rela ser276 phosphorylation is required for activation of a subset of nf b-dependent genes by...

Published Ahead of Print 24 March 2008. 10.1128/MCB.01152-07.

2008, 28(11):3623. DOI:Mol. Cell. Biol. R. Brasier

AllanBoldogh, Leoncio A. Vergara, Sanjeev Choudhary and David E. Nowak, Bing Tian, Mohammad Jamaluddin, Istvan Complexes Cyclin-Dependent Kinase 9/Cyclin T1 B-Dependent Genes by Recruiting

κfor Activation of a Subset of NF- Phosphorylation Is Required276RelA Ser

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MOLECULAR AND CELLULAR BIOLOGY, June 2008, p. 3623–3638 Vol. 28, No. 110270-7306/08/$08.00�0 doi:10.1128/MCB.01152-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

RelA Ser276 Phosphorylation Is Required for Activation of a Subset ofNF-�B-Dependent Genes by Recruiting Cyclin-Dependent Kinase

9/Cyclin T1 Complexes�†David E. Nowak,1‡ Bing Tian,1‡ Mohammad Jamaluddin,1 Istvan Boldogh,2,4 Leoncio A. Vergara,3

Sanjeev Choudhary,1 and Allan R. Brasier1,4*Departments of Medicine,1 Microbiology and Immunology,2 and Neuroscience and Cell Biology3 and The Sealy Center for

Molecular Medicine,4 The University of Texas Medical Branch, Galveston, Texas 77555-1060

Received 27 June 2007/Returned for modification 20 August 2007/Accepted 11 March 2008

NF-�B plays a central role in cytokine-inducible inflammatory gene expression. Previously we empiricallydetermined the identity of 92 members of the genetic network under direct NF-�B/RelA control that showmarked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigatethis network further, we have applied a recently developed two-step chromatin immunoprecipitation assay thataccurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducibleRelA binding occurs with similar kinetics on all NF-�B-dependent genes, serine 276 (Ser276)-phosphorylatedRelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF),including Gro-�, interleukin-8 (IL-8), and I�B�. Previous work has shown that TNF-inducible RelA Ser276

phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. Tofurther understand the role of phospho-Ser276 RelA in target gene expression, we inhibited its formation byROS scavengers and antioxidants, treatments that disrupt phospho-Ser276 formation but not the translocationand DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser276 RelA is required only foractivation of IL-8 and Gro-�, with I�B� being unaffected. These data were confirmed in experiments usingRelA�/� murine embryonic fibroblasts reconstituted with a RelA Ser276Ala mutation. In addition, we observethat phospho-Ser276 RelA binds the positive transcription elongation factor b (P-TEFb), a complex containingthe cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short inter-fering RNA (siRNA)-mediated knockdown shows that the phospho-Ser276 RelA–P-TEFb complex is requiredfor IL-8 and Gro-� gene activation but not for I�B� gene activation. These studies indicate that TNF inducestarget gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser276 RelA formation andchromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domainphosphorylation on the IL-8 and Gro-� genes. The second involves a phospho-Ser276 RelA-independentactivation of genes preloaded with Pol II, exemplified by the I�B� gene. Together, these data suggest that thebinding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlledby a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

Infectious and inflammatory stimuli induce expression ofgene networks controlling proinflammatory and innate im-mune responses. One important arm of the inflammatory re-sponse is mediated by monocyte-derived tumor necrosis factor(TNF), a cytokine that activates gene expression programs inadjacent epithelial cells to propagate mucosal inflammation(42, 51). Here, the spectrum of functional activities and themagnitude and timing of TNF-induced gene expression areimportant determinants of tissue homeostasis. A central me-diator of epithelial genomic response is nuclear factor-�B (NF-�B), an inducible transcription factor that controls the expres-sion of proinflammatory genes and whose dysregulation has

also been implicated in the pathogenesis of inflammatory, neo-plastic, and autoimmune diseases (4, 6, 17).

In most non-B-lymphoid cells, NF-�B is sequestered as aninactive complex in the cytoplasm by the inhibitors of �B(I�Bs). The mechanism by which NF-�B is activated by TNF,referred to as the “canonical” activation pathway, has beenextensively investigated (22). The canonical pathway is acti-vated by ligands of the TNF superfamily including TNF alpha,the prototypical macrophage-derived cytokine that binds to theubiquitously expressed TNF receptor 1 (TNFRI) (50). TNF-induced TNFRI trimerization induces the recruitment of cyto-plasmic signal adapters, including the TNF receptor-associateddeath domain (TRADD), TNF receptor-associated factor 2(TRAF2) and TRAF6, receptor-interacting protein (RIP), mi-togen/extracellular signal-regulated kinase kinase 3, and others(23–25). This activated submembranous TNFRI complex tran-siently recruits the I�B kinase (IKK) complex, resulting in thephosphorylation of the catalytic IKK� and -� kinases followedby its cytosolic release (15, 43). In the cytosol, activated IKKphosphorylates serine (Ser) residues 32 and 36 on the NH2terminal regulatory domain of I�B�, targeting I�B� for pro-teolytic destruction by ubiquitination and calpain pathways

* Corresponding author. Mailing address: Department of Medicine,The University of Texas Medical Branch, Galveston, TX 77555-1060.Phone: (409) 772-2824. Fax: (409) 772-8709. E-mail: [email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

‡ These authors contributed equally to this work.� Published ahead of print on 24 March 2008.

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(20, 29). I�B� degradation releases NF-�B to rapidly translo-cate into the nucleus.

In the nucleus, the activated NF-�B complex binds to highlyconserved regulatory sequences corresponding to the 5�-GGGRNNYYCC-3� consensus (30). Although NF-�B binding invitro is quite stable, with a half-life of 45 min or longer (8),fluorescence recovery after photobleaching and fluorescencelifetime measurements have shown that NF-�B interactingwith genomic targets within native chromatin is in a hyperdy-namic exchange, with an observed half-life of seconds (8).Studies of highly inducible promoters have suggested thatNF-�B interaction induces target gene activation through amechanism that induces the formation of a multiprotein com-plex known as an “enhanceosome” (34). The enhanceosomecontains non-DNA-binding chromatin-modifying proteins, in-cluding the CBP/p300 coactivator (49), a histone acetylase thatmodifies promoter-associated nucleosomes, as well as incom-pletely characterized methylases, kinases, and chromatin-orga-nizing proteins. This activity results in enhanced transcrip-tional initiation.

Although nuclear translocation is required for the activationof NF-�B-dependent genes, we have recently shown a require-ment for a TNF-inducible reactive oxygen species (ROS) path-way in licensing the transactivating potential of NF-�B (26, 58).In this pathway, TNF induces the formation of ROS, whoseformation occurs at times well after the occurrence of I�B�proteolysis and NF-�B nuclear translocation (58). This secondsignal activates the catalytic subunit of protein kinase A(PKAc), resulting in the selective phosphorylation of theRelA-transactivating subunit at Ser residue 276, a modificationthat permits complex formation with incompletely character-ized transcriptional activating proteins, including p300/CBP(60), and serves as a switch for additional posttranslationalmodifications, such as acetylation (12). We have recently foundthat the inhibition of the TNF-induced ROS prevents phospho-Ser276 RelA formation, resulting in the nuclear translocationof hypophosphorylated RelA and the formation of an unstableenhanceosome (26).

Despite intense study, a systematic definition of the NF-�B-dependent gene network has only recently been approached(53, 54, 56). High-density microarray analyses of cells express-ing a regulated dominant-negative NF-�B inhibitor have elu-cidated the genes under control of the canonical NF-�B acti-vation pathway in epithelial cells subjected to viral infections(40, 55) and cytokine stimulation (56) and those controlled byits distinct activation modes (54). These studies have shownthat in response to TNF stimulation, NF-�B binding controlsthe expression of a noncontiguous group of genes whose prod-ucts control a variety of biological processes, including leuko-cyte activation/chemotaxis, negative regulators of the TNF-IKK pathway, cellular metabolism, antigen processing, andothers (54).

In this study, we examine the detailed mechanisms for highlyinducible NF-�B-dependent genes by use of an efficient two-step chromatin immunoprecipitation (ChIP) assay (39). Weobserved indistinguishable patterns of inducible RelA bindingto all promoters irrespective of their degree of induction oractivation kinetics. However, distinct binding patterns ofSer276-phosphorylated RelA binding were observed, with rapidphospho-Ser276 RelA binding to the most highly inducible pro-

moters, and this was at times coincident with their maximalexpression. To establish the role of the phospho-Ser276 RelAmodification, we blocked phospho-Ser276 RelA formation us-ing ROS scavengers (dimethyl sulfoxide [DMSO]) and antioxi-dants (N-acetyl cysteine [NAC]), agents that blocked RelASer276 phosphorylation without affecting its translocation. Wefound that interleukin-8 (IL-8) and Gro-� expression werehighly dependent on phospho-Ser276 RelA binding, whereasI�B� was not. Further study of this mechanism revealed thatthe phospho-Ser276 RelA associates with the cyclin-dependentkinase 9 (CDK-9)–cyclin T1 (Ccn T1) complex, positive tran-scription elongation factor b (P-TEFb). Short interfering RNA(siRNA)-mediated knockdown showed that RelA recruitmentof P-TEFb activity is required only for a subset of NF-�B-dependent genes (the IL-8 and Gro-� genes), genes character-ized by TNF-induced polymerase II (Pol II) recruitment. To-gether, these data show for the first time that phospho-Ser276

RelA formation is responsible for the activation of a subset ofNF-�B-dependent genes via the P-TEFb transcriptional elon-gation complex.

