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Molecular and Cellular Biology, June 2008, p. 3623-3638, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.01152-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

RelA Ser²⁷⁶ Phosphorylation Is Required for Activation of a Subset of NF-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. Brasier^1,4*

Departments of Medicine,¹ Microbiology and Immunology,² Neuroscience and Cell Biology,³ The Sealy Center for Molecular Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1060⁴

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

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NF-B plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-B/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-B-dependent genes, serine 276 (Ser²⁷⁶)-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-β, interleukin-8 (IL-8), and IB. Previous work has shown that TNF-inducible RelA Ser²⁷⁶ phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser²⁷⁶ RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser²⁷⁶ formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser²⁷⁶ RelA is required only for activation of IL-8 and Gro-β, with IB being unaffected. These data were confirmed in experiments using RelA^–/– murine embryonic fibroblasts reconstituted with a RelA Ser²⁷⁶Ala mutation. In addition, we observe that phospho-Ser²⁷⁶ RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser²⁷⁶ RelA-P-TEFb complex is required for IL-8 and Gro-β gene activation but not for IB gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser²⁷⁶ RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-β genes. The second involves a phospho-Ser²⁷⁶ RelA-independent activation of genes preloaded with Pol II, exemplified by the IB gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infectious and inflammatory stimuli induce expression of gene networks controlling proinflammatory and innate immune responses. One important arm of the inflammatory response is mediated by monocyte-derived tumor necrosis factor (TNF), a cytokine that activates gene expression programs in adjacent epithelial cells to propagate mucosal inflammation (42, 51). Here, the spectrum of functional activities and the magnitude and timing of TNF-induced gene expression are important determinants of tissue homeostasis. A central mediator of epithelial genomic response is nuclear factor-B (NF-B), an inducible transcription factor that controls the expression of proinflammatory genes and whose dysregulation has also been implicated in the pathogenesis of inflammatory, neoplastic, and autoimmune diseases (4, 6, 17).

In most non-B-lymphoid cells, NF-B is sequestered as an inactive complex in the cytoplasm by the inhibitors of B (IBs). The mechanism by which NF-B is activated by TNF, referred to as the "canonical" activation pathway, has been extensively investigated (22). The canonical pathway is activated by ligands of the TNF superfamily including TNF alpha, the prototypical macrophage-derived cytokine that binds to the ubiquitously expressed TNF receptor 1 (TNFRI) (50). TNF-induced TNFRI trimerization induces the recruitment of cytoplasmic signal adapters, including the TNF receptor-associated death domain (TRADD), TNF receptor-associated factor 2 (TRAF2) and TRAF6, receptor-interacting protein (RIP), mitogen/extracellular signal-regulated kinase kinase 3, and others (23-25). This activated submembranous TNFRI complex transiently recruits the IB kinase (IKK) complex, resulting in the phosphorylation of the catalytic IKK and -β kinases followed by its cytosolic release (15, 43). In the cytosol, activated IKK phosphorylates serine (Ser) residues 32 and 36 on the NH2 terminal regulatory domain of IB, targeting IB for proteolytic destruction by ubiquitination and calpain pathways (20, 29). IB degradation releases NF-B to rapidly translocate into the nucleus.

In the nucleus, the activated NF-B complex binds to highly conserved regulatory sequences corresponding to the 5'-GGGRNNYYCC-3' consensus (30). Although NF-B binding in vitro is quite stable, with a half-life of 45 min or longer (8), fluorescence recovery after photobleaching and fluorescence lifetime measurements have shown that NF-B interacting with genomic targets within native chromatin is in a hyperdynamic exchange, with an observed half-life of seconds (8). Studies of highly inducible promoters have suggested that NF-B interaction induces target gene activation through a mechanism that induces the formation of a multiprotein complex known as an "enhanceosome" (34). The enhanceosome contains non-DNA-binding chromatin-modifying proteins, including the CBP/p300 coactivator (49), a histone acetylase that modifies promoter-associated nucleosomes, as well as incompletely characterized methylases, kinases, and chromatin-organizing proteins. This activity results in enhanced transcriptional initiation.

Although nuclear translocation is required for the activation of NF-B-dependent genes, we have recently shown a requirement for a TNF-inducible reactive oxygen species (ROS) pathway in licensing the transactivating potential of NF-B (26, 58). In this pathway, TNF induces the formation of ROS, whose formation occurs at times well after the occurrence of IB proteolysis and NF-B nuclear translocation (58). This second signal activates the catalytic subunit of protein kinase A (PKAc), resulting in the selective phosphorylation of the RelA-transactivating subunit at Ser residue 276, a modification that permits complex formation with incompletely characterized transcriptional activating proteins, including p300/CBP (60), and serves as a switch for additional posttranslational modifications, such as acetylation (12). We have recently found that the inhibition of the TNF-induced ROS prevents phospho-Ser²⁷⁶ RelA formation, resulting in the nuclear translocation of hypophosphorylated RelA and the formation of an unstable enhanceosome (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 expressing a regulated dominant-negative NF-B inhibitor have elucidated the genes under control of the canonical NF-B activation pathway in epithelial cells subjected to viral infections (40, 55) and cytokine stimulation (56) and those controlled by its distinct activation modes (54). These studies have shown that in response to TNF stimulation, NF-B binding controls the expression of a noncontiguous group of genes whose products control a variety of biological processes, including leukocyte activation/chemotaxis, negative regulators of the TNF-IKK pathway, cellular metabolism, antigen processing, and others (54).

