (ligand) superfamily, member 13b + w TNFSF14 tumor necrosis factor (ligand) superfamily, member 14 + w TNIP3 hypothetical protein FLJ21162 + TRA1 tumor rejection antigen (gp96) 1 + w TRAF1 TNF receptor-associated factor 1 + + + + + + X TRAPPC3 similar to yeast BET3 (S. cerevisiae) + + + + TRIM31 tripartite motif-containing 31 + + TRPM2 transient receptor potential cation channel, subfamily M + + + TTC17 hypothetical protein FLJ10890 + + + UBE2H ubiquitin-conjugating E2H + w UBE2L6 ubiquitin-conjugating enzyme E2L 6 + + + USP2 ubiquitin specific protease 2 + USP25 ubiquitin specific protease 25 + USP52 KIAA0710 gene product + + w VAV1 vav 1 oncogene + w + + w VSIG4 Ig superfamily protein + + w WBP4 WW domain binding protein 4 (formin binding protein 21) + + + + + YME1L1 YME1-like 1 (S. cerevisiae) + YWHAB tyrosine 3-monooxygenase, beta polypeptide + + ZBTB11 zinc finger protein + + ZDHHC6 hypothetical protein FLJ21952 + w w ZNF217 zinc finger protein 217 + ZNF221 zinc finger protein 221 + + + + + ZNF581 HSPC189 protein + ZNF76 zinc finger protein 76 (expressed in testis) + + w ZNHIT1 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) Table 4. Genes bound by E2F4 in unstimulated U937 cells