MATERIALS AND METHODS

Cell culture and reagents. HeLa S3 and A549 cells were from ATCC andmaintained as previously described (54, 56). HeLa expressing the nondegradableI�B� Mut inhibitor in Tet-Off (TtA) cells were described earlier (55, 56). ClonalHeLa cells stably expressing the enhanced green fluorescent protein (EGFP)-RelA fusion protein (33) were selected in G418 (250 �g/ml). In all experiments,unless specifically noted, cells were serum starved in Dulbecco’s modified Eagle’smedium containing 0.5% bovine serum albumin for 24 h prior to stimulation.Stimulation was performed by addition of recombinant TNF alpha (30 ng/ml;Peprotech) to the culture medium. Flavopiridol (FP) was used at 500 nM con-centrations for 1 h prior to TNF stimulation. For RelA site mutations, eukaryoticexpression vector encoding FLAG epitope-tagged RelA was generated by ligat-ing the monomeric strawberry cDNA (47) into a pcDNA3-FLAG backbone,generating the plasmid pcDNA-FLAG-Straw. Wild-type RelA (RelAWT) and aRelA (Ser276Ala) site mutation were generated by rolling-circle mutagenesis andcloned into pcDNA-FLAG-Straw. The transcription unit containing the cyto-megalovirus promoter and FLAG-mStraw-RelA coding sequences was excised asa BglII/XbaI fragment and ligated into pEF6/V5 plasmid digested with BglII/XbaI, producing pXFS-RelA. The pXFS plasmids confer blasticidin resistanceand were used to generate stable transfectants in a RelA�/� background. Forthis purpose, RelA�/� murine embryonic fibroblasts (MEFs) (a gift of D. Bal-timore [7]) were transfected with 8 �g of pXFS-RelA and 24 �l of Targefect-MEF transfection reagent (Targeting Systems Inc.) in Dulbecco’s modified Ea-gle’s medium containing 10% fetal bovine serum. After 24 h, cells weresubdivided and stable transfectants selected by the addition of blasticidin S (10�g/ml) for 3 weeks. Pools of cells were confirmed by fluorescence microscopy tohave similar levels of Straw fluorescence, and amounts of protein expressionwere confirmed by Western immunoblotting.

EMSA. Sucrose cushion-purified nuclear extracts were prepared as describedpreviously (19). Nuclear proteins (30 �g) were bound to a 40,000-cpm 32P-labeled duplex NF-�B site (27) in 1� binding buffer (10 mM HEPES, pH 7.9, 5mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 0.1 M KCl) with 1 �g poly(dA-dT) per sample. Binding reaction mixtures were incubated for 1 h at 4°C andfractionated by nondenaturing 6% Tris-borate-EDTA gels on ice. For super-shift assays, the same conditions were used except that 2 �g of the indicatedNF-�B isoform-specific antibody (Ab) (Santa Cruz) was preincubated for 1 hat 4°C prior to the addition of labeled probe. Supershift electromobility shiftassay (EMSA) samples were fractionated on a 5% acrylamide 1� Tris-borate-EDTA gel.

Western immunoblots. For measurement of phospho-Ser276 RelA formation,whole-cell extracts (WCEs) were prepared by lysis in 1� sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Protein from 3 �106 cells was fractionated by SDS-PAGE. For measurement of NF-�B subunitnuclear translocation, 75 �g of sucrose cushion-purified nuclear protein wasfractionated by SDS-PAGE. Gels were electrotransferred to polyvinylidene di-fluoride membranes (Millipore) overnight and blocked with 5% nonfat driedmilk in Tris-buffered saline (TBS)–0.1% Tween 20. Anti-RelA, -RelB, and -C-

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Rel Abs were from Santa Cruz. Phospho-Ser276 RelA was detected using anti-phospho-Ser276 RelA (1:1,000; Santa Cruz) in 5% bovine serum albumin-TBS-Tween 0.1% and incubated for 72 h at 4°C. Secondary Abs (IRD800 anti-rabbitimmunoglobulin G [IgG] or IRD680 anti-mouse IgG; Rockland) were diluted1:5,000 in TBS-Tween 0.1% and incubated for 1 h at 22°C, and the immunecomplexes were visualized by near-infrared fluorescence (Odyssey imaging sys-tem; LiCor BioSciences).

Gene expression analysis. Quantitative real-time PCR (Q-RT-PCR) was per-formed with SYBR green I by use of a MyiQ single-color RT-PCR detectionsystem and iQ SYBR green supermix (Bio-Rad, Hercules, CA) according to themanufacturer’s protocols. First-strand cDNA was prepared with an iScriptcDNA synthesis kit (Bio-Rad). cDNA (1 �l) was added to a reaction master mix(20 �l) containing 2.5 mM MgCl2, HotStart Taq DNA polymerase, SYBR greenI, deoxynucleoside triphosphates, fluorescein (10 nM), and gene-specific primers(200 nM each of forward and reverse primers). For each experimental sample,triplicate reactions were conducted in 96-well plates. PCR cycling conditionsconsisted of a hot-start activation of HotStart Taq DNA polymerase (94°C, 15min) and 40 cycles of denaturation (95°C, 15 s), annealing (55 to 58°C, 30 s), andextension (72°C, 30 s). Formation of PCR product was monitored in real time bymeasuring SYBR green I fluorescence at 72°C.

Primer sets used for human transcripts were as follows: for IL-8, 5�-ACTGAGAGTGATTGAGAGTGGAC-3� and 5�-AACCCTCTGCACCCAGTTTTC-3�; for Gro-�, 5�-AGAATGGGCAGAAAGCTTGTCT-3� and 5�-CAGCATCTTTTCGATGATTTTCTTAA-3�; for the I�B� 3� untranslated region, 5�-TGCCTAGCCCAAAACGTCTT-3� and 5�-CGTCCCCTACAAAAAGTTCACAA-3�;for I�B� coding sequences, 5�-CTCCGAGACTTTCGAGGAAATAC-3� and5�-GCCATTGTAGTTGGTAGCCTTCA-3�; for TNFAIP3/A20, 5�-AAGCTGTGAAGATACGGGAGA-3� and 5�-CGATGAGGGCTTTGTGGATGAT-3�;for Naf1, 5�-AGAGGACCGTACCGGATCTAC-3� and 5�-CCTTCACTAGGCGCTCAAAAG-3�; and for GAPDH, 5�-ATGGGGAAGGTGAAGGTCG-3�and 5�-GGGGTCATTGATGGCAACAATA-3�. Primers for mouse transcriptswere as follows: for mGro-�, 5�-CACTCTCAAGGGCGGTCAA-3� and 5�-TGGTTCTTCCGTTGAGGGAC-3�; and for mI�B�, 5�-TCCTGCACTTGGCAATCATC-3� and 5�-AGCCAGCTCTCAGAAGTGCC-3�. Relative gene expres-sion was determined using the 2�CT method (56). The mean threshold cycle(CT) of triplicate measures was computed for each sample. The sample mean CT

of cyclophilin (internal control) was subtracted from the sample mean CT of therespective gene of interest (CT). The mean CT of the control (untreated)sample was selected as a calibrator and subtracted from the mean CT of eachexperimental sample (CT). The severalfold change in gene expression (nor-malized to the internal control gene and relative to the control sample) wasexpressed as 2�CT.

Two-step ChIP assay. Two-step ChIP was performed as described previously(39). The Abs for RelA, RelB, c-Rel, NF-�B1, NF-�B2, total RNA Pol II,CDK-9, and Ccn T1 were those used in Western blotting. Mouse monoclonalAbs for phosphorylated RNA Pol II were from Covance Research Products.Monoclonal Ab-chromatin complexes were captured with a rabbit anti-mouseIgM Ab (Rockland) prior to binding to protein A magnetic beads (38). DNAbinding was detected by qualitative PCR using primers for IL-8, Gro-�, I�B�,Naf1, NF-�B2, and TRAF1 as described previously (39, 56). The primers usedfor TRAF1 were 5�-GATGTGCCCAGCGAAGTGG-3� and 5�-TGAGTCACAGCAGGGATGGAG-3�. For Q-RT-PCR, the primers used were as follows: forGro-�, 5�-TCGCCTTCCTTCCGAACTC-3� and 5�-CGAACCCCTTTTATGCATGGT-3�; and for Naf1, 5�-GGTCTAGGAAATCCCAGTCTGTTG-3� and5�-CGGGTGGGCAAATCCA-3�.

Intracellular ROS assay. Changes in intracellular ROS were determined asdescribed previously (26). Briefly, cells were suspended in phosphate-bufferedsaline (PBS) and loaded with 5 �M of 5 (and 6)-carboxy-2�,7�-dichlorodihydro-fluorescein diacetate (H2DCF-DA; Molecular Probes) for 15 min at 37°C. Afterremoval of excess H2DCF-DA, cells were TNF treated, and changes in DCFfluorescence were determined by flow cytometry (FACScan; Becton Dickinson).The fluorescences for 12,000 cells from three or more independent experimentswere analyzed and expressed as means standard errors of the means.

