table 1. antibodies used for chromatin immunoprecipitation...
TRANSCRIPT
Table 1. Antibodies used for chromatin immunoprecipitation (ChIP)
Protein Antibody Manufacturer ReferencesWestern blot ChIP
p65 sc-372 Santa CruzBiotechnology
(1, 2) (3-5)
p50 sc-1190 Santa CruzBiotechnology
(6) (7)
RelB sc-226 Santa CruzBiotechnology
(8) (5)
c-Rel sc-71 Santa CruzBiotechnology
(8) (5, 9)
p52 06-413 Upstate Biotechnology,Lake Placid, NY
(10)
E2F4 sc-1082 Santa CruzBiotechnology
(11) (12)
Pol II 8WG16 (13) (14)
The fact that the p65 dataset has no targets in unstimulated cells confirms the lack ofbackground enrichment from the Dynal magnetic beads. The number of p50 targets inunstimulated cells is higher than for the other subunits. We were concerned that this might be dueto the antibody used. Therefore, we tested another antibody raised against the N-terminus of theprotein (sc-1191). In test experiments, 90% of enriched gene promoters were shared between thetwo 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. USA97, 1014-1019.
3. Nissen, R. M. & Yamamoto, K. R. (2000) Genes 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. USA100, 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. Gene ontology (GO) categories enriched in the set of genes bound by NF-κB
GO system GO category Number ofbound genes
Total numberof genes incategory
P value
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 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 Antigen processing, endogenousantigen via MHC class I
3 7 0.00193
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 IIpromoter
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). Atotal of 5,707 genes present of the arrays were annotated by GO biological process information;227 of these were bound by NF-κB.
ABHD8ACTR3AF093680AF15Q14AIM2ALAS1AP1S2APBB1IPAPG-1AQP9ARF3ARHGAP4ARHGEF2ARL1ARL6IP5ARTS-1ASGR2ASH1ATF6ATP5G1BAT3BAZ1BBCL2A1BCL6BFBNIP1BPIC10orf117C11orf10C14orf111C20orf28C21orf45C21orf59C3C6orf109C6orf66C7orf10C9orf100CA11CAPZA1CARD15CATCCDC2CCL1CCL3CCL4CCL5CCNL1CCR1CCRL2CD37CD58CD69CDCA1CDH15CDKN1ACHI3L2CLCN1CLECSF14COX4I1CREB1CRYBA1CSF2CSF3CST7CTSCCYB5CYP51A1DAFDBIDBPDDX21DDX31DDX47DGCR6LDGUOKDJ434O14.3DKFZp434K1210DKFZp762L0311
hypothetical protein FLJ11743ARP3 actin-related protein 3 homolog similar to mouse Glt3 AF15q14 proteinabsent in melanoma 2aminolevulinate, delta-, synthase 1adaptor-related protein complex 1similar to proline-rich protein 48heat shock protein (hsp110 family)aquaporin 9ADP-ribosylation factor 3Rho GTPase activating protein 4rho/rac guanine nucleotide exchange factor ADP-ribosylation factor-like 1vitamin A responsive; cytoskeleton relatedtype 1 TNFR shedding aminopeptidase regulatorasialoglycoprotein receptor 2hypothetical protein ASH1activating transcription factor 6ATP synthase, H+ transportingHLA-B associated transcript 3bromodomain adjacent to zinc finger domain, 1BBCL2-related protein A1B-cell CLL/lymphoma 6 (zinc finger protein 51)B-factor, properdinBCL2/adenovirus E1B 19kD interacting protein 1bactericidal/permeability-increasing proteinAD24 proteinchromosome 11 open reading frame 10CGI-35 proteinchromosome 20 open reading frame 28chromosome 21 open reading frame 45chromosome 21 open reading frame 59complement component 3DKFZP566C243 proteinHSPC125 proteinchromosome 7 open reading frame 10hypothetical