EntrezGene Description EntrezGene Description symbol symbol AK2 adenylate kinase 2 ITGA2 integrin, alpha 2 (CD49B) AP2S1 adaptor-related protein complex 2, sigma 1 subunit KIAA0042 KIAA0042 gene product AP4B1 adaptor-related protein complex 4, beta 1 subunit KIAA0092 KIAA0092 gene product APG4C APG4 autophagy 4 homolog C (S. cerevisiae) KIAA0101 KIAA0101 gene product ARHGAP11A KIAA0013 gene product KIAA0410 KIAA0410 gene product ASF1B ASF1 anti-silencing function 1 homolog B KIAA0747 KIAA0747 protein ATF2 activating transcription factor 2 KIAA0872 KIAA0872 protein ATM ataxia telangiectasia mutated KIF20A kinesin family member 20A ATP5G3 ATP synthase, H+ transporting, mitochondrial F0 complex KIF22 kinesin family member 22 BAT4 HLA-B associated transcript 4 KIF2C kinesin family member 2C BAZ1B bromodomain adjacent to zinc finger domain, 1B KIF4A kinesin family member 4A BCAS1 breast carcinoma amplified sequence 1 KNSL7 kinesin-like 7 BLM Bloom syndrome KNTC2 kinetochore associated 2 BRD8 bromodomain containing 8 KPNB1 karyopherin (importin) beta 1 BUB3 BUB3 budding uninhibited by benzimidazoles 3 homolog LENG5 leukocyte receptor cluster (LRC) member 5 C10orf7 chromosome 10 open reading frame 76 LGALS1 lectin, galactoside-binding, soluble, 1 C11orf10 open reading frame 10 LOC55924 hypothetical protein C12orf5 chromosome 12 open reading frame 5 LOC81691 exonuclease NEF-sp C14orf123 chromosome 14 open reading frame 123 LSM4 U6 snRNA-associated Sm-like protein C20orf172 chromosome 20 open reading frame 172 LUC7L2 LUC7-like 2 (S. cerevisiae) C20orf77 chromosome 20 open reading frame 77 M96 putative DNA binding protein C21orf45 chromosome 21 open reading frame 45 MAPK7 mitogen-activated protein kinase 7 C6orf166 open reading frame 166 MASTL microtubule associated serine/threonine kinase-like C6orf18 chromosome 6 open reading frame 18 MCM3 MCM3 minichromosome maintenance deficient 3 C6orf211 chromosome 6 open reading frame 211 MDH1 malate dehydrogenase 1, NAD (soluble) C6orf82 chromosome 6 open reading frame 82 MDS009 x 009 protein C9orf76 chromosome 9 open reading frame 76 MGC5469 hypothetical protein MGC5469 CASP2 caspase 2, apoptosis-related cysteine protease MIS12 MIS12 homolog (yeast) CASP8AP2 CASP8 associated protein 2 MRP63 mitochondrial ribosomal protein 63 CBX3 chromobox homolog 3 (HP1 gamma homolog, Drosophila) MRPS11 mitochondrial ribosomal protein S11 CBX5 chromobox homolog 5 (HP1 alpha homolog, Drosophila) MRPS15 mitochondrial ribosomal protein S15 CCNB1 cyclin B1 MRPS18C mitochondrial ribosomal protein S18C CDC25A cell division cycle 25A MRPS27 mitochondrial ribosomal protein S27 CDC25C cell division cycle 25C MTAP methylthioadenosine phosphorylase CDC6 CDC6 cell division cycle 6 homolog (S. cerevisiae) MTRF1L mitochondrial translational release factor 1-like CDCA1 cell division cycle associated 1 MUT methylmalonyl Coenzyme A mutase CDCA8 cell division cycle associated 8 MYOHD1 myosin head domain containing 1 CENPF centromere protein F (350/400kD, mitosin) NAP1L1 nucleosome assembly protein 1-like 1 CKAP1 cytoskeleton-associated protein 1 NBR2 NBR2 CLSPN claspin homolog (Xenopus laevis) NDUFA1 NADH dehydrogenase 1 alpha subcomplex, 1 CSTF3 cleavage stimulation factor, 3' pre-RNA, subunit 3 NDUFB9 NADH dehydrogenase 1 beta subcomplex, 9 DCLRE1B DNA cross-link repair 1B (PSO2 homolog, S. cerevisiae) NEK2 NIMA (never in mitosis gene a)-related kinase 2 DDX10 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10 NFKBIB NF-κB inhibitor, beta DKFZP434B168 DKFZP434B168 protein NFYA nuclear transcription factor Y, alpha DLG7 discs, large homolog 7 (Drosophila) NFYC nuclear transcription factor Y, gamma EIF3S2 eukaryotic translation initiation factor 3, subunit 2 NUDT15 nudix-type motif 15 EXO1 exonuclease 1 NUP98 nucleoporin 98kD FANCL Fanconi anemia, complementation group L NUSAP1 nucleolar and spindle associated protein 1 FLJ10579 hypothetical protein FLJ10579 ORC3L origin recognition complex, subunit 3-like FLJ10922 hypothetical protein FLJ10922 