In this study, we examine the detailed mechanisms for highly inducible NF-B-dependent genes by use of an efficient two-step chromatin immunoprecipitation (ChIP) assay (39). We observed indistinguishable patterns of inducible RelA binding to all promoters irrespective of their degree of induction or activation kinetics. However, distinct binding patterns of Ser²⁷⁶-phosphorylated RelA binding were observed, with rapid phospho-Ser²⁷⁶ RelA binding to the most highly inducible promoters, and this was at times coincident with their maximal expression. To establish the role of the phospho-Ser²⁷⁶ RelA modification, we blocked phospho-Ser²⁷⁶ RelA formation using ROS scavengers (dimethyl sulfoxide [DMSO]) and antioxidants (N-acetyl cysteine [NAC]), agents that blocked RelA Ser²⁷⁶ phosphorylation without affecting its translocation. We found that interleukin-8 (IL-8) and Gro-β expression were highly dependent on phospho-Ser²⁷⁶ RelA binding, whereas IB was not. Further study of this mechanism revealed that the phospho-Ser²⁷⁶ RelA associates with the cyclin-dependent kinase 9 (CDK-9)-cyclin T1 (Ccn T1) complex, positive transcription elongation factor b (P-TEFb). Short interfering RNA (siRNA)-mediated knockdown showed that RelA recruitment of P-TEFb activity is required only for a subset of NF-B-dependent genes (the IL-8 and Gro-β genes), genes characterized by TNF-induced polymerase II (Pol II) recruitment. Together, these data show for the first time that phospho-Ser²⁷⁶ RelA formation is responsible for the activation of a subset of NF-B-dependent genes via the P-TEFb transcriptional elongation complex.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture and reagents. HeLa S3 and A549 cells were from ATCC and maintained as previously described (54, 56). HeLa expressing the nondegradable IB Mut inhibitor in Tet-Off (TtA) cells were described earlier (55, 56). Clonal HeLa cells stably expressing the enhanced green fluorescent protein (EGFP)-RelA fusion protein (33) were selected in G418 (250 µg/ml). In all experiments, unless specifically noted, cells were serum starved in Dulbecco's modified Eagle's medium 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 concentrations for 1 h prior to TNF stimulation. For RelA site mutations, eukaryotic expression vector encoding FLAG epitope-tagged RelA was generated by ligating the monomeric strawberry cDNA (47) into a pcDNA3-FLAG backbone, generating the plasmid pcDNA-FLAG-Straw. Wild-type RelA (RelAWT) and a RelA (Ser²⁷⁶Ala) site mutation were generated by rolling-circle mutagenesis and cloned into pcDNA-FLAG-Straw. The transcription unit containing the cytomegalovirus promoter and FLAG-mStraw-RelA coding sequences was excised as a BglII/XbaI fragment and ligated into pEF6/V5 plasmid digested with BglII/XbaI, producing pXFS-RelA. The pXFS plasmids confer blasticidin resistance and were used to generate stable transfectants in a RelA^–/– background. For this purpose, RelA^–/– murine embryonic fibroblasts (MEFs) (a gift of D. Baltimore [7]) were transfected with 8 µg of pXFS-RelA and 24 µl of Targefect-MEF transfection reagent (Targeting Systems Inc.) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After 24 h, cells were subdivided and stable transfectants selected by the addition of blasticidin S (10 µg/ml) for 3 weeks. Pools of cells were confirmed by fluorescence microscopy to have similar levels of Straw fluorescence, and amounts of protein expression were confirmed by Western immunoblotting.

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

Western immunoblots. For measurement of phospho-Ser²⁷⁶ RelA formation, whole-cell extracts (WCEs) were prepared by lysis in 1x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Protein from 3 x 10⁶ cells was fractionated by SDS-PAGE. For measurement of NF-B subunit nuclear translocation, 75 µg of sucrose cushion-purified nuclear protein was fractionated by SDS-PAGE. Gels were electrotransferred to polyvinylidene difluoride membranes (Millipore) overnight and blocked with 5% nonfat dried milk in Tris-buffered saline (TBS)-0.1% Tween 20. Anti-RelA, -RelB, and -C-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-rabbit immunoglobulin G [IgG] or IRD680 anti-mouse IgG; Rockland) were diluted 1:5,000 in TBS-Tween 0.1% and incubated for 1 h at 22°C, and the immune complexes were visualized by near-infrared fluorescence (Odyssey imaging system; LiCor BioSciences).

Gene expression analysis. Quantitative real-time PCR (Q-RT-PCR) was performed with SYBR green I by use of a MyiQ single-color RT-PCR detection system and iQ SYBR green supermix (Bio-Rad, Hercules, CA) according to the manufacturer's protocols. First-strand cDNA was prepared with an iScript cDNA synthesis kit (Bio-Rad). cDNA (1 µl) was added to a reaction master mix (20 µl) containing 2.5 mM MgCl₂, HotStart Taq DNA polymerase, SYBR green I, 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 conditions consisted of a hot-start activation of HotStart Taq DNA polymerase (94°C, 15 min) and 40 cycles of denaturation (95°C, 15 s), annealing (55 to 58°C, 30 s), and extension (72°C, 30 s). Formation of PCR product was monitored in real time by measuring 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-β, 5'-AGAATGGGCAGAAAGCTTGTCT-3' and 5'-CAGCATCTTTTCGATGATTTTCTTAA-3'; for the IB 3' untranslated region, 5'-TGCCTAGCCCAAAACGTCTT-3' and 5'-CGTCCCCTACAAAAAGTTCACAA-3'; for IB coding sequences, 5'-CTCCGAGACTTTCGAGGAAATAC-3' and 5'-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 transcripts were as follows: for mGro-β, 5'-CACTCTCAAGGGCGGTCAA-3' and 5'-TGGTTCTTCCGTTGAGGGAC-3'; and for mIB, 5'-TCCTGCACTTGGCAATCATC-3' and 5'-AGCCAGCTCTCAGAAGTGCC-3'. Relative gene expression was determined using the 2^–C_T method (56). The mean threshold cycle (C_T) of triplicate measures was computed for each sample. The sample mean C_T of cyclophilin (internal control) was subtracted from the sample mean C_T of the respective gene of interest (C_T). The mean C_T of the control (untreated) sample was selected as a calibrator and subtracted from the mean C_T of each experimental sample (C_T). The severalfold change in gene expression (normalized to the internal control gene and relative to the control sample) was expressed as 2^–C_T.