protein FLJ14642carbonic anhydrase XIcapping protein (actin filament) muscle Z-linecaspase recruitment domain family, member 15catalasecapillary morphogenesis protein 1small inducible cytokine A1, I-309small inducible cytokine A3, Mip-1-alphasmall inducible cytokine A4, Mip-1-betasmall inducible cytokine A5 (RANTES)cyclin L ania-6achemokine (C-C motif) receptor 1chemokine (C-C motif) receptor-like 2CD37 antigenCD58 antigen, (LFA 3)CD69 antigen (p60, early T-cell activation antigen)cell division cycle associated 1cadherin 15, M-cadherin (myotubule)cyclin-dependent kinase inhibitor 1A (p21, Cip1)chitinase 3-like 2chloride channel 1, skeletal muscle (Thomsen disease)macrophage lectin 2 (calcium dependent)cytochrome c oxidase subunit IV isoform 1cAMP responsive element binding protein 1crystallin, beta A1colony stimulating factor 2 (granulocyte-macrophage)colony stimulating factor 3 (granulocyte)cystatin F (leukocystatin)cathepsin Ccytochrome b-5cytochrome P450, 51 (lanosterol 14-alpha-demethylase)decay accelerating factor for complement diazepam binding inhibitor (GABA receptor modulator)D site of albumin promoter binding proteinDEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 21DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 31hqp0256 proteinDiGeorge syndrome critical region gene 6 likedeoxyguanosine kinasehypothetical protein dJ434O14.3hypothetical protein DKFZp434K1210hypothetical protein DKFZp762L0311
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EntrezGenesymbol
Description + LPS - LPS
p65
c-R
elR
elB
p50
p52
p65
c-R
elR
elB
p50
p52
X
X
X
X
X X X X
X
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X X
Table 3. Genes bound by NF-κB in U937 cells
Known targets
DMAP1DPP3DR1DXS9879EEFEMP2EFNB1EIF2S3ELF3ELK1EMP3ERBB2IPERCC1ETEXTL2FBXW3FCARFEN1FHL3FIBPFKSG87FLJ10420FLJ10687FLJ11082FLJ11773FLJ11838FLJ13195FLJ13639FLJ20080FLJ20257FLJ20477FLJ20825FLJ22353FLJ22555FLJ22573FLOT1FUT4GABPB2GADD45BGDF5GNA15GNG5GNL1GPR132GPR84GPX1GTF3C4GTPBP4GYPCHADHAHCSTHIST1H4IHIST1H4KHLA-BHLA-FHLA-GHMGN4HNMTHOXA7HSPA1BHSPC003HSPC052HSPC073HSPCBICAM1ICAM4IER3IFNGR1IL1BIL1F9IL2RGIL6STIL8ILKINHBEITGA5ITGAXITSN2IVNS1ABPJUN
DNA methyltransferase 1-associated protein 1dipeptidylpeptidase IIIdown-regulator of transcription 1, TBP-binding DNA segment on chromosome X, expressed sequenceEGF-containing fibulin-like extracellular matrix protein 2ephrin-B1eukaryotic translation initiation factor 2, subunit 3 E74-like factor 3 (ets domain transcription factor)ELK1, member of ETS oncogene familyepithelial membrane protein 3erbb2 interacting proteinexcision repair cross-complementing repair deficiencyhypothetical protein ETexostoses (multiple)-like 2F-box and WD-40 domain protein 3Fc fragment of IgA, receptor forflap structure-specific endonuclease 1four and a half LIM domains 3fibroblast growth factor (acidic) intracellular binding proteinFKSG87 proteinhypothetical protein FLJ10420hypothetical protein FLJ10687hypothetical protein FLJ11082hypothetical protein FLJ11773hypothetical protein FLJ11838hypothetical protein FLJ13195 similar to stromal antigen 3hypothetical protein FLJ13639hypothetical protein FLJ20080hypothetical protein FLJ20257hypothetical protein FLJ20477hypothetical protein FLJ20825hypothetical protein FLJ22353hypothetical protein FLJ22555hypothetical protein FLJ22573flotillin 