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• W2056512813 abstract "Phosphorylation of NF-κB plays an important role in modulating transcriptional activity of NF-κB independently of inhibitor of κB (IκB) proteins. For the p65 subunit, multiple phosphorylation sites have been mapped in and adjacent to both the N-terminal Rel homology domain and the C-terminal transactivation domain. Their impact on NF-κB-dependent transcription, however, has never been assessed at a broader level. In this study, we evaluate the importance of differential p65 phosphorylation on four serine acceptor sites in the Rel homology domain for the expression of an array of NF-κB-dependent genes in endothelial cells. We find that inhibition of p65 phosphorylation on these serine residues targets NF-κB activity to distinctive gene subsets in a κB enhancer element-specific context. We show that the phosphorylation-dependent alterations in gene and protein expression are reflective of the amount of p65 and phosphorylated RNA polymerase II (p-RNAP II) bound to respective gene promoter regions. Depending on the gene subset, impaired gene expression was either a result of decreased p65 promoter recruitment or of a failure of bound p65 to recruit p-RNAP II. In conclusion, our findings demonstrate that site-specific p65 phosphorylation targets NF-κB activity to particular gene subsets on a global level by influencing p65 and p-RNAP II promoter recruitment. Phosphorylation of NF-κB plays an important role in modulating transcriptional activity of NF-κB independently of inhibitor of κB (IκB) proteins. For the p65 subunit, multiple phosphorylation sites have been mapped in and adjacent to both the N-terminal Rel homology domain and the C-terminal transactivation domain. Their impact on NF-κB-dependent transcription, however, has never been assessed at a broader level. In this study, we evaluate the importance of differential p65 phosphorylation on four serine acceptor sites in the Rel homology domain for the expression of an array of NF-κB-dependent genes in endothelial cells. We find that inhibition of p65 phosphorylation on these serine residues targets NF-κB activity to distinctive gene subsets in a κB enhancer element-specific context. We show that the phosphorylation-dependent alterations in gene and protein expression are reflective of the amount of p65 and phosphorylated RNA polymerase II (p-RNAP II) bound to respective gene promoter regions. Depending on the gene subset, impaired gene expression was either a result of decreased p65 promoter recruitment or of a failure of bound p65 to recruit p-RNAP II. In conclusion, our findings demonstrate that site-specific p65 phosphorylation targets NF-κB activity to particular gene subsets on a global level by influencing p65 and p-RNAP II promoter recruitment. Gene expression regulation by NF-κB is fundamental for controlling many important cellular processes, including inflammatory and immune responses, cell proliferation, development, and apoptosis (1Barkett M. Gilmore T.D. Control of apoptosis by Rel/NF-κB transcription factors.Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1075) Google Scholar, 2Hayden M.S. West A.P. Ghosh S. NF-κB and the immune response.Oncogene. 2006; 25: 6758-6780Crossref PubMed Scopus (932) Google Scholar, 3Karin M. Lin A. NF-κB at the crossroads of life and death.Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2445) Google Scholar). In resting cells, NF-κB transactivators are sequestered in the cytosol by association with IκB proteins (4Baeuerle P.A. Baltimore D. IκB. A specific inhibitor of the NF-κB transcription factor.Science. 1988; 242: 540-546Crossref PubMed Scopus (1685) Google Scholar). Upon stimulation with pro-inflammatory inducers, IκB proteins are phosphorylated, ubiquitinated, and degraded (5Henkel T. Machleidt T. Alkalay I. Krönke M. Ben-Neriah Y. Baeuerle P.A. Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB.Nature. 1993; 365: 182-185Crossref PubMed Scopus (1035) Google Scholar, 6Spencer E. Jiang J. Chen Z.J. Signal-induced ubiquitination of IκBα by the F-box protein Slimb/β-TrCP.Genes Dev. 1999; 13: 284-294Crossref PubMed Scopus (372) Google Scholar, 7Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. Phosphorylation of human IκB-α on serines 32 and 36 controls IκB-α proteolysis and NF-κB activation in response to diverse stimuli.EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (931) Google Scholar). This event constitutes the major step for initiation of NF-κB-dependent transcription as it allows the transcription factor to enter the nucleus where it can bind to regulatory sequences in gene promoters/enhancers. However, it has been shown that this step alone is often not sufficient to initiate gene expression. Inducible post-translational modification of NF-κB subunits especially by phosphorylation has proven to be equally important for NF-κB to efficiently induce transcription of target genes (8Huang B. Yang X.D. Lamb A. Chen L.F. Post-translational modifications of NF-κB: another layer of regulation for NF-κB signaling pathway.Cell. Signal. 