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 monoclonal Abs for phosphorylated RNA Pol II were from Covance Research Products. Monoclonal Ab-chromatin complexes were captured with a rabbit anti-mouse IgM Ab (Rockland) prior to binding to protein A magnetic beads (38). DNA binding was detected by qualitative PCR using primers for IL-8, Gro-β, IB, Naf1, NF-B2, and TRAF1 as described previously (39, 56). The primers used for TRAF1 were 5'-GATGTGCCCAGCGAAGTGG-3' and 5'-TGAGTCACAGCAGGGATGGAG-3'. For Q-RT-PCR, the primers used were as follows: for Gro-β, 5'-TCGCCTTCCTTCCGAACTC-3' and 5'-CGAACCCCTTTTATGCATGGT-3'; and for Naf1, 5'-GGTCTAGGAAATCCCAGTCTGTTG-3' and 5'-CGGGTGGGCAAATCCA-3'.

Intracellular ROS assay. Changes in intracellular ROS were determined as described previously (26). Briefly, cells were suspended in phosphate-buffered saline (PBS) and loaded with 5 µM of 5 (and 6)-carboxy-2',7'-dichlorodihydro-fluorescein diacetate (H2DCF-DA; Molecular Probes) for 15 min at 37°C. After removal of excess H2DCF-DA, cells were TNF treated, and changes in DCF fluorescence were determined by flow cytometry (FACScan; Becton Dickinson). The fluorescences for 12,000 cells from three or more independent experiments were analyzed and expressed as means ± standard errors of the means.

Changes in levels of carbonylated proteins were determined as described previously (31). Briefly, TNF-stimulated HeLa cells were lysed in radioimmunoprecipitation assay buffer containing 5 mM dithiothreitol and protease inhibitors. The carbonyl groups in the side chains of proteins (20 µg) were derivatized to 2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophenylhydrazine. The reaction was neutralized with neutralization buffer provided by the Chemicon OxyBlot assay kit. The 2,4-dinitrophenyl-derivatized proteins were fractionated on an SDS-10% polyacrylamide gel and proteins transferred to polyvinylidene difluoride membranes (Amersham, Inc.). After transfer, the membranes were blocked 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 added for 1 h. Detection was performed by enhanced chemiluminescence (ECL; Amersham, Inc.).

RelA-CDK-9 colocalization analysis. HeLa S3 cells stably expressing the EGFP-RelA fusion protein were plated on glass coverslips. Once the cultures reached 50 to 70% confluence, individual coverslips were exposed to TNF for 0 (control), 30, and 60 min in triplicate and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Immunofluorescence staining was done using rabbit anti-CDK-9 Ab as the primary Ab (1:100) and the Alexa Fluor 568 F(ab')₂ fragment of goat anti-rabbit IgG (heavy plus light chains) (Invitrogen, Eugene, OR) as the secondary Ab (5 µg/ml). Then, the cells were counterstained with 500 nM TO-PRO-3 (Invitrogen) and mounted on glass slides by use of Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluorescence imaging was performed with a Zeiss LSM510META confocal microscope with a Plan-Apochromat 63x 1.4-numerical-aperture oil immersion lens. The GFP, Alexa 568, and TO-PRO-3 image components were obtained using 488-nm (argon ion), 543-nm (green He/Ne), and 633-nm (red He/Ne) laser excitation lines; the corresponding emission collection ranges were 505 to 530 nm, 560 to 620 nm, and longer than 650 nm, respectively. At the image collection settings 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 µm and a pixel size of 0.1 µm, respectively. Images were acquired using a bit depth of 12- and 16-frame Kallman averaging. At least five fields were captured per coverslip.

For image processing and analysis, the .lsm files generated by the Zeiss confocal 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) was used for background correction and image segmentation and to quantify translocation. The TO-PRO-3 stain was used to create segmentation masks separating individual nuclei. Dilation of these masks was used to select perinuclear cytoplasmic rings for each cell. Average intensity measurements within each nuclear region and the corresponding cytoplasmic ring were used to quantify RelA translocation. To quantify colocalization, each nucleus was analyzed separately. First, an intensity threshold was used to exclude the nucleolus, an area which showed low levels of both RelA and CDK-9 stains. Each masked nuclear image was exported to Imaris (Bitplane, Zurich, Switzerland) and the degree of colocalization between RelA and CDK-9 was assessed using the "colocalization analysis module." This module uses correlation analysis first to test the significance of true colocalization over random color overlap and then to quantify the degree of colocalization by use of an automatic threshold search algorithm to avoid the bias of visual interpretation. The algorithm used in this software was described previously (13). Results are expressed as percentages of EGFP colocalized with the Alexa-568 signal.

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

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Kinetics of NF-B-dependent gene transcription. To explore the genetic networks under NF-B control, we have developed a tetracycline (Tet)-regulated cell system (Tet-Off [18]) in which a nondegradable FLAG epitope-tagged IB (IB Ser³²Ala/Ser³⁶Ala, termed FLAG-IB Mut) under control of the Tet operator was stably introduced into cells expressing the Tet transactivator (tTA). FLAG-IB Mut contains site mutations in the serine phosphoacceptor sites of IKKβ that block its inducible degradation and functions as a potent dominant-negative inhibitor of the canonical NF-B activation pathway (11, 55). In these cells, doxycycline (Dox) inhibits FLAG-IB Mut expression; upon Dox withdrawal, FLAG-IB Mut expression is strongly upregulated (Fig. 1A). To confirm that FLAG-IB Mut expression was inert to TNF-induced proteolysis and can inhibit NF-B activation, a time course of TNF stimulation was performed. In cells cultured in the continuous presence of Dox, little detectable FLAG-IB Mut is detected (Fig. 1B). Within 15 min of TNF stimulation, endogenous IB is rapidly proteolyzed to undetectable levels, and by 60 min, endogenous IB is resynthesized. In contrast, upon Dox withdrawal, FLAG-IB Mut is strongly expressed at amounts similar to those for endogenous IB and does not degrade in response to TNF (Fig. 1B, right).