1fucosyltransferase 4 (alpha (1,3) fucosyltransferase)GA binding protein transcription factor, beta subunit 2 growth arrest and DNA-damage-inducible, betagrowth differentiation factor 5guanine nucleotide binding protein (G protein), alpha 15 guanine nucleotide binding protein (G protein), gamma 5guanine nucleotide binding protein-like 1G protein-coupled receptorG protein-coupled receptor 84glutathione peroxidase 1general transcription factor IIIC, polypeptide 4 (90kD)G protein-binding protein CRFGglycophorin C (Gerbich blood group)hydroxyacyl-Coenzyme A dehydrogenaseDNAX-activation protein 10H4 histone family, member MH4 histone family, member Dmajor histocompatibility complex, class I, Bmajor histocompatibility complex, class I, FHLA-G histocompatibility antigen, class I, Ghigh-mobility group protein 17-like 3histamine N-methyltransferasehomeo box A7heat shock 70kD protein 1BHSPC003 proteinHSPC052 proteinHSPC073 proteinheat shock 90kD protein 1, betaintercellular adhesion molecule 1 (CD54)intercellular adhesion molecule 4immediate early response 3interferon gamma receptor 1interleukin 1, betainterleukin-1 homolog 1interleukin 2 receptor, gamma interleukin 6 signal transducer (gp130)interleukin 8integrin-linked kinasehypothetical protein MGC4638integrin, alpha 5 (fibronectin receptor)integrin, alpha X, alpha polypeptide)intersectin 2NS1-binding proteinv-jun sarcoma virus 17 oncogene homolog (avian)
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Entrez Genesymbol
Description + LPS - LPS
p65
c-R
elR
elB
p50
p52
p65
c-R
elR
elB
p50
p52
Known targets
X
X
X
X X
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KIAA0186KIAA0218KIAA0319KIAA0409KIAA0669KIAA1008KIAA1056KLF15LGALS1LIM2LIMK1LIPT1LMNALOC113201LOC116211LOC51015LOC51321LOC55580LOC92106LRRC19LRSAM1M96MAN2B1MAPK6MASTLMCCC2MCPMDH1MEF2BMGC10960MGC10966MGC14141MGC4677MGC4771MGC5242MID1IP1MLLT10MMABMMP19MMP8MMP9MRPL30MRPL34MRPS7MX2NAG73NAP1L1NAPANCR1NFATC2NFKB2NFKBIBNKG7NME1NOLC1NOVA1NR1D1NRASNRXN3NUP107OAS3OAZ1OAZ2ORMDL1P4HA1PAFAH2PAK4PALMPDE6DPEX13PHLDA1PILRAPILRBPKM2PLK1PLXNB1PMAIP1PMS1PMS2L8
KIAA0186 gene productKIAA0218 gene productKIAA0319 gene productKIAA0409 proteinKIAA0669 gene productmitotic control protein dis3 homologKIAA1056 proteinKruppel-like factor 15lectin, galactoside-binding, soluble, 1 (galectin 1)lens intrinsic membrane protein 2 (19kD)LIM domain kinase 1lipoyltransferaselamin A/Chypothetical protein BC012124hypothetical protein BC013113CGI-111 proteinhypothetical proteinhypothetical proteinhypothetical protein BC008322leucine rich repeat containing 19leucine rich repeat and sterile alpha motif containing 1putative DNA binding proteinmannosidase, alpha, class 2B, member 1mitogen-activated protein kinase 6hypothetical protein FLJ14813methylcrotonoyl-Coenzyme A carboxylase 2 (beta)membrane cofactor protein (CD46)malate dehydrogenase 1, NAD (soluble)MADS box transcription factor 2, polypeptide B hypothetical protein MGC10960hypothetical protein MGC10966hypothetical protein MGC14141hypothetical protein MGC4677hypothetical protein MGC4771hypothetical protein MGC5242hypothetical protein STRAIT11499myeloid/lymphoid leukemia translocated to 10hypothetical protein MGC20496matrix metalloproteinase 19matrix metalloproteinase 8 (neutrophil collagenase)matrix metalloproteinase 9 (gelatinase B, 92kD gelatinase)mitochondrial