ORMDL1 ORM1-like 1 (S. cerevisiae) FLJ11029 hypothetical protein FLJ11029 PAI-RBP1 PAI-1 mRNA-binding protein FLJ11222 hypothetical protein FLJ11222 PCNA proliferating cell nuclear antigen FLJ13912 hypothetical protein FLJ13912 PCNP PEST-containing nuclear protein FLJ20354 hypothetical protein FLJ20354 PDZK11 PDZ domain containing 11 FLJ20508 hypothetical protein FLJ20508 PELO pelota homolog (Drosophila) GHITM growth hormone inducible transmembrane protein PEX12 peroxisomal biogenesis factor 12 GPHN gephyrin PIGC phosphatidylinositol glycan, class C H2AFG H2A histone family, member G PLCG2 phospholipase C, gamma 2 H2AFP H2A histone family, member P PLK1 Polo like Kinase 1 HAGHL hydroxyacylglutathione hydrolase-like PLK4 Polo like Kinase 4 HIST1H2AJ histone 1, H2aj PMS1 PMS1 postmeiotic segregation increased 1 HIST1H2AL histone 1, H2al POLA polymerase (DNA directed), alpha HIST1H2BJ histone 1, H2bj POLD1 polymerase (DNA directed), delta 1, catalytic subunit HIST1H2BK histone 1, H2bk POLE3 polymerase (DNA directed), epsilon 3 (p17 subunit) HIST1H2BL histone 1, H2bl POLR3K polymerase (RNA) III (DNA directed) polypeptide K HIST1H2BM histone 1, H2bm PRPS1 phosphoribosyl pyrophosphate synthetase 1 HIST1H3B histone 1, H3b QPCTL glutaminyl-peptide cyclotransferase-like HIST1H4C histone 1, H4c RAD51 RAD51 homolog (RecA homolog, E. coli) HIST1H4K histone 1, H4k RAMP RA-regulated nuclear matrix-associated protein HMGB2 high-mobility group box 2 RBBP4 retinoblastoma binding protein 4 HNRPA1 heterogeneous nuclear ribonucleoprotein A1 RBL1 retinoblastoma-like 1 (p107) HPCL2 2-hydroxyphytanoyl-CoA lyase RBM15 RNA binding motif protein 15 HSPC052 HSPC052 protein RFC5 replication factor C (activator 1) 5 (36.5kD) HSPC150 HSPC150 protein similar to ubiquitin-conjugating enzyme RNASE4 ribonuclease, RNase A family, 4 HT007 uncharacterized hypothalamus protein HT007 RNPC2 RNA-binding region (RNP1, RRM) containing 2 IER5 immediate early response 5 RPGR retinitis pigmentosa GTPase regulator IRF3 interferon regulatory factor 3 RPL26 ribosomal protein L26 EntrezGene Description symbol RPL37 ribosomal protein L37 RPL39L ribosomal protein L39-like RQCD1 RCD1 required for cell differentiation1 homolog RRM1 ribonucleotide reductase M1 polypeptide RXRB , beta SAMHD1 SAM domain and HD domain, 1 SF3A3 splicing factor 3a, subunit 3, 60kD SFRS1 splicing factor, arginine/serine-rich 1 SFRS2 splicing factor, arginine/serine-rich 2 SLC2A4 solute carrier family 2, member 4 SLC31A1 solute carrier family 31, member 1 SLC35A5 solute carrier family 35, member A5 SLC39A9 solute carrier family 39, member 9 SLC8A3 solute carrier family 8, member 3 SMC1L1 SMC1 structural maintenance of 1-like 1 SMC2L1 SMC2 structural maintenance of chromosomes 2-like 1 SMC4L1 SMC4 structural maintenance of chromosomes 4-like 1 SMN2 survival of motor neuron 2, centromeric SNRPD2 small nuclear ribonucleoprotein D2 polypeptide (16.5kD) SNX13 sorting nexin 13 SPOCK3 sparc, cwcv and kazal-like domains proteoglycan 3 STOML2 stomatin (EPB72)-like 2 SUCLG1 succinate-CoA ligase, GDP-forming, alpha subunit SULT1A3 sulfotransferase family, cytosolic, 1A, phenol-preferring 3 SUMO1 SMT3 suppressor of mif two 3 homolog 1 TCF19 transcription factor 19 (SC1) TDRD3 tudor domain containing 3 TIMELESS timeless homolog (Drosophila) TMEM5 transmembrane protein 5 TOPK T-LAK cell-originated protein kinase TTF2 transcription termination factor, RNA polymerase II TTRAP TRAF and TNF receptor associated protein TU12B1-TY TU12B1-TY protein TUSC4 tumor suppressor candidate 4 TYMS thymidylate synthetase UCHL5 ubiquitin carboxyl-terminal hydrolase L5 USP1 ubiquitin specific protease 1 VILL villin-like VPS29 vacuolar protein sorting 29 (yeast) WDR19 WD repeat domain 19 XRCC5 X-ray repair complementing defective repair in Chinese hamster cells 5 YWHAB tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein ZIC1 Zic family member 1 (odd-paired homolog, Drosophila) ZNF451 zinc finger protein 451 Table 5. Genes bound by four or five NF-κB subunits