View larger version (44K): [in this window] [in a new window]   FIG. 1. Dox-regulated NF-B-dependent gene expression. (A) Tet-regulated IB Mut expression. HeLa Tet-Off cells expressing FLAG epitope-tagged IB Mut (Ser32Ala/Ser36Ala) were cultured in the presence (+) or absence (–) of 2 µg/ml Dox. Cytoplasmic extracts were extracted and assayed by Western blotting. (Top) Anti-FLAG Ab; (bottom) β-actin used as a loading control. (B) IB Mut is TNF resistant. Tet-regulated FLAG-IB Mut cells cultured in the presence or absence of Dox were TNF stimulated for the indicated times (min). (Top) Cytoplasmic extracts were assayed for IB abundance using anti-IB Ab. The endogenous and IB Mut isoforms are shown. (Bottom) β-Actin staining. (C) NF-B binding in EMSA. Nuclear extracts from the experiment shown in panel B were assayed for NF-B binding using EMSA. Shown are the bound complexes. The RelA/NF-B1 (p50)-containing complex is indicated. (D) Supershift assay in EMSA. EMSA was 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 translocation dependent. RNA from a time series of TNF-stimulated HeLa IB Mut cells was analyzed by Q-RT-PCR. (The IB primers were designed to hybridize selectively to the 3' untranslated region of the endogenous gene and do not detect IB Mut expression.) Shown are mRNA expression profiles in the presence or absence of Dox. For each gene, change is determined relative to unstimulated values.

The effects of FLAG-IB Mut on TNF-induced endogenous gene expression was measured for four NF-B-dependent genes (the Gro-β, IL-8, IB, and Naf-1 genes) by use of Q-RT-PCR. In the presence of Dox, TNF stimulation rapidly induces IL-8, Gro-β, and IB gene expression, peaking within 30 min to 1 h after stimulation at 900-fold, 23-fold, and 22-fold, respectively, over the control level (Fig. 1E). In the absence of Dox, the TNF-induced mRNA expression is almost completely abrogated, 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 also NF-B dependent.

Kinetics of NF-B subunit recruitment to the target genes in chromatin. To better characterize the kinetics of NF-B recruitment on individual promoters within their native chromatin context, we developed a highly efficient two-step ChIP assay that first immobilizes RelA by N-hydroxysuccinimide ester-mediated protein-protein cross-linking prior to a second formaldehyde-mediated protein-DNA cross-linking step (39). The protein cross-linking step was necessary because fluorescence photobleaching experiments of NF-B interacting with its multimeric binding sites in vivo have shown that RelA is in rapid exchange with its target DNA, being completely replaced within 30 s (8). Although we earlier demonstrated that this assay was robust for distinct NF-B isoforms and captured targets at levels greater than seen for IgG controls (39), the performance of this modified ChIP assay to enrich for RelA binding to target genes was further examined. First, to establish whether the two-step ChIP specifically measured RelA binding to target genes containing NF-B sites, chromatin from 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 reversal of the cross-links, the DNA was subjected to PCR using primers 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 RelA spanning nucleotides –1042 to –826 [IL-8 (Dist) (9, 38)], and primers spanning an unrelated U6 snRNA spanning nucleotides –245 to +85, an RNA Pol III-driven gene. We observed specific PCR products only in the IL-8 (Prox) samples spanning the high-affinity RelA site, with a faint signal being detected at time zero and a strong induction 30 and 60 min after TNF administration relative to what was seen for input DNA used as a loading control (Fig. 2A). Specificity was indicated by a lack of signal produced by the IL-8 (Dist) and U6 primers (Fig. 2A).

View larger version (69K): [in this window] [in a new window]   FIG. 2. Recruitment kinetics of NF-B/RelA. (A) Specificity of ChIP assay. Two-step ChIP assay was performed on a time series of TNF-stimulated cells (39). Chromatin was subjected to IP using anti-RelA Ab. Eluted DNA was subjected to PCR using primers spanning the high-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-8 not 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 the IL-8 (Prox) primers produce a TNF-inducible signal. (B) EMSA of TNF-stimulated cells. HeLa cells were exposed to either a 15-min pulse or continuous (tonic) stimulation. Nuclear extracts were subjected to EMSA using a high-affinity RelA-NF-B1 binding site (10). Shown is an autoradiograph of the specifically bound species. (Top) Pulse stimulation; (bottom) tonic stimulation. (C) ChIP of tonic versus pulse TNF stimulation protocols. HeLa cells were stimulated for the indicated times after 15-min pulse (top) or tonic (bottom) TNF stimulation. Chromatin was 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 chromatin immunoprecipitated with anti-RelA Ab. Shown is an ethidium bromide-stained gel for PCR of the IL-8, IB, Gro-β, Naf1, NF-B2, and TRAF1 genes 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 positive control (PC). The negative control (NC) for the IP was IgG.

The two-step ChIP was applied to chromatin prepared from cells induced by the two different stimulation protocols. The cross-linked chromatin was immunoprecipitated using anti-RelA Ab, and Gro-β binding was detected by qualitative PCR. In response to pulsatile TNF stimulation, we observed that RelA binding was rapidly induced at 15 to 30 min, whereupon it dissociated from the Gro-β promoter coincidentally with its cytoplasmic recapture (Fig. 2C, top). By contrast, in response to tonic TNF stimulation, RelA binding to Gro-β was stable over the entire time course (Fig. 2C, bottom). These findings indicated that the two-step ChIP could detect the association and dissociation of RelA binding with its endogenous gene targets and that there was no detectable lag in RelA binding between RelA's appearance in the nucleoplasm (detected by EMSA) and its association with chromatin (detected by ChIP) on the Gro-β promoter. This experimental result is further compatible with previous findings that nuclear RelA is in rapid exchange with its target sites in chromatin (8). Finally, this result validated that the modified two-step ChIP was capable of measuring both the association and the dissociation of RelA on its endogenous binding sites.