ribosomal protein L30mitochondrial ribosomal protein L34mitochondrial ribosomal protein S7myxovirus (influenza virus) resistance 2 (mouse)NPC-related protein NAG73nucleosome assembly protein 1-like 1N-ethylmaleimide-sensitive factor attachment protein, alphalymphocyte antigen 94 homolog, NK-p46, (mouse)nuclear factor of activated T-cells, calcineurin-dependent 2NF-κB p52/p100NF-κB inhibitor, betanatural killer cell group 7 sequencenon-metastatic cells 1, protein (NM23A) expressed innucleolar and coiled-body phosphprotein 1neuro-oncological ventral antigen 1nuclear receptor subfamily 1, group D, member 1neuroblastoma RAS viral (v-ras) oncogene homologneurexin 3nuclear pore complex protein2'-5'-oligoadenylate synthetase 3 (100 kD)ornithine decarboxylase antizyme 1ornithine decarboxylase antizyme 2hypothetical proteinprocollagen-proline, 2-oxoglutarate 4-dioxygenase alpha platelet-activating factor acetylhydrolase 2 (40kD)p21(CDKN1A)-activated kinase 4paralemminphosphodiesterase 6D, cGMP-specific, rod, deltaperoxisome biogenesis factor 13pleckstrin homology-like domain, family A, member 1paired immunoglobulin-like receptor alphapaired immunoglobulin-like receptor betapyruvate kinase, musclepolo-like kinase (Drosophila)plexin B1phorbol-12-myristate-13-acetate-induced protein 1PMS1 postmeiotic segregation increased 1 (S. cerevisiae)postmeiotic segregation increased 2-like 8
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EntrezGenesymbol
Description + LPS - LPS
p65
c-R
elR
elB
p50
p52
p65
c-R
elR
elB
p50
p52
X
X
X
Known targets
POLBPOLD4POLR1CPOLR2APOLR3EPPFIBP1PPP1R15BPPP1R3BPPP5CPRDM1PRDM11PRDM8PRKAB2PTGS2PTPN7PTX3PXMP3QTRTD1RAB7L1RABL2ARABL2BRAC2RB1RB1CC1RBBP4RFPRFXANKRGC32RGL1RND1RNF4RPL27ARPL39RPL39LRPS28RPS8RS1S100A12S100A3S100A4S100A9S100ZSASSCARB1SDF2L1SELPLGSEMA7ASEPW1SERPINB2SERPINE1SFRS2SIRT2SLAMF8SLC1A3SLC25A12SLC30A7SLC39A6SLC7A1SMPD1SOX8SPATA2SQSTM1SREBF2SRFSSSCA1STARD10STAT1STAT5ASTAT6STATIP1STXBP3SVILSYNPOTAGLN2TAPBPTFAP2ATFDP3
polymerase (DNA directed), betapolymerase (DNA-directed), delta 4RNA polymerase I subunitpolymerase (RNA) II (DNA directed) polypeptide Ahypothetical protein FLJ10509PTPRF interacting protein, binding protein 1 protein phosphatase 1, regulatory subunit 15Bprotein phosphatase 1, regulatory subunit 3Bprotein phosphatase 5, catalytic subunitPR domain containing 1, with ZNF domainPR domain containing 11PR domain containing 8protein kinase, AMP-activated, beta 2 subunitprostaglandin-endoperoxide synthase 2, COX-2protein tyrosine phosphatase, non-receptor type 7pentaxin-related gene, rapidly induced by IL-1 betaperoxisomal membrane protein 3 (Zellweger syndrome)hypothetical protein FLJ12960RAB7, member RAS oncogene family-like 1RAB, member of RAS oncogene family-like 2ARAB, member of RAS oncogene family-like 2Bras-related C3 botulinum toxin substrate 2 (rho family)retinoblastoma 1 (including osteosarcoma)RB1-inducible coiled-coil 1retinoblastoma binding protein 4ret finger proteinregulatory factor X-associated ankyrin-containing proteinRGC32 proteinRalGDS-like geneGTP-binding proteinring finger protein 4ribosomal protein L27aribosomal protein L39ribosomal protein L39-likeribosomal protein S28ribosomal protein S8retinoschisis (X-linked, juvenile) 1S100 calcium binding protein A12 (calgranulin C)S100 calcium binding protein A3S100 calcium binding