EntrezGene Description symbol

4 subunits

APBB1IP similar to proline-rich protein 48 ASH1 hypothetical protein ASH1 BCL2A1 BCL2-related protein A1 C3 complement component 3 CCL4 small inducible cytokine A4 CCRL2 chemokine (C-C motif) receptor-like 2 CD69 CD69 antigen (p60, early T-cell activation antigen) CREB1 cAMP responsive element binding protein 1 DBI diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein) EMP3 epithelial membrane protein 3 FLJ10420 hypothetical protein FLJ10420 FLJ22555 hypothetical protein FLJ22555 HSPCB heat shock 90kD protein 1, beta IL1F9 interleukin-1 homolog 1 MAN2B1 mannosidase, alpha, class 2B, member 1 OAS3 2'-5'-oligoadenylate synthetase 3 (100 kD) P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I PPP1R15B protein phosphatase 1, regulatory (inhibitor) subunit 15B TAPBP TAP binding protein (tapasin) TIMP1 tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor) TNF tumor necrosis factor (TNF superfamily, member 2) WBP4 WW domain binding protein 4 (formin binding protein 21)

5 subunits

AQP9 aquaporin 9 ARHGEF2 rho/rac guanine nucleotide exchange factor (GEF) 2 CCL3 small inducible cytokine A3, Mip-1-alpha CCNL1 cyclin L ania-6a DAF decay accelerating factor for complement (CD55) FLJ20477 hypothetical protein FLJ20477 FLOT1 flotillin 1 GADD45B growth arrest and DNA-damage-inducible, beta GPX1 glutathione peroxidase 1 GYPC glycophorin C (Gerbich blood group) ICAM1 intercellular adhesion molecule 1 (CD54) IL2RG interleukin 2 receptor, gamma NFKB2 p52/p100 SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal TRAF1 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 y-

axis 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 p-

value 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 fold- enrichment 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 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, 1475-

1489). 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% NP-

40 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 SDS-

polyacrylamide 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

0 targets 193 targets 4 4 HLA-B HLA-B NFKB2 p50 NFKB2 p65 NFKBIB NFKBIB -LPS +LPS P4HA1

3 l o g ( I P ) 3 l o g ( I P ) CCL1 CCL1 P4HA1 CSF2 PTX3 RAC2 CSF2 β-actin PTX3 β-actin 2 RAC2 2

1 1

0 0 0 1 2 3 44 0 1 2 3 4 log(WCE) log(WCE)

Supporting Information Figure 8

Cytoplasm Nucleus 06030 180 06030 180 p65

p105

p50

RelB

c-Rel

p100

p52 AB Supporting Information Figure9 Supporting Information 1/2 3/5 3/2 5/3 2/3 2 Change in Change N o II RNA Pol

p50 binding p52 NF-

p65 κ B RelB c-Rel <1/8 1/4 1/2 >8 1 4 2

0 hrs ingene Change expression 1 hr 3 hrs 9 hrs 27 hrs

p50 binding p52 NF-

p65 κ B RelB c-Rel Supporting Information Figure 10

RB1 BCL6 IL8 JUN TFAP2A VAV1 ICAM1 STAT6 CDKNA1 RB1 BCL6 IL8 JUN TFAP2A VAV1 ICAM1 STAT6 CDKNA1

+ LPS p50 p52 p65 RelB c-Rel p50 p52 p65 RelB c-Rel

PRDM1 TAPBP STAT1 NFKBIB CREB1 CCL3 CCL4 SRF NFKB2 PRDM1 TAPBP STAT1 NFKBIB CREB1 CCL3 CCL4 SRF NFKB2