We next sought to determine RelA binding kinetics on target genes showing a rapid transcriptional spike. Using qualitative PCR, we could detect changes in target DNA over a concentration range of 0.2 to 20 ng, as shown for IL-8, Gro-β, and IB (see Fig. S1 in the supplemental material). For all genes examined, we found that the kinetics of TNF-induced RelA binding was rapid and was maximal within 15 to 30 min after stimulation (Fig. 2D). Interestingly, for genes that are strongly induced by TNF, such as the IL-8, IB, and Gro-β genes, as well as genes that are not, such as the Naf1, NF-B1, and TRAF2 genes, RelA binding was rapidly inducible, peaking 15 min after stimulation. Moreover, the binding profiles were indistinguishable in nature for the duration of the stimulation. To confirm these findings, we measured RelA-associated target genes by use of Q-RT-PCR, where a three- to fourfold change in RelA binding for Gro-β and Naf1 was observed at identical times (see Fig. S2 in the supplemental material). These findings were surprising because previous work has suggested that RelA is occluded from accessing chromatin sites in late-response genes in monocytes (45).

Also of note is that RelA binding to the IL-8, IB, and Gro-β promoters was persistent over the 9-h time course, even though mRNA expression by these genes was terminating (cf. Fig. 1E and 2D). These findings suggested to us that the differential patterns of NF-B-dependent gene expression were not due to different rates of RelA association.

Promoter-specific phospho-Ser²⁷⁶ RelA recruitment. Because the binding of bulk RelA did not accurately track with the kinetics of transcriptional activation (e.g., Naf1, TRAF1, and NF-B2) or termination (IL-8, IB and Gro-β), we next examined the kinetics of formation and chromatin binding of phospho-Ser²⁷⁶, as this phosphorylation results in a potent activating species required for IL-8 gene induction (26, 59). Western blots were performed on a time series of TNF-stimulated cells extracted by denaturing lysis using a phospho-Ser²⁷⁶ RelA-specific Ab (Fig. 3A). In unstimulated cells, low amounts of phospho-Ser²⁷⁶ RelA could be detected, a form which migrates aberrantly at 80 kDa, consistent with the findings of others (59, 60). TNF induced rapid formation of phospho-Ser²⁷⁶ RelA, peaking 15 to 30 min later at 3-fold induction, which rapidly returned to unstimulated levels 1 h after stimulation (Fig. 3A). To determine whether this isoform translocated into the nucleus, Western blots of cytoplasmic and sucrose cushion-purified nuclear extracts were performed. Phospho-Ser²⁷⁶ RelA was highly enriched in the nuclear fractions, with little detectable cytoplasmic staining (Fig. 3B). The kinetics of nuclear accumulation parallel those of the whole-cell lysate. Together, these data indicate that phospho-Ser²⁷⁶ RelA is rapidly translocated into the nucleus.

View larger version (40K): [in this window] [in a new window]   FIG. 3. (A) Time course of phospho-Ser276 RelA formation. WCEs prepared from a time course of TNF-stimulated cells were fractionated by SDS-PAGE and blotted with anti-phospho-Ser276 RelA (top), anti-RelA (middle), and β-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 into cytoplasmic and nuclear fractions. (Top) Western immunoblotting using anti-phospho-Ser276 RelA Ab. (Middle) Blot was reprobed for RelA as a loading control. Note the inducible accumulation of RelA in the nuclear fraction within 15 min of stimulation. (Bottom) β Lamin staining as a nuclear fraction control. The nuclear fractions stain strongly for β 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 course of phospho-Ser276 RelA binding in A549 cells. Experiment is as described for panel C. A549 cells show similar patterns of phospho-Ser276 RelA recruitment.

To determine whether distinct differential phospho-Ser²⁷⁶ RelA recruitment was cell type dependent, we performed the same experiment on TNF-stimulated type II alveolar epithelial cells (A549). As seen in Fig. 3D, similar results were obtained, with an apparent peak in phospho-Ser²⁷⁶ RelA binding to IL-8 and IB 15 to 30 min after stimulation and binding to Naf1 and TRAF1 3 h later. The mechanisms controlling the expression of late genes will require further study. For the purposes of this work, we sought to evaluate the role of phospho-Ser²⁷⁶ RelA in the expression of the rapidly inducible genes because phospho-Ser²⁷⁶ RelA binding was coincident with gene expression.

Requirement of phospho-Ser²⁷⁶ binding in NF-B-dependent gene expression. Previous work in our laboratory has shown that a TNF-induced ROS signaling pathway mediates the phosphorylation of Ser²⁷⁶ on RelA in monocytic cells (26). Here, intracellular ROS activate IB-RelA-associated PKAc to phosphorylate RelA selectively on Ser²⁷⁶. We further showed that phospho-Ser²⁷⁶ RelA formation can be selectively disrupted by a variety of chemically unrelated antioxidants, including the ROS scavenger DMSO, an agent that inhibits PKAc but does not inhibit TNF-induced Jun N-terminal protein kinase activation or mitogen-stress related kinase or induce cellular toxicity (26).