Changes in levels of carbonylated proteins were determined as describedpreviously (31). Briefly, TNF-stimulated HeLa cells were lysed in radioimmuno-precipitation assay buffer containing 5 mM dithiothreitol and protease inhibitors.The carbonyl groups in the side chains of proteins (20 �g) were derivatized to2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophenylhydrazine. The re-action was neutralized with neutralization buffer provided by the ChemiconOxyBlot assay kit. The 2,4-dinitrophenyl-derivatized proteins were fractionatedon an SDS-10% polyacrylamide gel and proteins transferred to polyvinylidenedifluoride membranes (Amersham, Inc.). After transfer, the membranes wereblocked with 5% dry milk in PBS-Tween 20 (0.1%) for 3 h at room temperature

than incubated with primary Ab to NAD (Chemicon, Inc.). After the washing,horseradish peroxidase-conjugated secondary Ab (Amersham, Inc.) was addedfor 1 h. Detection was performed by enhanced chemiluminescence (ECL; Am-ersham, Inc.).

RelA–CDK-9 colocalization analysis. HeLa S3 cells stably expressing theEGFP-RelA fusion protein were plated on glass coverslips. Once the culturesreached 50 to 70% confluence, individual coverslips were exposed to TNF for 0(control), 30, and 60 min in triplicate and fixed with 4% paraformaldehyde inPBS at room temperature for 20 min. Immunofluorescence staining was doneusing rabbit anti-CDK-9 Ab as the primary Ab (1:100) and the Alexa Fluor 568F(ab�)2 fragment of goat anti-rabbit IgG (heavy plus light chains) (Invitrogen,Eugene, OR) as the secondary Ab (5 �g/ml). Then, the cells were counterstainedwith 500 nM TO-PRO-3 (Invitrogen) and mounted on glass slides by use ofVectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluo-rescence imaging was performed with a Zeiss LSM510META confocal micro-scope with a Plan-Apochromat 63� 1.4-numerical-aperture oil immersion lens.The GFP, Alexa 568, and TO-PRO-3 image components were obtained using488-nm (argon ion), 543-nm (green He/Ne), and 633-nm (red He/Ne) laserexcitation lines; the corresponding emission collection ranges were 505 to 530nm, 560 to 620 nm, and longer than 650 nm, respectively. At the image collectionsettings used (laser intensity and detection gain), no bleed-through was observed.The pinhole size and scan settings were adjusted for a section thickness of 0.7 �mand a pixel size of 0.1 �m, respectively. Images were acquired using a bit depthof 12- and 16-frame Kallman averaging. At least five fields were captured percoverslip.

For image processing and analysis, the .lsm files generated by the Zeiss con-focal microscope were translated into TIFF files by use of Image J (http://rsb.info.nih.gov/ij/) and the LSM reader plug-in (http://rsb.info.nih.gov/ij/plugins/lsm-reader.html). Metamorph V6.1 (Molecular Devices, Downingtown, PA) wasused for background correction and image segmentation and to quantify trans-location. The TO-PRO-3 stain was used to create segmentation masks separatingindividual nuclei. Dilation of these masks was used to select perinuclear cyto-plasmic rings for each cell. Average intensity measurements within each nuclearregion and the corresponding cytoplasmic ring were used to quantify RelAtranslocation. To quantify colocalization, each nucleus was analyzed separately.First, an intensity threshold was used to exclude the nucleolus, an area whichshowed low levels of both RelA and CDK-9 stains. Each masked nuclear imagewas exported to Imaris (Bitplane, Zurich, Switzerland) and the degree of colo-calization between RelA and CDK-9 was assessed using the “colocalizationanalysis module.” This module uses correlation analysis first to test the signifi-cance of true colocalization over random color overlap and then to quantify thedegree of colocalization by use of an automatic threshold search algorithm toavoid the bias of visual interpretation. The algorithm used in this software wasdescribed previously (13). Results are expressed as percentages of EGFP colo-calized with the Alexa-568 signal.

siRNA-mediated CDK-9 knockdown. For CDK-9 knockdown, control andCDK-9 siRNAs were from Ambion, Inc. (Austin, TX). siRNAs were transfectedat a 100 nM concentration into A549 cells by use of TransIT-TKO transfectionreagent (Mirus Bio Corp., Madison, WI). For Western blotting, 72 h later, cellswere lysed in modified radioimmunoprecipitation assay buffer; for RNA extrac-tion, cells were TNF stimulated for the indicated times and lysed in TRI reagent(Sigma-Aldrich Inc., St. Louis, MO).

RESULTS

Kinetics of NF-�B-dependent gene transcription. To ex-plore the genetic networks under NF-�B control, we havedeveloped a tetracycline (Tet)-regulated cell system (Tet-Off[18]) in which a nondegradable FLAG epitope-tagged I�B�(I�B� Ser32Ala/Ser36Ala, termed FLAG-I�B� Mut) undercontrol of the Tet operator was stably introduced into cellsexpressing the Tet transactivator (tTA). FLAG-I�B� Mut con-tains site mutations in the serine phosphoacceptor sites ofIKK� that block its inducible degradation and functions as apotent dominant-negative inhibitor of the canonical NF-�Bactivation pathway (11, 55). In these cells, doxycycline (Dox)inhibits FLAG-I�B� Mut expression; upon Dox withdrawal,FLAG-I�B� Mut expression is strongly upregulated (Fig. 1A).To confirm that FLAG-I�B� Mut expression was inert to

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FIG. 1. Dox-regulated NF-�B-dependent gene expression. (A) Tet-regulated I�B� Mut expression. HeLa Tet-Off cells expressing FLAGepitope-tagged I�B� Mut (Ser32Ala/Ser36Ala) were cultured in the presence (�) or absence (�) of 2 �g/ml Dox. Cytoplasmic extracts wereextracted and assayed by Western blotting. (Top) Anti-FLAG Ab; (bottom) �-actin used as a loading control. (B) I�B� Mut is TNF resistant.Tet-regulated FLAG-I�B� Mut cells cultured in the presence or absence of Dox were TNF stimulated for the indicated times (min). (Top)Cytoplasmic extracts were assayed for I�B� abundance using anti-I�B� Ab. The endogenous and I�B� Mut isoforms are shown. (Bottom)�-Actin staining. (C) NF-�B binding in EMSA. Nuclear extracts from the experiment shown in panel B were assayed for NF-�B binding usingEMSA. Shown are the bound complexes. The RelA/NF-�B1 (p50)-containing complex is indicated. (D) Supershift assay in EMSA. EMSAwas performed on control (�) or TNF-stimulated (�) nuclear extracts in the absence or presence of indicated Ab (top). (Top) Long exposure(exp) of the supershifted (SShift) bands (indicated by �); (bottom) short exposure to demonstrate depletion of the TNF-inducible complex.The TNF-inducible NF-�B complex is composed predominantly of RelA � p50 heterodimers. (E) mRNA expression is NF-�B transloca-tion dependent. RNA from a time series of TNF-stimulated HeLa I�B� Mut cells was analyzed by Q-RT-PCR. (The I�B� primers weredesigned to hybridize selectively to the 3� untranslated region of the endogenous gene and do not detect I�B� Mut expression.) Shown aremRNA expression profiles in the presence or absence of Dox. For each gene, change is determined relative to unstimulated values.



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TNF-induced proteolysis and can inhibit NF-�B activation, atime course of TNF stimulation was performed. In cells cul-tured in the continuous presence of Dox, little detectableFLAG-I�B� Mut is detected (Fig. 1B). Within 15 min of TNFstimulation, endogenous I�B� is rapidly proteolyzed to unde-tectable levels, and by 60 min, endogenous I�B� is resynthe-sized. In contrast, upon Dox withdrawal, FLAG-I�B� Mut isstrongly expressed at amounts similar to those for endogenousI�B� and does not degrade in response to TNF (Fig. 1B,right).

We sought to establish whether this level of I�B� Mut ex-pression was sufficient to inhibit NF-�B activation by perform-ing EMSA on nuclear extracts prepared from the same exper-iment. In the presence of TNF, a rapid and potent induction ofthe RelA–NF-�B1 (p50) heterodimer binding was observed,and its abundance peaked at 15 min (Fig. 1C). In the absenceof Dox, no TNF-inducible changes in NF-�B binding abovewhat were seen for the unstimulated control were observed byEMSA (Fig. 1C, right). The rapidly induced DNA-bindingcomplex is composed primarily of RelA–NF-�B1(p50) sub-units, because the complex is disrupted by anti-RelA and anti-NF-�B1 Abs (Fig. 1D). Although a strong supershift of NF-�B1 is seen, only a faint supershift of RelA is seen, a findingconsistent with the destabilization of the Ab-RelA complex inEMSA (19).