2010; 22: 1282-1290Crossref PubMed Scopus (238) Google Scholar). For the p65 subunit, which constitutes the most potent transcriptional activator of the NF-κB protein family (9Schmitz M.L. Baeuerle P.A. The p65 subunit is responsible for the strong transcription activating potential of NF-κB.EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar), 12 phospho-acceptor sites have been mapped. Five sites (Ser-205, Thr-254, Ser-276, Ser-281, and Ser-311) are located in or adjacent to the N-terminal RHD, 2The abbreviations used are: RHDRel homology domainp-RNAP IIphosphorylated RNA polymerase II. and seven residues (Thr-435, Ser-468, Thr-505, Ser-529, Ser-535, Ser-536, and Ser-547) are contained in the C-terminal transactivation domain. Dependent on the stimulus and the modification site, phosphorylation of p65 regulates NF-κB transcriptional activity by different mechanisms. Ser-276 and Ser-311 phosphorylation in the RHD is enhancing p65 interaction with the transcriptional co-activator cAMP-response element-binding binding protein, thus activating NF-κB-dependent gene expression (10Duran A. Diaz-Meco M.T. Moscat J. Essential role of RelA Ser-311 phosphorylation by ζPKC in NF-κB transcriptional activation.EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (269) Google Scholar, 11Zhong H. Voll R.E. Ghosh S. Phosphorylation of NF-κB p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300.Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar). Phosphorylation in the transactivation domain at Thr-435, Thr-505, or Ser-547 can either activate or inhibit NF-κB-dependent transcription by impeding or promoting the interaction with histone deacetylase-1 (HDAC1) (12O'Shea J.M. Perkins N.D. Thr-435 phosphorylation regulates RelA (p65) NF-κB subunit transactivation.Biochem. J. 2010; 426: 345-354Crossref PubMed Scopus (17) Google Scholar, 13Rocha S. Garrett M.D. Campbell K.J. Schumm K. Perkins N.D. Regulation of NF-κB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor.EMBO J. 2005; 24: 1157-1169Crossref PubMed Scopus (138) Google Scholar, 14Sabatel H. Di Valentin E. Gloire G. Dequiedt F. Piette J. Habraken Y. Phosphorylation of p65(RelA) on Ser(547) by ATM represses NF-κB-dependent transcription of specific genes after genotoxic stress.PLoS ONE. 2012; 7e38246 Crossref PubMed Scopus (32) Google Scholar). Phosphorylation at Ser-529 and Ser-536 was shown to alter the association with basal components of the transcriptional machinery (15Buss H. Dörrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-κB at serine 536 is mediated by multiple protein kinases including IκB kinase (IKK)-α, IKKβ, IKKϵ, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription.J. Biol. Chem. 2004; 279: 55633-55643Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 16Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. Tumor necrosis factor α-induced phosphorylation of RelA/p65 on Ser-529 is controlled by casein kinase II.J. Biol. Chem. 2000; 275: 32592-32597Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), and Ser-205, Ser-276, Ser-281, and Ser-536 phosphorylation regulates p65 subcellular localization (17Bohuslav J. Chen L.F. Kwon H. Mu Y. Greene W.C. p53 induces NF-κB activation by an IκB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1.J. Biol. Chem. 2004; 279: 26115-26125Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 18Drier E.A. Huang L.H. Steward R. Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation.Genes Dev. 1999; 13: 556-568Crossref PubMed Scopus (80) Google Scholar, 19Hochrainer K. Racchumi G. Anrather J. Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis.FEBS Lett. 2007; 581: 5493-5499Crossref PubMed Scopus (15) Google Scholar). Attachment of phospho-groups to Thr-254 and Ser-468 is determining the binding to the suppressor of cytokine signaling 1 (SOCS1) ubiquitin ligase complex thereby regulating p65 protein stability (20Geng H. Wittwer T. Dittrich-Breiholz O. Kracht M. Schmitz M.L. Phosphorylation of NF-κB p65 at Ser-468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination.EMBO Rep. 2009; 10: 381-386Crossref PubMed Scopus (121) Google Scholar, 21Ryo A. Suizu F. Yoshida Y. Perrem K. Liou Y.C. Wulf G. Rottapel R. Yamaoka S. Lu K.P. Regulation of NF-κB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA.Mol. Cell. 2003; 12: 1413-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Rel homology domain phosphorylated RNA polymerase II. Whereas degradation of IκB and subsequent nuclear translocation of NF-κB serves as a general step in NF-κB activation that