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

View larger version (31K): [in this window] [in a new window]   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%). Fluorescence measurements were performed using a fluorescence-activated cell sorter at indicated times. Symbols: , TNF; , TNF plus 2% DMSO; , TNF plus 0.5% DMSO; •, untreated. TNF induces detectable ROS formation within 15 min of stimulation. (Inset) Histograms of fluorescence-activated cell sorter analysis at 0, 30, and 60 min of treatment. (B) Kinetics of TNF-induced protein carbonylation. HeLa cells were stimulated with TNF for the times indicated. Shown is a Western blot for carbonylated proteins. Inducible carbonylation is seen within 15 min of stimulation (indicated by arrow). (C) Selective inhibition of TNF-induced phospho-Ser276 RelA formation by DMSO. HeLa cells were stimulated with TNF for 30 min the 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 total RelA recruitment to target promoters. HeLa cells were stimulated for indicated times in the absence or presence of DMSO. Shown is a two-step ChIP 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) Effect of 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 an internal control. **, P < 0.01, t test. (F) Inhibition of TNF-induced phospho-Ser276 RelA formation by NAC. HeLa cells were stimulated with TNF for 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 absence or 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 blocks phospho-Ser276 RelA recruitment. HeLa cells were TNF stimulated for the indicated times in the absence or presence of DMSO. Shown is a two-step ChIP assay using anti-phospho-Ser276 RelA Ab as the immunoprecipitating Ab. DMSO completely blocks TNF-inducible phospho-Ser276 RelA binding.

To exclude the possibility that DMSO interfered with bulk NF-B binding to its sites in chromatin, ChIP assays were performed on a time course of TNF-stimulated cells in the presence or absence of DMSO by use of an anti-RelA Ab that binds both phosphorylated and nonphosphorylated ("bulk") RelA (Fig. 4D). Again, TNF stimulation alone induced robust bulk RelA binding. Importantly, bulk RelA binding was not affected in the presence of DMSO (Fig. 4D, right). From these data, we concluded that inhibition of the ROS pathway has no effect 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 an examination of the role of phospho-Ser²⁷⁶ RelA in NF-B-dependent gene expression. For this purpose, TNF-induced expression of Gro-β, IB, and IL-8 mRNA was determined by Q-RT-PCR in the absence or presence of DMSO. We found that DMSO completely inhibits TNF-inducible Gro-β and IL-8 expression but does not affect IB expression (Fig. 4E). These data suggest that Gro-β and IL-8 require phospho-Ser²⁷⁶ RelA for inducible gene expression but IB does not.

To confirm these observations using a chemically unrelated antioxidant, we performed TNF stimulations using NAC at concentrations that inhibited TNF-induced DCF oxidation (58). NAC blocked phospho-Ser²⁷⁶ formation (Fig. 4F) and significantly inhibited Gro-β and IL-8 gene expression without affecting IB (Fig. 4G).

To confirm that DMSO reduced phospho-Ser²⁷⁶ RelA binding, two-step ChIP was conducted using a phospho-Ser²⁷⁶ RelA Ab. In this experiment, we plated the cells at a twofold increase in cell density to maximize the detection of phospho-Ser²⁷⁶ RelA signal by the two-step ChIP assay. As a result, a basal signal of phospho-Ser²⁷⁶ RelA binding could be detected for Gro-β and, to a lesser extent, for IL-8 in unstimulated cells due to the increase in DNA input into the PCR (Fig. 4H). Moreover, for reasons not yet completely understood, the kinetics of phospho-RelA binding were slightly delayed by 15 min relative to the experimental results shown earlier in Fig. 3C, suggesting that the kinetics of ROS formation are cell density dependent. Nevertheless, and most importantly, inducible phospho-Ser²⁷⁶ RelA binding to Gro-β, IL-8, and IB was completely inhibited (Fig. 4H). These data indicated that the inhibition of the ROS pathway selectively ablates the phospho-Ser²⁷⁶ RelA formation and recruitment of phospho-Ser²⁷⁶ RelA, but not bulk RelA, to target genes. Taken together, 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 the expression of a subset of NF-B-dependent genes, including the Gro-β and IL-8 genes.

RelA Ser²⁷⁶ mediates differential patterns of gene expression. To further determine the requirement of RelA Ser²⁷⁶ phosphorylation in the activation of a subset of NF-B-dependent genes, we examined the effect of expressing a RelA Ser-Ala site mutation at residue 276 (RelA Ser²⁷⁶Ala). RelA^–/– MEFs were transfected with FLAG epitope-tagged strawberry-RelAWT or RelA Ser²⁷⁶Ala expression vectors and pools of stable transfectants isolated. Cells were then TNF stimulated for a 6-h time course, and Q-RT-PCR was performed. We selected for measurement the mouse homologs of the Gro-β and IB genes (note that mice do not have an IL-8 gene). Although RelAWT potently transactivated the mGro-β gene, the RelA Ser²⁷⁶Ala mutant did not (Fig. 5). Conversely, both proteins strongly transactivated the mIB gene by 5-fold. These data confirmed our earlier findings that phospho-Ser²⁷⁶ RelA controls a subset of NF-B gene expression.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Effect of RelA Ser276Ala mutation on target gene expression. RelA–/– MEFs were transfected with expression vectors encoding a FLAG epitope-tagged monomeric strawberry fusion with RelAWT or RelA Ser276Ala, and pooled transfectants were isolated. Cells were stimulated for indicated times with TNF, and mRNA was extracted. Q-RT-PCR was performed to determine the activation of endogenous NF-B-dependent genes.