The effects of FLAG-I�B� Mut on TNF-induced endoge-nous gene expression was measured for four NF-�B-depen-dent genes (the Gro-�, IL-8, I�B�, and Naf-1 genes) by use ofQ-RT-PCR. In the presence of Dox, TNF stimulation rapidlyinduces IL-8, Gro-�, and I�B� gene expression, peaking within30 min to 1 h after stimulation at 900-fold, 23-fold, and 22-fold,respectively, over the control level (Fig. 1E). In the absence ofDox, the TNF-induced mRNA expression is almost completelyabrogated, indicating that these genes are NF-�B dependent.Although the expression of the Naf1 gene is kinetically distinct,peaking 6 to 9 h after TNF administration, its expression is alsoNF-�B dependent.

Kinetics of NF-�B subunit recruitment to the target genesin chromatin. To better characterize the kinetics of NF-�Brecruitment on individual promoters within their native chro-matin context, we developed a highly efficient two-step ChIPassay that first immobilizes RelA by N-hydroxysuccinimide es-ter-mediated protein-protein cross-linking prior to a secondformaldehyde-mediated protein-DNA cross-linking step (39).The protein cross-linking step was necessary because fluores-cence photobleaching experiments of NF-�B interacting withits multimeric binding sites in vivo have shown that RelA is inrapid exchange with its target DNA, being completely replacedwithin 30 s (8). Although we earlier demonstrated that thisassay was robust for distinct NF-�B isoforms and capturedtargets at levels greater than seen for IgG controls (39), theperformance of this modified ChIP assay to enrich for RelAbinding to target genes was further examined. First, to estab-lish whether the two-step ChIP specifically measured RelAbinding to target genes containing NF-�B sites, chromatinfrom a time series of TNF-stimulated HeLa cells was cross-linked, sonicated, and used as input for immunoprecipitation(IP) using anti-RelA Ab. After capture, washing, and reversalof the cross-links, the DNA was subjected to PCR using prim-ers spanning the high-affinity RelA site located at nucleotides

�99 to �61 in the human IL-8 promoter [IL-8 (Prox); Fig. 2A],primers to an upstream region of IL-8 not known to bind RelAspanning nucleotides �1042 to �826 [IL-8 (Dist) (9, 38)], andprimers spanning an unrelated U6 snRNA spanning nucleo-tides �245 to �85, an RNA Pol III-driven gene. We observedspecific PCR products only in the IL-8 (Prox) samples span-ning the high-affinity RelA site, with a faint signal being de-tected at time zero and a strong induction 30 and 60 min afterTNF administration relative to what was seen for input DNAused as a loading control (Fig. 2A). Specificity was indicated bya lack of signal produced by the IL-8 (Dist) and U6 primers(Fig. 2A).

To determine whether the two-step ChIP assay could detectdynamic differences in the association and dissociation ofRelA, we exploited the findings that distinct NF-�B activationkinetics can be produced by different TNF stimulation proto-cols. Previous work using dynamic fluorescence imaging hasshown that RelA can be induced to translocate in separatemodes, depending on the nature of the TNF stimulation pro-tocol and the nucleus-cytoplasm volumetric ratio (8, 32, 36,54). If the TNF is applied as a transient pulse (15 min), theinitial NF-�B translocation is monophasic. After a rapid initialtranslocation, RelA induces the resynthesis of its I�B� inhib-itor, resulting in its cytoplasmic reaccumulation (19, 52). Uponreaccumulation of I�B�, nuclear RelA is captured and relo-cated to the cytoplasm, resulting in a single spike in nuclearNF-�B binding. Conversely, if the TNF agonist is applied tothe cells without removal (tonic stimulation), RelA is consti-tutively nuclear in cells with large nuclear volumes and oscil-latory in cells with small nuclear volumes. The tonic exposureof agonist results in continuous I�B� degradation, in spite ofits nuclear resynthesis. To illustrate, nuclear extracts preparedfrom TNF-stimulated HeLa cells for various times were boundto a radiolabeled NF-�B consensus site in EMSA (Fig. 2B). Inresponse to pulsatile TNF stimulation, NF-�B DNA-bindingactivity was rapidly induced, peaking from 15 to 60 min anddeclining rapidly thereafter to control levels (32, 36, 54–56). Bycontrast, in response to tonic TNF stimulation, NF-�B bindingactivity was induced and persisted over 9 h of stimulation (Fig.2B). Together, these data indicated that we could induce dis-tinct RelA activation modes using different TNF stimulationprotocols.

The two-step ChIP was applied to chromatin prepared fromcells induced by the two different stimulation protocols. Thecross-linked chromatin was immunoprecipitated using anti-RelA Ab, and Gro-� binding was detected by qualitative PCR.In response to pulsatile TNF stimulation, we observed thatRelA binding was rapidly induced at 15 to 30 min, whereuponit dissociated from the Gro-� promoter coincidentally with itscytoplasmic recapture (Fig. 2C, top). By contrast, in responseto tonic TNF stimulation, RelA binding to Gro-� was stableover the entire time course (Fig. 2C, bottom). These findingsindicated that the two-step ChIP could detect the associationand dissociation of RelA binding with its endogenous genetargets and that there was no detectable lag in RelA bindingbetween RelA’s appearance in the nucleoplasm (detected byEMSA) and its association with chromatin (detected by ChIP)on the Gro-� promoter. This experimental result is furthercompatible with previous findings that nuclear RelA is in rapidexchange with its target sites in chromatin (8). Finally, this



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result validated that the modified two-step ChIP was capableof measuring both the association and the dissociation of RelAon its endogenous binding sites.

We next sought to determine RelA binding kinetics on tar-get genes showing a rapid transcriptional spike. Using qualita-tive PCR, we could detect changes in target DNA over aconcentration range of 0.2 to 20 ng, as shown for IL-8, Gro-�,and I�B� (see Fig. S1 in the supplemental material). For allgenes examined, we found that the kinetics of TNF-inducedRelA binding was rapid and was maximal within 15 to 30 minafter stimulation (Fig. 2D). Interestingly, for genes that arestrongly induced by TNF, such as the IL-8, I�B�, and Gro-�genes, as well as genes that are not, such as the Naf1, NF-�B1,and TRAF2 genes, RelA binding was rapidly inducible, peak-ing 15 min after stimulation. Moreover, the binding profileswere indistinguishable in nature for the duration of the stim-ulation. To confirm these findings, we measured RelA-associ-

ated target genes by use of Q-RT-PCR, where a three- tofourfold change in RelA binding for Gro-� and Naf1 wasobserved at identical times (see Fig. S2 in the supplementalmaterial). These findings were surprising because previouswork has suggested that RelA is occluded from accessing chro-matin sites in late-response genes in monocytes (45).

Also of note is that RelA binding to the IL-8, I�B�, andGro-� promoters was persistent over the 9-h time course, eventhough mRNA expression by these genes was terminating (cf.Fig. 1E and 2D). These findings suggested to us that the dif-ferential patterns of NF-�B-dependent gene expression werenot due to different rates of RelA association.

Promoter-specific phospho-Ser276 RelA recruitment. Be-cause the binding of bulk RelA did not accurately track withthe kinetics of transcriptional activation (e.g., Naf1, TRAF1,and NF-�B2) or termination (IL-8, I�B� and Gro-�), we nextexamined the kinetics of formation and chromatin binding of

FIG. 2. Recruitment kinetics of NF-�B/RelA. (A) Specificity of ChIP assay. Two-step ChIP assay was performed on a time series ofTNF-stimulated cells (39). Chromatin was subjected to IP using anti-RelA Ab. Eluted DNA was subjected to PCR using primers spanning thehigh-affinity RelA site located at nucleotides �99 to �61 in the human IL-8 promoter [IL-8 (Prox)] (top), primers to an upstream region of IL-8not known to bind RelA and spanning nucleotides �1042 to �826 [(IL-8(Dist)] (middle), and primers spanning the Pol III-driven gene, U6 snRNA(bottom). Input DNA was used as a loading control. Shown is an ethidium bromide-stained agarose gel of the PCR products. Note that only theIL-8 (Prox) primers produce a TNF-inducible signal. (B) EMSA of TNF-stimulated cells. HeLa cells were exposed to either a 15-min pulse orcontinuous (tonic) stimulation. Nuclear extracts were subjected to EMSA using a high-affinity RelA-NF-�B1 binding site (10). Shown is anautoradiograph of the specifically bound species. (Top) Pulse stimulation; (bottom) tonic stimulation. (C) ChIP of tonic versus pulse TNFstimulation protocols. HeLa cells were stimulated for the indicated times after 15-min pulse (top) or tonic (bottom) TNF stimulation. Chromatinwas prepared using two-step ChIP and immunoprecipitated with anti-RelA Ab. Shown is an ethidium bromide-stained gel of the PCR products.Input DNA was used as an IP control. (D) Time course of RelA recruitment to target genes. Cells were stained for indicated times and chromatinimmunoprecipitated with anti-RelA Ab. Shown is an ethidium bromide-stained gel for PCR of the IL-8, I�B�, Gro-�, Naf1, NF-�B2, and TRAF1genes by use of gene-specific primers flanking a high-affinity NF-�B binding site (56). Each PCR was run with 25 ng of genomic DNA as a positivecontrol (PC). The negative control (NC) for the IP was IgG.