protein A4 (calvasculin)S100 calcium binding protein A9 (calgranulin B)S100Z proteinsarcoma amplified sequenceCD36 antigen (thrombospondin receptor)-like 1stromal cell-derived factor 2-like 1selectin P ligandsema domain, immunoglobulin domain, semaphorin 7Aselenoprotein W, 1serine (or cysteine) proteinase inhibitor, clade B serine (or cysteine) proteinase inhibitor, clade E splicing factor, arginine/serine-rich 2sirtuin silent mating type information regulation 2 B lymphocyte activator macrophage expressedsolute carrier family 1 (glutamate transporter), member 3solute carrier family 25, member 12zinc transporter like 2LIV-1 protein, estrogen regulatedsolute carrier family 7, member 1sphingomyelin phosphodiesterase 1, acid lysosomal SRY (sex determining region Y)-box 8spermatogenesis associated 2sequestosome 1sterol regulatory element binding transcription factor 2serum response factor Sjogren's syndrome/scleroderma autoantigen 1serologically defined colon cancer antigen 28signal transducer and activator of transcription 1signal transducer and activator of transcription 5Asignal transducer and activator of transcription 6elongator protein 2syntaxin binding protein 3supervillinsynaptopodintransgelin 2TAP binding protein (tapasin)transcription factor AP-2 alpha E2F-like protein
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EntrezGenesymbol
Description + LPS - LPS
p65
c-R
elR
elB
p50
p52
p65
c-R
elR
elB
p50
p52
X
X
X X
X X
X
X
X
Known targets
TFE3TIMP1TJP2TLN1TMPITTNFTNFRSF1ATNFSF13TNFSF13BTNFSF14TNIP3TRA1TRAF1TRAPPC3TRIM31TRPM2TTC17UBE2HUBE2L6USP2USP25USP52VAV1VSIG4WBP4YME1L1YWHABZBTB11ZDHHC6ZNF217ZNF221ZNF581ZNF76ZNHIT1
transcription factor binding to IGHM enhancer 3tissue inhibitor of metalloproteinase 1 tight junction protein 2 (zona occludens 2)talin 1transmembrane protein induced by tumor TNFtumor necrosis factor (TNF superfamily, member 2)tumor necrosis factor receptor superfamily, member 1Atumor necrosis factor (ligand) superfamily, member 13tumor necrosis factor (ligand) superfamily, member 13btumor necrosis factor (ligand) superfamily, member 14hypothetical protein FLJ21162tumor rejection antigen (gp96) 1TNF receptor-associated factor 1similar to yeast BET3 (S. cerevisiae)tripartite motif-containing 31transient receptor potential cation channel, subfamily Mhypothetical protein FLJ10890ubiquitin-conjugating enzyme E2H ubiquitin-conjugating enzyme E2L 6ubiquitin specific protease 2ubiquitin specific protease 25KIAA0710 gene productvav 1 oncogeneIg superfamily proteinWW domain binding protein 4 (formin binding protein 21)YME1-like 1 (S. cerevisiae)tyrosine 3-monooxygenase, beta polypeptidezinc finger proteinhypothetical protein FLJ21952zinc finger protein 217zinc finger protein 221HSPC189 proteinzinc finger protein 76 (expressed in testis)putative cyclin G1 interacting protein
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EntrezGenesymbol
Description + LPS - LPS
p65
c-R
elR
elB
p50
p52
p65
c-R
elR
elB
p50
p52
X
X
Known targets
+, Indicates high confidence binding (p< 0.002), at least one subunit had to match this criterionw, Indicates weaker binding events (p< 0.01)
EntrezGenesymbol
Description
Table 4. Genes bound by E2F4 in unstimulated U937 cells