View larger version (47K): [in this window] [in a new window]   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-RelA were stimulated in the absence (control [Con]) or presence of TNF (30 min). Cells were fixed and stained with rabbit anti-CDK-9 Ab and Alexa Fluor 568-labeled goat anti-rabbit IgG. Nuclei were counterstained with TO-PRO-3 and are shown as overlays with differential interference contrast. (Bottom left) Sequential stages in image analysis with masking and colocalized pixels; (bottom right) corresponding two-dimensional histogram of EGFP-RelA and CDK-9 correlation. Shown is least-squares correlation analysis and the extent of colocalization for this image. After analysis 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 presence of DMSO as indicated. RelA-associated complexes were immunoprecipitated using anti-FLAG Ab, and the association of CDK-9 was detected by immunoblotting. (Bottom) IB of FLAG-RelA as a recovery control for the immunoprecipitation (IP). CDK-9-RelA complex association is TNF inducible and is reduced by DMSO treatment. (D) Coimmunoprecipitation assay for RelA-P-TEFb complex formation. HeLa cells were stimulated with TNF for 0 and 0.5 h in the absence or presence of antioxidant (DMSO). Nuclear extracts were prepared and immunoprecipitated with indicated 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-TEFb complex is quantitatively prevented by DMSO treatment. (E) Inducible P-TEFb binding. HeLa cells were stimulated with TNF for 0, 0.25, and 0.5 h in 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-β, and IB as indicated (left). TNF-inducible recruitment of each member of the pTEF complex is rapid and indistinguishable. Inducible P-TEFb recruitment is significantly inhibited by DMSO.

To examine whether phospho-Ser²⁷⁶ RelA associated with CDK-9, nondenaturing coimmunoprecipitation experiments were performed. Control or TNF-stimulated nuclear extracts were immunoprecipitated with anti CDK-9 Ab, and the presence 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 comigrated with the 80-kDa species that was produced by anti-phospho-Ser²⁷⁶-RelA Ab (Fig. 6C and reference 12). The binding of phospho-RelA to Ccn T1 and CDK-9 in the presence of TNF stimulation was significantly inhibited by the presence of DMSO (Fig. 6D). Together, these data suggest that the TNF-inducible Ser²⁷⁶ phosphorylation of RelA is required for association with the P-TEFb Pol II CTD kinase.

TNF-induced P-TEFb recruitment to target genes is dependent on phospho-Ser²⁷⁶ RelA formation. To determine whether TNF induced P-TEFb recruitment on NF-B-dependent genes, and if so, whether this recruitment required Ser²⁷⁶-phosphorylated RelA, a time course experiment using a ChIP assay of TNF stimulation in the presence or absence of DMSO was conducted. As seen in Fig. 6E, TNF-inducible CDK-9 and Ccn T1 binding was observed for IL-8, Gro-β, and IB. Here, P-TEFb binding occurred rapidly, within 15 min, at times coincident with target gene expression. Importantly, TNF-inducible CDK-9 and Ccn T1 binding was significantly inhibited in cells TNF stimulated in the presence of DMSO. Together, these data indicate that the P-TEFb complex is inducibly recruited to target promoters in a phospho-Ser²⁷⁶ RelA-dependent manner.

TNF-induced NF-B-dependent gene expression requires CDK activity. To further establish the functional role of CDK in NF-B-dependent gene expression, we examined the effect of the CDK inhibitor FP (48). FP is a highly selective CDK inhibitor that potently inhibits the kinase activity of CDK-9 (37). In this experiment, vehicle- or FP (500 nM)-pretreated cells were stimulated with TNF, and the induction of NF-B-dependent genes was measured by Q-RT-PCR. FP completely blocked the expression of IL-8 and Gro-β (Fig. 7A).

View larger version (17K): [in this window] [in a new window]   FIG. 7. CDK inhibitor FP inhibits NF-B-dependent gene expression by disrupting Pol II phospho-Ser2 CTD formation. (A) Gene expression HeLa cells were pretreated with vehicle or FP (500 nM) for 1 h prior to 30 min of TNF stimulation. mRNA expression was measured by Q-RT-PCR. Shown is change in mRNA normalized by 18S relative to unstimulated values. (B) FP inhibits P-TEFb-induced phospho-Ser Pol II CTD formation. HeLa cells were pretreated with vehicle 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 immunoprecipitating Ab; (bottom) anti-phospho-Ser Pol II Ab was used. Although FP has no effect on inducible RelA binding, it significantly inhibits phospho-Ser Pol II recruitment.

CDK-9 subunit is required for optimal expression of Gro-β/IL-8 genes. To more specifically implicate the role of CDK-9 in NF-B-dependent gene expression, we conducted a series of experiments using siRNA knockdown. Relative to those transfected with control, cells transfected with CDK-9 siRNA showed a reduction of 50% in normalized CDK-9 steady-state levels (Fig. 8A). Under these conditions of CDK-9 knockdown, a time course of TNF stimulation was performed and gene expression measured by Q-RT-PCR. In contrast to what was seen for control transfectants, we observed a significant reduction in the peak expression of Gro-β and IL-8 mRNA but not in that of IB (Fig. 8B). We interpret this result to mean that CDK-9 is an important mediator of phospho-Ser²⁷⁶ RelA-dependent gene expression.

View larger version (16K): [in this window] [in a new window]   FIG. 8. Effect of siRNA-mediated knockdown of CDK-9 on NF-B-dependent gene expression. (A) Magnitude of siRNA-mediated knockdown. Cells were transfected with control (Con) or anti-CDK-9 siRNA prior to lysis and analysis of CDK-9 expression by Western blotting. Duplicate plates are shown for reproducibility. (Top) Staining with anti-CDK-9 Ab; the specific 42-kDa band is shown. (Bottom) Blot was reprobed with anti-β-actin Ab as a loading control. When normalized to β-actin, CDK-9 abundance is downregulated by 50%. (B) Effect of CDK-9 knockdown on inducible gene expression. Cells were transfected with control or CDK-9-targeted siRNA for 72 h prior to TNF stimulation for the indicated times. mRNA expression of early genes was measured by Q-RT-PCR. Shown is change in mRNA normalized by 18S relative to unstimulated values. *, P < 0.01, two-tailed t test.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NF-B subunits are nuclear phosphoproteins responsive to diverse signaling pathways (35). Specifically, the potent RelA-transactivating subunit can be phosphorylated at multiple sites, including Ser²⁷⁶ by PKAc (26, 60) and mitogen and stress kinase 1 (57) and Ser⁵³⁶ by IKKβ, NK-B-inducing kinase, and c-Src (28, 46). These phosphorylation events lead to an increase in RelA nuclear translocation, DNA binding, and/or transcriptional activity, through the mechanisms not completely understood. Of relevance to this study is the fact that RelA Ser²⁷⁶ phosphorylation occurs upon the activation of the canonical NF-B pathway and cytoplasmic release from IB. Although the relative roles of PKAc and mitogen and stress kinase 1 have yet to be fully understood, we have recently observed that PKAc mediates TNF-induced phospho-Ser²⁷⁶ RelA formation via an intracellular ROS signal (26). This second ROS-PKAc pathway is completely independent of that controlling RelA translocation and is necessary for the acquisition of full transcriptional activity (26). The consequence of Ser²⁷⁶ phosphorylation is thought to be a destabilization of the intermolecular association of the NH₂- and COOH-terminal ends of RelA (60), producing a conformational change that may produce a more stable association with CBP/p300 and other coactivators (60). As a result, phospho-Ser²⁷⁶ RelA becomes a substrate for acetylation, resulting in enhanced transcriptional activity (12).