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phospho-Ser276, as this phosphorylation results in a potentactivating species required for IL-8 gene induction (26, 59).Western blots were performed on a time series of TNF-stim-ulated cells extracted by denaturing lysis using a phospho-Ser276 RelA-specific Ab (Fig. 3A). In unstimulated cells, lowamounts of phospho-Ser276 RelA could be detected, a formwhich migrates aberrantly at 80 kDa, consistent with the find-ings of others (59, 60). TNF induced rapid formation of phospho-Ser276 RelA, peaking 15 to 30 min later at �3-fold induc-tion, which rapidly returned to unstimulated levels 1 h afterstimulation (Fig. 3A). To determine whether this isoformtranslocated into the nucleus, Western blots of cytoplasmic andsucrose cushion-purified nuclear extracts were performed.Phospho-Ser276 RelA was highly enriched in the nuclear frac-tions, with little detectable cytoplasmic staining (Fig. 3B). Thekinetics of nuclear accumulation parallel those of the whole-cell lysate. Together, these data indicate that phospho-Ser276

RelA is rapidly translocated into the nucleus.We next measured the binding kinetics of phospho-Ser276

RelA to NF-�B-dependent genes. Chromatin from a timecourse of TNF stimulation was prepared and two-step ChIPperformed using anti-phospho-Ser276 RelA Ab. Here we foundthat in the absence of stimulation, phospho-Ser276 RelA bind-ing was very low, but phospho-Ser276 RelA was rapidly re-cruited to the IL-8, I�B�, and Gro-� promoters, peaking 15 to30 min after stimulation (Fig. 3C). Importantly, Ser276-phos-phorylated RelA binding to IL-8, I�B�, and Gro-� gene pro-

moters occurred at times corresponding to their peak tran-scriptional activations (compare Fig. 1A and 3C). In contrast,phospho-Ser276 RelA binding to Naf1 and NF-�B2 promoterswas distinct from this pattern, with no detectable change 15 to30 min after stimulation, but we did note a gradual increase inbinding over 3 to 9 h that was significantly reduced in magni-tude (Fig. 3C, bottom).

To determine whether distinct differential phospho-Ser276

RelA recruitment was cell type dependent, we performed thesame experiment on TNF-stimulated type II alveolar epithelialcells (A549). As seen in Fig. 3D, similar results were obtained,with an apparent peak in phospho-Ser276 RelA binding to IL-8and I�B� 15 to 30 min after stimulation and binding to Naf1and TRAF1 �3 h later. The mechanisms controlling the ex-pression of late genes will require further study. For the pur-poses of this work, we sought to evaluate the role of phospho-Ser276 RelA in the expression of the rapidly inducible genesbecause phospho-Ser276 RelA binding was coincident withgene expression.

Requirement of phospho-Ser276 binding in NF-�B-depen-dent gene expression. Previous work in our laboratory hasshown that a TNF-induced ROS signaling pathway mediatesthe phosphorylation of Ser276 on RelA in monocytic cells (26).Here, intracellular ROS activate I�B�–RelA-associated PKActo phosphorylate RelA selectively on Ser276. We furthershowed that phospho-Ser276 RelA formation can be selectivelydisrupted by a variety of chemically unrelated antioxidants,

FIG. 3. (A) Time course of phospho-Ser276 RelA formation. WCEs prepared from a time course of TNF-stimulated cells were fractionated bySDS-PAGE and blotted with anti-phospho-Ser276 RelA (top), anti-RelA (middle), and �-actin as an internal control (bottom). (B) Nucleus-cytoplasm distribution of phospho-Ser276 RelA. HeLa cells were stimulated with TNF for the times indicated at the top and fractionated intocytoplasmic and nuclear fractions. (Top) Western immunoblotting using anti-phospho-Ser276 RelA Ab. (Middle) Blot was reprobed for RelA asa loading control. Note the inducible accumulation of RelA in the nuclear fraction within 15 min of stimulation. (Bottom) � Lamin staining as anuclear fraction control. The nuclear fractions stain strongly for � lamin. (C) Time course of phospho-Ser276 RelA recruitment to NF-�B-dependent genes. HeLa cells were subjected to a time course of TNF stimulation and chromatin was prepared. Shown is an ethidium bromide-stained gel of the PCR products. IgG, negative control for IP using nonimmune IgG. Each row represents a separate PCR assay. (D) Time courseof phospho-Ser276 RelA binding in A549 cells. Experiment is as described for panel C. A549 cells show similar patterns of phospho-Ser276 RelArecruitment.



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FIG. 4. Inhibition of phospho-Ser276 RelA formation by antioxidant. (A) Kinetics of TNF-induced ROS formation and inhibition by DMSO.HeLa cells were loaded with DCFDA and stimulated with TNF (20 ng/ml) in the absence or presence of DMSO (0.5%, 2%). Fluorescencemeasurements were performed using a fluorescence-activated cell sorter at indicated times. Symbols: �, TNF; �, TNF plus 2% DMSO; Œ, TNFplus 0.5% DMSO; F, untreated. TNF induces detectable ROS formation within 15 min of stimulation. (Inset) Histograms of fluorescence-activated



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including the ROS scavenger DMSO, an agent that inhibitsPKAc but does not inhibit TNF-induced Jun N-terminal pro-tein kinase activation or mitogen-stress related kinase or in-duce cellular toxicity (26).

To confirm that DMSO interferes with TNF-induced ROSformation in HeLa cells, DCFDA-loaded HeLa cells werestimulated in the absence or presence of various concentra-tions of DMSO. TNF induces a detectable increase in ROSformation within 15 min and produces a broad peak from 30 toover 90 min after stimulation (Fig. 4A). These kinetics of ROSformation are consistent with previous studies (3, 21). Al-though a significant inhibition of ROS was observed with 0.5%DMSO, complete inhibition was observed using 2% DMSO(Fig. 4A).

As an independent confirmation of TNF-inducible oxidativestress, TNF-induced protein carbonylation was assayed. Car-bonylation is an irreversible oxidation of Arg, Lys, and Pro sidechains that targets proteins for proteasomal degradation (31).TNF stimulation resulted in rapid carbonylation within 15 minof treatment (Fig. 4B). These data confirm that TNF rapidlyinduces biologically significant oxidative stress and are consis-tent with our previous findings of enhanced 8-oxoguanosineadduct formation on DNA (16). To test whether the TNF-induced ROS pathway was required for phospho-Ser276 RelAformation in HeLa cells, a Western blot on TNF-stimulatedcells in the absence or presence of antioxidant was performed.Here we found that the highly inducible phospho-Ser276 RelAformation after TNF stimulation was completely abrogatedafter DMSO treatment (Fig. 4C, top), whereas there was noeffect on phospho-Ser536 RelA formation (Fig. 4C, bottom).DMSO, therefore, selectively interfered with the formation ofSer276-phosphorylated RelA without apparently inhibiting itsother phosphorylation steps or nuclear translocation (26).

To exclude the possibility that DMSO interfered with bulkNF-�B binding to its sites in chromatin, ChIP assays wereperformed on a time course of TNF-stimulated cells in thepresence or absence of DMSO by use of an anti-RelA Ab thatbinds both phosphorylated and nonphosphorylated (“bulk”)RelA (Fig. 4D). Again, TNF stimulation alone induced robustbulk RelA binding. Importantly, bulk RelA binding was notaffected in the presence of DMSO (Fig. 4D, right). From thesedata, we concluded that inhibition of the ROS pathway has noeffect on total RelA nuclear translocation or DNA binding

to target genes, findings consistent with our earlier studies(26, 58).

Inhibition of the ROS pathway by DMSO, then, allows anexamination of the role of phospho-Ser276 RelA in NF-�B-dependent gene expression. For this purpose, TNF-inducedexpression of Gro-�, I�B�, and IL-8 mRNA was determinedby Q-RT-PCR in the absence or presence of DMSO. We foundthat DMSO completely inhibits TNF-inducible Gro-� and IL-8expression but does not affect I�B� expression (Fig. 4E).These data suggest that Gro-� and IL-8 require phospho-Ser276 RelA for inducible gene expression but I�B� does not.

To confirm these observations using a chemically unrelatedantioxidant, we performed TNF stimulations using NAC atconcentrations that inhibited TNF-induced DCF oxidation(58). NAC blocked phospho-Ser276 formation (Fig. 4F) andsignificantly inhibited Gro-� and IL-8 gene expression withoutaffecting I�B� (Fig. 4G).

To confirm that DMSO reduced phospho-Ser276 RelA bind-ing, two-step ChIP was conducted using a phospho-Ser276

RelA Ab. In this experiment, we plated the cells at a twofoldincrease in cell density to maximize the detection of phospho-Ser276 RelA signal by the two-step ChIP assay. As a result, abasal signal of phospho-Ser276 RelA binding could be detectedfor Gro-� and, to a lesser extent, for IL-8 in unstimulated cellsdue to the increase in DNA input into the PCR (Fig. 4H).Moreover, for reasons not yet completely understood, the ki-netics of phospho-RelA binding were slightly delayed by 15min relative to the experimental results shown earlier in Fig.3C, suggesting that the kinetics of ROS formation are celldensity dependent. Nevertheless, and most importantly, induc-ible phospho-Ser276 RelA binding to Gro-�, IL-8, and I�B�was completely inhibited (Fig. 4H). These data indicated thatthe inhibition of the ROS pathway selectively ablates thephospho-Ser276 RelA formation and recruitment of phospho-Ser276 RelA, but not bulk RelA, to target genes. Taken to-gether, this series of experiments shows that phospho-Ser276

RelA is rapidly formed after TNF stimulation in an oxidant-dependent manner and that its binding is necessary for theexpression of a subset of NF-�B-dependent genes, includingthe Gro-� and IL-8 genes.