AK2AP2S1AP4B1APG4CARHGAP11AASF1BATF2ATMATP5G3BAT4BAZ1BBCAS1BLMBRD8BUB3C10orf7C11orf10C12orf5C14orf123C20orf172C20orf77C21orf45C6orf166C6orf18C6orf211C6orf82C9orf76CASP2CASP8AP2CBX3CBX5CCNB1CDC25ACDC25CCDC6CDCA1CDCA8CENPFCKAP1CLSPNCSTF3DCLRE1BDDX10DKFZP434B168DLG7EIF3S2EXO1FANCLFLJ10579FLJ10922FLJ11029FLJ11222FLJ13912FLJ20354FLJ20508GHITMGPHNH2AFGH2AFPHAGHLHIST1H2AJHIST1H2ALHIST1H2BJHIST1H2BKHIST1H2BLHIST1H2BMHIST1H3BHIST1H4CHIST1H4KHMGB2HNRPA1HPCL2HSPC052HSPC150HT007IER5IRF3
adenylate kinase 2adaptor-related protein complex 2, sigma 1 subunitadaptor-related protein complex 4, beta 1 subunitAPG4 autophagy 4 homolog C (S. cerevisiae)KIAA0013 gene productASF1 anti-silencing function 1 homolog Bactivating transcription factor 2ataxia telangiectasia mutatedATP synthase, H+ transporting, mitochondrial F0 complexHLA-B associated transcript 4bromodomain adjacent to zinc finger domain, 1Bbreast carcinoma amplified sequence 1Bloom syndromebromodomain containing 8BUB3 budding uninhibited by benzimidazoles 3 homolog chromosome 10 open reading frame 76chromosome 11 open reading frame 10chromosome 12 open reading frame 5chromosome 14 open reading frame 123chromosome 20 open reading frame 172chromosome 20 open reading frame 77chromosome 21 open reading frame 45chromosome 6 open reading frame 166chromosome 6 open reading frame 18chromosome 6 open reading frame 211chromosome 6 open reading frame 82chromosome 9 open reading frame 76caspase 2, apoptosis-related cysteine proteaseCASP8 associated protein 2chromobox homolog 3 (HP1 gamma homolog, Drosophila)chromobox homolog 5 (HP1 alpha homolog, Drosophila)cyclin B1cell division cycle 25Acell division cycle 25CCDC6 cell division cycle 6 homolog (S. cerevisiae)cell division cycle associated 1cell division cycle associated 8centromere protein F (350/400kD, mitosin)cytoskeleton-associated protein 1claspin homolog (Xenopus laevis)cleavage stimulation factor, 3' pre-RNA, subunit 3DNA cross-link repair 1B (PSO2 homolog, S. cerevisiae)DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10 DKFZP434B168 proteindiscs, large homolog 7 (Drosophila)eukaryotic translation initiation factor 3, subunit 2 exonuclease 1Fanconi anemia, complementation group Lhypothetical protein FLJ10579hypothetical protein FLJ10922hypothetical protein FLJ11029hypothetical protein FLJ11222hypothetical protein FLJ13912hypothetical protein FLJ20354hypothetical protein FLJ20508growth hormone inducible transmembrane proteingephyrinH2A histone family, member GH2A histone family, member Phydroxyacylglutathione hydrolase-likehistone 1, H2ajhistone 1, H2alhistone 1, H2bjhistone 1, H2bkhistone 1, H2blhistone 1, H2bmhistone 1, H3bhistone 1, H4chistone 1, H4khigh-mobility group box 2heterogeneous nuclear ribonucleoprotein A12-hydroxyphytanoyl-CoA lyaseHSPC052 proteinHSPC150 protein similar to ubiquitin-conjugating enzymeuncharacterized hypothalamus protein HT007immediate early response 5interferon regulatory factor 3
ITGA2KIAA0042KIAA0092KIAA0101KIAA0410KIAA0747KIAA0872KIF20AKIF22KIF2CKIF4AKNSL7KNTC2KPNB1LENG5LGALS1LOC55924LOC81691LSM4LUC7L2M96MAPK7MASTLMCM3MDH1MDS009MGC5469MIS12MRP63MRPS11MRPS15MRPS18CMRPS27MTAPMTRF1LMUTMYOHD1NAP1L1NBR2NDUFA1NDUFB9NEK2NFKBIBNFYANFYCNUDT15NUP98NUSAP1ORC3LORMDL1PAI-RBP1PCNAPCNPPDZK11PELOPEX12PIGCPLCG2PLK1PLK4PMS1POLAPOLD1POLE3POLR3KPRPS1QPCTLRAD51RAMPRBBP4RBL1RBM15RFC5RNASE4RNPC2RPGRRPL26