Our findings extend the range of protein interactions involving activated RelA, as we have observed that Ser²⁷⁶ phosphorylation is also required for interaction with the P-TEFb complex. This finding is important because it suggests that phospho-Ser²⁷⁶ RelA binding can mediate transcriptional elongation by recruiting P-TEFb to target genes as an additional mechanism for inducible gene expression over that mediated by p300/CBP recruitment. Importantly, the requirement for P-TEFb recruitment is not a uniform feature of NF-B-dependent gene activation. Rather, our data suggest that phospho-Ser²⁷⁶ RelA plays only a restricted role in the activation of two NF-B-dependent targets, the IL-8 and Gro-β genes (but not the IB gene). Our ChIP studies indicate that the IL-8 and Gro-β genes are highly inducible through a mechanism involving Pol II recruitment, whereas the IB gene is one whose magnitude of expression is not as highly inducible and is stably engaged with Pol II in the absence of TNF stimulation (Fig. 6A). Previously, using genomic footprinting of the IL-8 gene by ligation-mediated PCR, we found that NF-B activation produced dramatic modifications of the proteins binding the TATA box and transcriptional initiation site, suggesting that NF-B activated this gene by a promoter recruitment mechanism (9). Here, we extend this mechanism to include both Pol II and P-TEFb recruitment. In response to CDK-9-mediated CTD phosphorylation, recruited Pol II is able to produce full-length transcripts. In the case of the IB gene, others have shown that the gene is preloaded with RNA polymerase, making it able to rapidly respond to NF-B binding without needing time-dependent formation of a preinitiation complex (1). Together, these studies indicate that NF-B activates its downstream gene targets through pleiotropic mechanisms.

By comparing the kinetics of bulk RelA binding and those of phospho-Ser²⁷⁶ RelA binding to genomic targets, this study represents the first demonstration that there is a dissociation in the timing between phospho- and non-phospho-RelA recruitment. Specifically, our data show that phospho-Ser²⁷⁶ RelA binding is the functionally important complex in Gro-β and IL-8 gene expression because (i) phospho-Ser²⁷⁶ RelA binding temporally coincides with peak transcriptional activation and peak steady-state mRNA abundance, (ii) inhibition of phospho-Ser²⁷⁶ formation by antagonism of the ROS pathway selectively blocks phospho-RelA (but not bulk RelA) binding and transcriptional activation of IL-8/Gro-β, and (iii) a phosphorylation-deficient Ser-to-Ala site mutation at residue 276 is unable to efficiently transactivate Gro-β in a RelA-deficient MEFs. Others have that shown Ser²⁷⁶ phosphorylation was necessary for a strong response to the NF-B-inducing stimuli TNF and IL-1 (2, 12, 60). It is possible that the size or shape of this multiprotein complex or the nature of chromatin where the NF-B binding site is found may restrict the access of the phospho-Ser²⁷⁶ RelA complex to certain genes. It will be interesting to compare protein complexes formed by phospho-Ser²⁷⁶ RelA and nonphosphorylated RelA.

Although the Ser²⁷⁶ RelA complex plays a key functional role in IL-8 and Gro-β gene expression, it is not required for IB expression, even though our ChIP assays show that phospho-Ser²⁷⁶ RelA rapidly and inducibly binds to this promoter. This conclusion is made based on the ability of hypo-Ser²⁷⁶-phosphorylated RelA to transactivate IB after the inhibition of the ROS-PKAc pathway, the ability of the RelA Ser²⁷⁶Ala mutant to transactivate IB in RelA^–/– MEFs, and the lack of effect of CDK-9 downregulation on IB expression. Therefore, although we observed an induction of phospho-Ser²⁷⁶ RelA binding to IB in the ChIP assay, this RelA modification and P-TEFb recruitment were not required to activate productive transcription. In this regard, we note earlier studies that have shown phospho-defective RelA at residues Ser²⁰⁵, Ser²⁷⁶, and Ser²⁸¹ are unable to mediate a subset of NF-B-dependent genes (the ICAM-1, VCAM-1, and MIP-2 genes) in response to lipopolysaccharide and gamma interferon, whereas induction of two other NF-B-dependent genes, the major histocompatibility complex class I and Mn superoxide genes, are essentially unaffected (2). These data suggest to us that a phosphorylation code controls NF-B's ability to activate subnetworks of target genes, where distinct groups are induced by particular forms of phospho-RelA and the protein complexes that they form. Elucidation of the complete spectrum of genes under phospho-Ser²⁷⁶ RelA-dependent 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 factor mediating human immunodeficiency virus TAT-dependent transactivation (5) and the activation of heat shock genes in Drosophila melanogaster (37). P-TEFb mediates transcriptional elongation by its ability to phosphorylate several targets in the paused Pol II-dependent promoter, including serine 2 in the hept