RelA Ser276 mediates differential patterns of gene expres-sion. To further determine the requirement of RelA Ser276

phosphorylation in the activation of a subset of NF-�B-depen-

cell sorter analysis at 0, 30, and 60 min of treatment. (B) Kinetics of TNF-induced protein carbonylation. HeLa cells were stimulated with TNFfor the times indicated. Shown is a Western blot for carbonylated proteins. Inducible carbonylation is seen within 15 min of stimulation (indicatedby arrow). (C) Selective inhibition of TNF-induced phospho-Ser276 RelA formation by DMSO. HeLa cells were stimulated with TNF for 30 minthe absence or presence of nontoxic concentrations of DMSO (2%) (26, 58). (Top) WCEs were prepared and blotted with anti-phospho-Ser276

RelA or pan anti-RelA Abs; (bottom) WCEs were blotted with anti-phospho-Ser536 RelA or anti-RelA Abs. (D) Antioxidant does not affect totalRelA recruitment to target promoters. HeLa cells were stimulated for indicated times in the absence or presence of DMSO. Shown is a two-stepChIP assay using pan-anti-RelA Ab in the IP. Input is the loading control for the IP. DMSO has no impact on inducible RelA binding. (E) Effectof DMSO on NF-�B-dependent gene expression. mRNA was prepared from TNF-stimulated HeLa cells in the absence or presence of DMSO.Q-RT-PCR analysis was performed for the indicated genes. Shown is change relative to level at time zero and normalized to 18S RNA as aninternal control. **, P � 0.01, t test. (F) Inhibition of TNF-induced phospho-Ser276 RelA formation by NAC. HeLa cells were stimulated with TNFfor 30 min in the absence or presence of 10 mM NAC (26, 58). WCEs were prepared and blotted with anti-phospho-Ser276 RelA or anti-RelA Abs.(G) Effect of NAC on NF-�B-dependent gene expression. mRNA was prepared from a time course of TNF-stimulated HeLa cells in the absenceor presence of NAC (10 mM). Q-RT-PCR analysis was performed as described for panel E. *, P � 0.05; **, P � 0.01, t test. (H) Antioxidant blocksphospho-Ser276 RelA recruitment. HeLa cells were TNF stimulated for the indicated times in the absence or presence of DMSO. Shown is atwo-step ChIP assay using anti-phospho-Ser276 RelA Ab as the immunoprecipitating Ab. DMSO completely blocks TNF-inducible phospho-Ser276

RelA binding.



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dent genes, we examined the effect of expressing a RelASer-Ala site mutation at residue 276 (RelA Ser276Ala).RelA�/� MEFs were transfected with FLAG epitope-taggedstrawberry–RelAWT or RelA Ser276Ala expression vectorsand pools of stable transfectants isolated. Cells were then TNFstimulated for a 6-h time course, and Q-RT-PCR was per-formed. We selected for measurement the mouse homologs ofthe Gro-� and I�B� genes (note that mice do not have an IL-8gene). Although RelAWT potently transactivated the mGro-�gene, the RelA Ser276Ala mutant did not (Fig. 5). Conversely,both proteins strongly transactivated the mI�B� gene by �5-fold. These data confirmed our earlier findings that phospho-Ser276 RelA controls a subset of NF-�B gene expression.

The CDK-9–Ccn T1 complex, P-TEFb, inducibly bindsphospho-Ser276 RelA. Our data suggested that induciblephospho-Ser276 RelA binding is important for the activation ofthe IL-8 and Gro-� genes but not for that of the I�B� gene. Ofthese, the IL-8 and Gro-� genes show inducible RNA Pol IIrecruitment, whereas the I�B� gene is preloaded with Pol II(Fig. 6A). This finding suggested to us that phospho-Ser276

RelA may regulate transcriptional elongation of the I�B�gene. In this process, inducible association of RelA with spe-cific kinases that phosphorylate the Pol II carboxy-terminaldomain (CTD) allows it to enter a processive phase of mRNAtranscription. Previous work has shown that P-TEFb, a com-plex containing CDK-9–Ccn T1, is responsible for phosphor-

ylating RNA Pol II on the Ser2 CTD, licensing Pol II to enterthe active transcriptional elongation mode (37, 44).

We therefore investigated whether RelA spatially colocal-ized with CDK-9 by use of fluorescence confocal microscopy.For this purpose, cells stably expressing a TNF-regulatedEGFP-RelA fusion protein were stimulated in the presence orabsence of TNF. Staining with anti-CDK-9 Ab was then usedto visualize endogenous CDK-9, tagging it with Alexa 568 (Fig.6B). In the absence of stimulation, EGFP-RelA was stronglycytoplasmic; in response to TNF, EGFP-RelA was translo-cated into the nucleus in 100% of cells in a nonrandom distri-bution, excluding nucleoli (Fig. 6B). By contrast, CDK-9 wasconstitutively nuclear and did not change in response to TNF.To rigorously demonstrate colocalization, we analyzed theirnuclear spatial distributions for correlation in excess of ran-dom overlap by use of a validated statistical algorithm (13),where the observed correlation between two color channels iscompared against a probability distribution of random overlapgenerated by repetitive image scrambling (13). The extent ofcolocalization is calculated by using an automatic thresholdsearch method that avoids the subjective bias that usually oc-curs with other methods that rely more in visual inspection.Using this approach, we observed that 56.65% 19.9% ofEGFP-RelA was colocalized with CDK-9, an association thatwas highly statistically significant (P � 0.001). These data sug-gested that CDK-9 colocalized with activated nuclear RelA.

To examine whether phospho-Ser276 RelA associated withCDK-9, nondenaturing coimmunoprecipitation experimentswere performed. Control or TNF-stimulated nuclear extractswere immunoprecipitated with anti CDK-9 Ab, and the pres-ence of RelA isoforms was detected by Western blotting (Fig.6C). We detected twofold-increased binding of phospho-Ser276

RelA in the CDK-9 complexes (Fig. 6C). RelA comigratedwith the 80-kDa species that was produced by anti-phospho-Ser276-RelA Ab (Fig. 6C and reference 12). The binding ofphospho-RelA to Ccn T1 and CDK-9 in the presence of TNFstimulation was significantly inhibited by the presence ofDMSO (Fig. 6D). Together, these data suggest that the TNF-inducible Ser276 phosphorylation of RelA is required for asso-ciation with the P-TEFb Pol II CTD kinase.

TNF-induced P-TEFb recruitment to target genes is depen-dent on phospho-Ser276 RelA formation. To determinewhether TNF induced P-TEFb recruitment on NF-�B-depen-dent genes, and if so, whether this recruitment required Ser276-phosphorylated RelA, a time course experiment using a ChIPassay of TNF stimulation in the presence or absence of DMSOwas conducted. As seen in Fig. 6E, TNF-inducible CDK-9 andCcn T1 binding was observed for IL-8, Gro-�, and I�B�. Here,P-TEFb binding occurred rapidly, within 15 min, at times co-incident with target gene expression. Importantly, TNF-induc-ible CDK-9 and Ccn T1 binding was significantly inhibited incells TNF stimulated in the presence of DMSO. Together,these data indicate that the P-TEFb complex is inducibly re-cruited to target promoters in a phospho-Ser276 RelA-depen-dent manner.

TNF-induced NF-�B-dependent gene expression requiresCDK activity. To further establish the functional role of CDKin NF-�B-dependent gene expression, we examined the effectof the CDK inhibitor FP (48). FP is a highly selective CDKinhibitor that potently inhibits the kinase activity of CDK-9

FIG. 5. Effect of RelA Ser276Ala mutation on target gene expres-sion. RelA�/� MEFs were transfected with expression vectors encod-ing a FLAG epitope-tagged monomeric strawberry fusion withRelAWT or RelA Ser276Ala, and pooled transfectants were isolated.Cells were stimulated for indicated times with TNF, and mRNA wasextracted. Q-RT-PCR was performed to determine the activation ofendogenous NF-�B-dependent genes.



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(37). In this experiment, vehicle- or FP (500 nM)-pretreatedcells were stimulated with TNF, and the induction of NF-�B-dependent genes was measured by Q-RT-PCR. FP completelyblocked the expression of IL-8 and Gro-� (Fig. 7A).