integrin, alpha 2 (CD49B)KIAA0042 gene productKIAA0092 gene productKIAA0101 gene productKIAA0410 gene productKIAA0747 proteinKIAA0872 proteinkinesin family member 20Akinesin family member 22kinesin family member 2Ckinesin family member 4Akinesin-like 7kinetochore associated 2karyopherin (importin) beta 1leukocyte receptor cluster (LRC) member 5lectin, galactoside-binding, soluble, 1 hypothetical proteinexonuclease NEF-spU6 snRNA-associated Sm-like proteinLUC7-like 2 (S. cerevisiae)putative DNA binding proteinmitogen-activated protein kinase 7microtubule associated serine/threonine kinase-likeMCM3 minichromosome maintenance deficient 3 malate dehydrogenase 1, NAD (soluble)x 009 proteinhypothetical protein MGC5469MIS12 homolog (yeast)mitochondrial ribosomal protein 63mitochondrial ribosomal protein S11mitochondrial ribosomal protein S15mitochondrial ribosomal protein S18Cmitochondrial ribosomal protein S27methylthioadenosine phosphorylasemitochondrial translational release factor 1-likemethylmalonyl Coenzyme A mutasemyosin head domain containing 1nucleosome assembly protein 1-like 1NBR2NADH dehydrogenase 1 alpha subcomplex, 1 NADH dehydrogenase 1 beta subcomplex, 9 NIMA (never in mitosis gene a)-related kinase 2NF-κB inhibitor, beta nuclear transcription factor Y, alphanuclear transcription factor Y, gammanudix-type motif 15nucleoporin 98kDnucleolar and spindle associated protein 1origin recognition complex, subunit 3-likeORM1-like 1 (S. cerevisiae)PAI-1 mRNA-binding proteinproliferating cell nuclear antigenPEST-containing nuclear proteinPDZ domain containing 11pelota homolog (Drosophila)peroxisomal biogenesis factor 12phosphatidylinositol glycan, class Cphospholipase C, gamma 2 Polo like Kinase 1Polo like Kinase 4PMS1 postmeiotic segregation increased 1 polymerase (DNA directed), alphapolymerase (DNA directed), delta 1, catalytic subunit polymerase (DNA directed), epsilon 3 (p17 subunit)polymerase (RNA) III (DNA directed) polypeptide K phosphoribosyl pyrophosphate synthetase 1glutaminyl-peptide cyclotransferase-likeRAD51 homolog (RecA homolog, E. coli) RA-regulated nuclear matrix-associated proteinretinoblastoma binding protein 4retinoblastoma-like 1 (p107)RNA binding motif protein 15replication factor C (activator 1) 5 (36.5kD)ribonuclease, RNase A family, 4RNA-binding region (RNP1, RRM) containing 2retinitis pigmentosa GTPase regulatorribosomal protein L26
EntrezGenesymbol
Description
EntrezGenesymbol
Description
RPL37RPL39LRQCD1RRM1RXRBSAMHD1SF3A3SFRS1SFRS2SLC2A4SLC31A1SLC35A5SLC39A9SLC8A3SMC1L1SMC2L1SMC4L1SMN2SNRPD2SNX13SPOCK3STOML2SUCLG1SULT1A3SUMO1TCF19TDRD3TIMELESSTMEM5TOPKTTF2TTRAPTU12B1-TYTUSC4TYMSUCHL5USP1VILLVPS29WDR19XRCC5YWHABZIC1ZNF451
ribosomal protein L37ribosomal protein L39-likeRCD1 required for cell differentiation1 homolog ribonucleotide reductase M1 polypeptideretinoid X receptor, betaSAM domain and HD domain, 1splicing factor 3a, subunit 3, 60kDsplicing factor, arginine/serine-rich 1 splicing factor, arginine/serine-rich 2solute carrier family 2, member 4solute carrier family 31, member 1solute carrier family 35, member A5solute carrier family 39, member 9solute carrier family 8, member 3SMC1 structural maintenance of chromosomes 1-like 1SMC2 structural maintenance of chromosomes 2-like 1SMC4 structural maintenance of chromosomes 4-like 1 survival of motor neuron 2, centromericsmall nuclear ribonucleoprotein D2 polypeptide (16.5kD)sorting nexin 