affects expression of all dependent genes, there is strong evidence that the phosphorylation of p65 regulates the activity of NF-κB in a gene-specific context. Several studies have shown that reconstitution of p65-deficient mouse embryonic fibroblasts with p65 S205A, S276A, S281A, T435A, S468A, and S536A mutants leads to reduction of NF-κB transcriptional activity confined to certain genes (12O'Shea J.M. Perkins N.D. Thr-435 phosphorylation regulates RelA (p65) NF-κB subunit transactivation.Biochem. J. 2010; 426: 345-354Crossref PubMed Scopus (17) Google Scholar, 20Geng H. Wittwer T. Dittrich-Breiholz O. Kracht M. Schmitz M.L. Phosphorylation of NF-κB p65 at Ser-468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination.EMBO Rep. 2009; 10: 381-386Crossref PubMed Scopus (121) Google Scholar, 22Anrather J. Racchumi G. Iadecola C. cis-Acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB.J. Biol. Chem. 2005; 280: 244-252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Dong J. Jimi E. Zhong H. Hayden M.S. Ghosh S. Repression of gene expression by unphosphorylated NF-κB p65 through epigenetic mechanisms.Genes Dev. 2008; 22: 1159-1173Crossref PubMed Scopus (114) Google Scholar, 24Moreno R. Sobotzik J.M. Schultz C. Schmitz M.L. Specification of the NF-κB transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of IKKϵ.Nucleic Acids Res. 2010; 38: 6029-6044Crossref PubMed Scopus (91) Google Scholar, 25Nowak D.E. Tian B. Jamaluddin M. Boldogh I. Vergara L.A. Choudhary S. Brasier A.R. RelA Ser-276 phosphorylation is required for activation of a subset of NF-κB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes.Mol. Cell. Biol. 2008; 28: 3623-3638Crossref PubMed Scopus (141) Google Scholar). Also, loss of glycogen synthase kinase-3β (GSK-3β), which phosphorylates p65 at Ser-468 (26Buss H. Dörrie A. Schmitz M.L. Frank R. Livingstone M. Resch K. Kracht M. Phosphorylation of serine 468 by GSK-3β negatively regulates basal p65 NF-κB activity.J. Biol. Chem. 2004; 279: 49571-49574Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), has profound effects on the expression of interleukin-6 (IL-6) and chemokine (CC motif) ligand 2 (CCL2) genes, but it only minimally impacts IκBα (NFKBIA) and chemokine (CXC motif) ligand 2 (CXCL2) expression (27Steinbrecher K.A. Wilson 3rd, W. Cogswell P.C. Baldwin A.S. Glycogen synthase kinase 3β functions to specify gene-specific, NF-κB-dependent transcription.Mol. Cell. Biol. 2005; 25: 8444-8455Crossref PubMed Scopus (175) Google Scholar). Silencing of NF-κB-dependent transcription by nuclear IκB is dependent on p65 Ser-536 phosphorylation and targets TNF and IL-6 genes but not the interleukin-8 (IL-8) gene (28Ghosh C.C. Ramaswami S. Juvekar A. Vu H.Y. Galdieri L. Davidson D. Vancurova I. Gene-specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα.J. Immunol. 2010; 185: 3685-3693Crossref PubMed Scopus (50) Google Scholar). Because existing reports only include a limited amount of NF-κB responsive genes, it is still unclear, however, to what extent differential phosphorylation of NF-κB leads to changes in its transcriptional profile at a global level. In this study, we investigated the effects of impaired p65 serine phosphorylation in and adjacent to the RHD on the expression of an array of NF-κB-dependent genes. The mouse endothelial cell line bEND.3 was obtained from ATCC. Mouse embryonic fibroblasts (3T3) isolated from p65−/− mice were a kind gift of Dr. A. Beg (Moffitt Cancer Center, Tampa, FL) and were described previously (29Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB.Nature. 1995; 376: 167-170Crossref PubMed Scopus (1634) Google Scholar). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 100 units/ml penicillin G and 100 μg/ml streptomycin B (both Cellgro) in a humidified atmosphere containing 5% CO2. Tumor necrosis factor (TNF; Invitrogen) was used at a final concentration of 10 ng/ml. Stable shRNA cell pools and p65 transfectants were selected in the presence of 1.5 μg/ml puromycin (Sigma) and 250 μg/ml hygromycin B (InvivoGen), respectively. Sequence and cloning of murine p65 and luciferase shRNAs in pSiren-RetroX (Clontech) were described elsewhere (30Anrather J. Racchumi G. Iadecola C. NF-κB regulates phagocytic NADPH oxidase by inducing the expression of gp91phox.J. Biol. Chem. 2006; 281: 5657-5667Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The bicistronic retroviral vector pLXIH was obtained by exchanging the neomycin resistance cassette of pLXIN (Clontech) with the hygromycin resistance gene. Insertion of human p65 WT, S205A, S276A, S281A, and S311A into pLXIH was carried out by standard cloning procedures. Endogenous p65 expression was suppressed in bEND.3 by retrovirally delivered shRNA. The resulting bEND.3 p65 knockdown cells as well as 3T3 p65−/− cells were retrovirally