We next investigated whether CDK kinase activity was re-quired for TNF-induced Ser2 Pol II CTD phosphorylation.Chromatin prepared from control or TNF-stimulated cells inthe absence or presence of FP was assayed for RelA and

phospho-Pol II recruitment. Consistent with its selective effecton CDK-9 activity, FP had no effect on inducible RelA binding(Fig. 7B). By contrast, TNF-inducible phospho-Ser2 Pol IICTD binding was significantly inhibited, indicating that CDKactivity was required to mediate this key posttranslational stepin transforming Pol II to its transcriptional elongation mode.These data suggested that phospho-Ser276 RelA-mediatedcomplex formation with P-TEFb mediates transcriptional elon-

FIG. 6. TNF-inducible Pol II recruitment and RelA-P-TEFb complex formation. (A) Effect of TNF on Pol II loading. ChIP from TNF-stimulated HeLa cells, which were immunoprecipitated with anti-Pol II Ab. (B) Colocalization analysis. HeLa cells stably expressing EGFP-RelAwere stimulated in the absence (control [Con]) or presence of TNF (30 min). Cells were fixed and stained with rabbit anti-CDK-9 Ab and AlexaFluor 568-labeled goat anti-rabbit IgG. Nuclei were counterstained with TO-PRO-3 and are shown as overlays with differential interferencecontrast. (Bottom left) Sequential stages in image analysis with masking and colocalized pixels; (bottom right) corresponding two-dimensionalhistogram of EGFP-RelA and CDK-9 correlation. Shown is least-squares correlation analysis and the extent of colocalization for this image. Afteranalysis of 39 individual cells, the Pearson’s correlation coefficient was 0.428 0.127, and the association was highly statistically significant (P �0.001). (C) CDK-9 recruitment to RelA. FLAG-RelA-expressing HeLa cells were stimulated with TNF for 0 and 0.5 h in the absence or presenceof DMSO as indicated. RelA-associated complexes were immunoprecipitated using anti-FLAG Ab, and the association of CDK-9 was detectedby immunoblotting. (Bottom) IB of FLAG-RelA as a recovery control for the immunoprecipitation (IP). CDK-9–RelA complex association is TNFinducible and is reduced by DMSO treatment. (D) Coimmunoprecipitation assay for RelA-P-TEFb complex formation. HeLa cells were stimulatedwith TNF for 0 and 0.5 h in the absence or presence of antioxidant (DMSO). Nuclear extracts were prepared and immunoprecipitated withindicated anti-CDK-9 or Ccn T1 Abs. The presence of RelA in the immune complexes was then detected using anti-RelA Ab in the immunoblot(IB). A TNF-inducible complex was detected between Ser276-phosphorylated RelA, CDK-9, and Ccn T1. (Right) The inducible RelA–P-TEFbcomplex is quantitatively prevented by DMSO treatment. (E) Inducible P-TEFb binding. HeLa cells were stimulated with TNF for 0, 0.25, and 0.5 hin the absence or presence of DMSO as indicated. (Top) Two-step ChIP assay using anti-CDK-9 Ab as the immunoprecipitating Ab; (bottom)anti-Ccn T1 Ab was used. Shown is an ethidium bromide-stained gel of PCRs for IL-8, Gro-�, and I�B� as indicated (left). TNF-induciblerecruitment of each member of the pTEF complex is rapid and indistinguishable. Inducible P-TEFb recruitment is significantly inhibited by DMSO.



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gation, a process important in NF-�B-dependent Gro-� ex-pression.

CDK-9 subunit is required for optimal expression of Gro-�/IL-8 genes. To more specifically implicate the role of CDK-9in NF-�B-dependent gene expression, we conducted a series ofexperiments using siRNA knockdown. Relative to those trans-

fected with control, cells transfected with CDK-9 siRNAshowed a reduction of �50% in normalized CDK-9 steady-state levels (Fig. 8A). Under these conditions of CDK-9 knock-down, a time course of TNF stimulation was performed andgene expression measured by Q-RT-PCR. In contrast to whatwas seen for control transfectants, we observed a significantreduction in the peak expression of Gro-� and IL-8 mRNA butnot in that of I�B� (Fig. 8B). We interpret this result to meanthat CDK-9 is an important mediator of phospho-Ser276 RelA-dependent gene expression.

DISCUSSION

TNF is a pleiotropic cytokine that mediates the pulmonarycytokine cascade, the hepatic acute-phase response, and theregulator of leukocyte activation and apoptosis. Upon its liga-tion to cell surface receptors, TNF induces protein recruitmentto cytoplasmic death domains, assembling a signaling complexcomposed of TRADD, FADD, and TRAF2 (and others) thatactivates divergent intracellular signals, including the Jun N-terminal protein kinase–AP-1 and the IKK–NF-�B pathwaysto induce genomic responses in the target cell (23, 24). Al-though the canonical IKK–NF-�B pathway is critical for induc-ing tissue inflammation and preventing TNF-induced pro-grammed cell death, surprisingly little is known about itsmechanism for activating downstream gene targets. We haveused high-density DNA microarrays of Tet-regulated domi-nant-negative cell lines to systematically identify the networkof genes downstream of the NF-�B transcription factor (54–56). Surprisingly, these studies indicate that genes under directNF-�B control are expressed in temporally distinct waves, witheach group affecting a different biological process (54). In thisstudy, we have exploited our recent developments in a quan-titative two-step ChIP assay (39), an assay sensitive to bindingand dissociation of NF-�B, to probe the diverse mechanismsgoverning NF-�B-inducible gene expression.

NF-�B subunits are nuclear phosphoproteins responsive todiverse signaling pathways (35). Specifically, the potent RelA-transactivating subunit can be phosphorylated at multiple sites,including Ser276 by PKAc (26, 60) and mitogen and stresskinase 1 (57) and Ser536 by IKK�, NK-�B-inducing kinase, andc-Src (28, 46). These phosphorylation events lead to an in-crease in RelA nuclear translocation, DNA binding, and/ortranscriptional activity, through the mechanisms not com-pletely understood. Of relevance to this study is the fact thatRelA Ser276 phosphorylation occurs upon the activation of thecanonical NF-�B pathway and cytoplasmic release from I�B�.Although the relative roles of PKAc and mitogen and stresskinase 1 have yet to be fully understood, we have recentlyobserved that PKAc mediates TNF-induced phospho-Ser276

RelA formation via an intracellular ROS signal (26). Thissecond ROS-PKAc pathway is completely independent of thatcontrolling RelA translocation and is necessary for the acqui-sition of full transcriptional activity (26). The consequence ofSer276 phosphorylation is thought to be a destabilization of theintermolecular association of the NH2- and COOH-terminalends of RelA (60), producing a conformational change thatmay produce a more stable association with CBP/p300 andother coactivators (60). As a result, phospho-Ser276 RelA be-

FIG. 7. CDK inhibitor FP inhibits NF-�B-dependent gene expres-sion by disrupting Pol II phospho-Ser2 CTD formation. (A) Geneexpression HeLa cells were pretreated with vehicle or FP (500 nM) for1 h prior to 30 min of TNF stimulation. mRNA expression was mea-sured by Q-RT-PCR. Shown is change in mRNA normalized by 18Srelative to unstimulated values. (B) FP inhibits P-TEFb-induced phos-pho-Ser Pol II CTD formation. HeLa cells were pretreated with vehi-cle or FP (500 nM) for 1 h prior to 30 min of TNF stimulation. (Top)Two-step ChIP assay using anti-RelA Ab as the immunoprecipitatingAb; (bottom) anti-phospho-Ser Pol II Ab was used. Although FP hasno effect on inducible RelA binding, it significantly inhibits phospho-Ser Pol II recruitment.



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comes a substrate for acetylation, resulting in enhanced tran-scriptional activity (12).

Our findings extend the range of protein interactions involv-ing activated RelA, as we have observed that Ser276 phosphor-ylation is also required for interaction with the P-TEFbcomplex. This finding is important because it suggests thatphospho-Ser276 RelA binding can mediate transcriptionalelongation by recruiting P-TEFb to target genes as an addi-tional mechanism for inducible gene expression over that me-diated by p300/CBP recruitment. Importantly, the requirementfor P-TEFb recruitment is not a uniform feature of NF-�B-dependent gene activation. Rather, our data suggest thatphospho-Ser276 RelA plays only a restricted role in the activa-tion of two NF-�B-dependent targets, the IL-8 and Gro-�genes (but not the I�B� gene). Our ChIP studies indicate thatthe IL-8 and Gro-� genes are highly inducible through a mech-anism involving Pol II recruitment, whereas the I�B� gene isone whose magnitude of expression is not as highly inducibleand is stably engaged with Pol II in the absence of TNF stim-ulation (Fig. 6A). Previously, using genomic footprinting of theIL-8 gene by ligation-mediated PCR, we found that NF-�Bactivation produced dramatic modifications of the proteinsbinding the TATA box and transcriptional initiation site, sug-gesting that NF-�B activated this gene by a promoter recruit-ment mechanism (9). Here, we extend this mechanism to in-clude both Pol II and P-TEFb recruitment. In response toCDK-9-mediated CTD phosphorylation, recruited Pol II isable to produce full-length transcripts. In the case of the I�B�gene, others have shown that the gene is preloaded with RNApolymerase, making it able to rapidly respond to NF-�B bind-ing without needing time-dependent formation of a preinitia-tion complex (1). Together, these studies indicate that NF-�Bactivates its downstream gene targets through pleiotropicmechanisms.