13sparc, cwcv and kazal-like domains proteoglycan 3stomatin (EPB72)-like 2succinate-CoA ligase, GDP-forming, alpha subunitsulfotransferase family, cytosolic, 1A, phenol-preferring 3SMT3 suppressor of mif two 3 homolog 1transcription factor 19 (SC1)tudor domain containing 3timeless homolog (Drosophila)transmembrane protein 5T-LAK cell-originated protein kinasetranscription termination factor, RNA polymerase IITRAF and TNF receptor associated proteinTU12B1-TY proteintumor suppressor candidate 4thymidylate synthetaseubiquitin carboxyl-terminal hydrolase L5ubiquitin specific protease 1villin-likevacuolar protein sorting 29 (yeast)WD repeat domain 19X-ray repair complementing defective repair in Chinese hamster cells 5tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteinZic family member 1 (odd-paired homolog, Drosophila)zinc finger protein 451
APBB1IPASH1BCL2A1C3CCL4CCRL2CD69CREB1DBIEMP3FLJ10420FLJ22555HSPCBIL1F9MAN2B1OAS3P4HA1PPP1R15BTAPBPTIMP1TNFWBP4
AQP9ARHGEF2CCL3CCNL1DAFFLJ20477FLOT1GADD45BGPX1GYPCICAM1IL2RGNFKB2SMPD1TRAF1
similar to proline-rich protein 48hypothetical protein ASH1BCL2-related protein A1complement component 3small inducible cytokine A4chemokine (C-C motif) receptor-like 2CD69 antigen (p60, early T-cell activation antigen)cAMP responsive element binding protein 1diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein)epithelial membrane protein 3hypothetical protein FLJ10420hypothetical protein FLJ22555heat shock 90kD protein 1, betainterleukin-1 homolog 1mannosidase, alpha, class 2B, member 12'-5'-oligoadenylate synthetase 3 (100 kD)procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide Iprotein phosphatase 1, regulatory (inhibitor) subunit 15BTAP 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)
aquaporin 9rho/rac guanine nucleotide exchange factor (GEF) 2small inducible cytokine A3, Mip-1-alphacyclin L ania-6adecay accelerating factor for complement (CD55)hypothetical protein FLJ20477flotillin 1growth arrest and DNA-damage-inducible, betaglutathione peroxidase 1glycophorin C (Gerbich blood group)intercellular adhesion molecule 1 (CD54)interleukin 2 receptor, gammap52/p100sphingomyelin phosphodiesterase 1, acid lysosomalTNF receptor-associated factor 1
Table 5. Genes bound by four or five NF-κB subunits
DescriptionEntrezGenesymbol
4 subunits
5 subunits
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 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, 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.
0
2
4
0 2 4
log(
IP)
log(WCE)
p50+LPS
193 targets
β-actin
Supporting Information Figure 6
4
log(WCE)
log(
IP)
p65-LPS
0
2
4
0 2 4
0 targets
β-actin
1
1
1
1
3
3
3
3
HLA-BNFKB2
NFKBIB
CSF2
P4HA1CCL1
RAC2PTX3
HLA-BNFKB2
NFKBIB
CSF2
P4HA1CCL1
RAC2
PTX3
Supporting Information Figure 8
0 6030 180Cytoplasm Nucleus
p65
c-Rel
RelB
p52
p50
p100
p105
0 6030 180
p50
p52
p65
Rel
Bc-
Rel
NF-κBbinding
p50
p52
p65
Rel
Bc-
Rel
NF-κBbinding
0 hr
s1
hr3
hrs
9 hr
s27
hrs
Change in geneexpression
Change in RNA Pol II
A B
5/3
2
3/2
2/3
3/5
1/2
24
>8
11/21/4
<1/8
Supporting Information Figure 9
IL8RB1 STAT6JUNBCL6 TFAP2A
Supporting Information Figure 10
TAPBP CCL3NFKBIB CCL4PRDM1 SRF
VAV1 ICAM1
STAT1 CREB1
p50 p65 RelB c-Relp52+ LPS
p50 p65 RelB c-Relp52
CDKNA1
NFKB2
IL8RB1 STAT6JUNBCL6 TFAP2A
TAPBP CCL3NFKBIB CCL4PRDM1 SRF
VAV1 ICAM1
STAT1 CREB1
CDKNA1
NFKB2