By comparing the kinetics of bulk RelA binding and those ofphospho-Ser276 RelA binding to genomic targets, this studyrepresents the first demonstration that there is a dissociation inthe timing between phospho- and non-phospho-RelA recruit-ment. Specifically, our data show that phospho-Ser276 RelAbinding is the functionally important complex in Gro-� andIL-8 gene expression because (i) phospho-Ser276 RelA bindingtemporally coincides with peak transcriptional activation andpeak steady-state mRNA abundance, (ii) inhibition of phospho-Ser276 formation by antagonism of the ROS pathway selec-tively blocks phospho-RelA (but not bulk RelA) binding andtranscriptional activation of IL-8/Gro-�, and (iii) a phosphor-ylation-deficient Ser-to-Ala site mutation at residue 276 is un-able to efficiently transactivate Gro-� in a RelA-deficientMEFs. Others have that shown Ser276 phosphorylation wasnecessary for a strong response to the NF-�B-inducing stimuliTNF and IL-1 (2, 12, 60). It is possible that the size or shape ofthis multiprotein complex or the nature of chromatin wherethe NF-�B binding site is found may restrict the access of thephospho-Ser276 RelA complex to certain genes. It will be in-teresting to compare protein complexes formed by phospho-Ser276 RelA and nonphosphorylated RelA.

Although the Ser276 RelA complex plays a key functionalrole in IL-8 and Gro-� gene expression, it is not required forI�B� expression, even though our ChIP assays show thatphospho-Ser276 RelA rapidly and inducibly binds to this pro-

FIG. 8. Effect of siRNA-mediated knockdown of CDK-9 on NF-�B-dependent gene expression. (A) Magnitude of siRNA-mediatedknockdown. Cells were transfected with control (Con) or anti-CDK-9siRNA prior to lysis and analysis of CDK-9 expression by Westernblotting. Duplicate plates are shown for reproducibility. (Top) Stainingwith anti-CDK-9 Ab; the specific 42-kDa band is shown. (Bottom) Blotwas reprobed with anti-�-actin Ab as a loading control. When normal-ized to �-actin, CDK-9 abundance is downregulated by �50%. (B) Ef-fect of CDK-9 knockdown on inducible gene expression. Cells weretransfected with control or CDK-9-targeted siRNA for 72 h prior toTNF stimulation for the indicated times. mRNA expression of earlygenes was measured by Q-RT-PCR. Shown is change in mRNA nor-malized by 18S relative to unstimulated values. �, P � 0.01, two-tailedt test.



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moter. This conclusion is made based on the ability of hypo-Ser276-phosphorylated RelA to transactivate I�B� after theinhibition of the ROS-PKAc pathway, the ability of the RelASer276Ala mutant to transactivate I�B� in RelA�/� MEFs, andthe lack of effect of CDK-9 downregulation on I�B� expres-sion. Therefore, although we observed an induction of phospho-Ser276 RelA binding to I�B� in the ChIP assay, this RelAmodification and P-TEFb recruitment were not required toactivate productive transcription. In this regard, we note ear-lier studies that have shown phospho-defective RelA at resi-dues Ser205, Ser276, and Ser281 are unable to mediate a subsetof NF-�B-dependent genes (the ICAM-1, VCAM-1, andMIP-2 genes) in response to lipopolysaccharide and gammainterferon, whereas induction of two other NF-�B-dependentgenes, the major histocompatibility complex class I and Mnsuperoxide genes, are essentially unaffected (2). These datasuggest to us that a phosphorylation code controls NF-�B’sability to activate subnetworks of target genes, where distinctgroups are induced by particular forms of phospho-RelA andthe protein complexes that they form. Elucidation of the com-plete spectrum of genes under phospho-Ser276 RelA-depen-dent control will require further investigation.

CDK-9, along with Ccn T1, are core constituents of P-TEFb,a complex that functions as a transcriptional elongation factormediating human immunodeficiency virus TAT-dependenttransactivation (5) and the activation of heat shock genes inDrosophila melanogaster (37). P-TEFb mediates transcriptionalelongation by its ability to phosphorylate several targets in thepaused Pol II-dependent promoter, including serine 2 in theheptad repeat in the Pol II CTD, as well as inhibitory factorssuch as negative elongation factor (NELF) and DRB sensitiv-ity-inducing factor (DRIF) (41). Our studies extend the under-standing of inducible phospho-RelA protein interactions andsuggest for the first time that phospho-Ser276 RelA is requiredfor complex formation with the P-TEFb kinase. Our findingsusing a highly potent and specific CDK inhibitor and siRNA-mediated knockdown both suggest that IL-8 and Gro-� areactivated by a mechanism involving P-TEFb recruitment. ThatCDK kinase activity mediates Pol II CTD phosphorylation isindicated by the reduction in CTD phosphorylation in re-sponse to FP (Fig. 6B). Further studies are needed to moreprecisely identify the spectrum of proteins modified by CDK-9on NF-�B-dependent promoters, particularly NELF and DRIF.

Our fluorescence colocalization studies suggest that a signif-icant fraction of RelA colocalizes with CDK-9 upon nuclearentry. Although spatial colocalization is subject to randomerror, especially in this case, where the concentration of oneinteracting protein is increasing in the cellular compartment ofinterest, we have analyzed these data using a formal statisticalrandomization algorithm which distinguishes random coloroverlap due to compartmentalization from that arising due tospecific colocalization (13). Using this technique, we find thata significant fraction of RelA associated with CDK-9 in thenucleus. Because much of the nuclear fraction of RelA is notassociated with chromatin, we believe that RelA–CDK-9 in-teraction occurs prior to promoter binding. To this end, we cancoimmunoprecipitate NF-�B/RelA–CDK-9 from the nuclei ofTNF-stimulated cells.

Recent work using fluorescence microscopy (photobleachingand fluorescence lifetime measurements) has shown that

NF-�B transiently binds its chromatin targets with an exchangerate of seconds (8), resulting in the appreciation that chroma-tin-bound NF-�B is in dynamic exchange with its non-DNA-associated nucleoplasmic pool. This finding suggests that con-centration changes in phospho-NF-�B isoforms entering thenuclear compartment can rapidly exchange with promoter-associated NF-�B. This phenomenon has been experimentallyverified by us using Ang II, a ligand that activates phospho-Ser536 RelA formation without affecting total RelA nuclearabundance. In this case, an increase in the fraction of nuclearphospho-Ser536 RelA can rapidly exchange with promoter-bound hypophosphorylated RelA, producing a transition ingene expression from an inactive state to an activated one (14).Our ChIP analysis of phospho-Ser276 RelA binding, however,indicates that this isoform does not rapidly exchange with thehypophosphorylated RelA bound to the Naf1 gene. We sus-pect that phospho-Ser276 RelA complexed with p300/CBP andP-TEFb is a macromolecular complex whose promoter acces-sibility is dependent on the architecture and topology of thetarget promoters.

The findings of this study indicate that NF-�B controlsdownstream genes through at least two distinct mechanisms,schematically illustrated in Fig. 9. The first mechanism, exem-plified by IL-8 and Gro-�, controls genes that are not signifi-cantly engaged with RNA Pol II in the absence of stimulationand require phospho-Ser276 RelA binding and P-TEFb recruit-ment for inducible Pol II recruitment and activation of tran-

FIG. 9. Model of NF-�B-dependent initiation of transcription. Thisschematic represents the steps involved in the two mechanisticallydistinct pathways involved in the transcriptional initiation of NF-�B-dependent gene expression mediated by inducible Pol II loading. Inthe cytoplasm, TNF stimulation releases RelA from sequestered cyto-plasmic sites by inducing I�B� proteolysis. A delayed ROS signalmediates phospho-Ser276 RelA formation on a subset of proteins.Phospho-Ser276 RelA binds the CDK-9–Ccn T1 P-TEFb complex, re-cruiting it to IL-8 and Gro-� gene promoters. Here, P-TEFb is in-volved in phosphorylation of Ser2 in the Pol II CTD, NELF, and DSIF,resulting in productive transcriptional elongation. Transcriptional ac-tivation of the IL-8 and Gro-� genes is sensitive to antioxidant DMSOby its ability to disrupt RelA phosphorylation at Ser276 and therebydisrupt association with P-TEFb complexes. Transcriptional activationof the IL-8 and Gro-� genes is disrupted by FP and CDK-9 siRNA byblocking the serine phosphorylation of RNA Pol II CTD. Althoughphospho-Ser276 RelA binds I�B�, it is not functionally required forgene expression.



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scriptional elongation. The second mechanism, exemplified byI�B�, binds with RNA Pol II in the absence of stimulation.Although I�B� inducibly binds phospho-Ser276 RelA and P-TEFb, this complex is not required for promoter activation.

Epithelial cells play important roles in the initiation andmaintenance of innate immunity. For example, in the airway,epithelial cells respond to a variety of viral, environmental, andhormonal (cytokine) stimuli to activate NF-�B-dependent cy-tokine cascades important in the genesis and maintenance ofairway inflammation (42, 51, 55, 56). Our delineation of dis-tinct mechanisms by which NF-�B activates target genes hasimportant implications for targeted anti-inflammatory therapyat epithelial surfaces, where distinct biological responses couldbe modified. For example, inhibition of the ROS-dependentphospho-Ser276 RelA-P-TEFb pathway may be useful to con-trol the cytokine cascade without influencing other NF-�B-mediated antiapoptotic responses.

ACKNOWLEDGMENTS

We thank the NCI Developmental Therapeutics Program and Se-rono-Aventis for the gift of FP.

This project was supported by NIAID grants R01 AI40218 and P01AI062885 (to A.R.B.) and a J. W. McLaughlin predoctoral fellowship(to D.E.N.). Core laboratory support was from NIEHS grant P30ES06676 (to J. Halpert, UTMB).

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