reconstituted with p65 WT or p65 phosphorylation-deficient mutants. Retroviral production, cellular infection with retrovirus-containing supernatants, and selection for positive cell pools was performed as described (22Anrather J. Racchumi G. Iadecola C. cis-Acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB.J. Biol. Chem. 2005; 280: 244-252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). For immunoblotting, cells were lysed in RIPA buffer (50 mm Tris-HCl, pH 8, 150 mm sodium chloride, 1 mm EDTA, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1× protease inhibitors (Roche Applied Sciences)), and equal volumes were mixed with SDS sample buffer, boiled, and analyzed on 10% SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes (Millipore), blocked with 5% milk in TBS, 0.1% Tween 20 (TBST), and incubated with polyclonal anti-p65 (C-20; Santa Cruz Biotechnology) or monoclonal anti-β-actin antibody (AC-74; Sigma). Membranes were washed in TBST and incubated with donkey anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology), and protein bands were visualized with Chemiluminescence Reagent (Santa Cruz Biotechnology) on a Kodak Image Station 2000R. For immunofluorescence, cells were stimulated with TNF for 0, 30, and 60 min and processed as described previously (19Hochrainer K. Racchumi G. Anrather J. Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis.FEBS Lett. 2007; 581: 5493-5499Crossref PubMed Scopus (15) Google Scholar). The ratio of cytoplasmic to nuclear p65 localization was determined as described elsewhere (31Hochrainer K. Racchumi G. Zhang S. Iadecola C. Anrather J. Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation.Cell. Mol. Life Sci. 2012; 69: 2057-2073Crossref PubMed Scopus (22) Google Scholar). Transcriptional profiles of NF-κB-dependent genes were determined by quantitative real time PCR under untreated conditions and after 3 h of TNF stimulation. Genes were compiled after ranking induction levels in seven datasets derived from short term cytokine-induced endothelial cells deposited in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov). The following datasets were used: GSE973, GSE973, GSE2639, GSE2638, GSE5883, GSE8166, and GSE9647. The first 200 ranked genes were tested for inclusion in two curated gene lists for NF-κB-regulated genes (Dr. T. Gilmore laboratory, Boston University and Bonsai Bioinformatics Software Server, Lille University of Science and Technology, France). Only genes listed in at least one of the databases were considered to be NF-κB-dependent. Hence, mRNA expression levels of 70 potentially NF-κB-regulated genes and two housekeeping genes used for normalization were analyzed by quantitative RT-PCR using SYBR Green chemistry (Fermentas) on a Chromo4 continuous fluorescence monitoring thermocycler (MJ Research) as described previously (22Anrather J. Racchumi G. Iadecola C. cis-Acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB.J. Biol. Chem. 2005; 280: 244-252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A complete list of tested genes as well as primer sequences can be found in the supplemental Table 1. Three independent experiments were performed. In the final analysis, only genes were included that were called in all TNF-induced datasets and showed at least 2-fold induction of mRNA levels in p65 WT-reconstituted cells over the untreated or TNF-stimulated empty vector control. A total of 37 genes fulfilled all criteria (see Fig. 2). In 3T3 cells, eight genes were tested for induction of mRNA expression (see supplemental Fig. 1). Cells were collected in 1× citric saline (135 mm potassium chloride, 15 mm sodium citrate), spun down, and incubated with Alexa Fluor 647 anti-mouse CD54 (ICAM-1), PerCP/Cy5.5 anti-mouse CD106 (VCAM-1), and FITC anti-mouse H-2Kd antibodies (all Biolegend) for 20 min on ice. Antibodies were titrated using mouse splenocytes or bone marrow cells to achieve optimal signal-to-noise ratio. Fluorochrome-matched isotype controls were used to validate detection specificity. Analysis was performed on a six-channel (Accuri C6, BD Biosciences) cytometer. Cells were gated according to their forward and side scatters to eliminate debris and dead cells. Gates were validated by TOPRO-3 and fluorescein-diacetate labeling to identify dead and live cells, respectively. Analysis of median fluorescent intensity was performed on 20,000 live cells. ChIP analysis was carried out as described (30Anrather J. Racchumi G. Iadecola C. NF-κB regulates phagocytic NADPH oxidase by inducing the expression of gp91phox.J. Biol. Chem. 2006; 281: 5657-5667Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) with the following variations. bEND.3 cells required 16 cycles of 15-s sonication pulses at 20% amplitude to yield 0.3–1.5-kb DNA fragments. For immunoprecipitation, 2 μg of anti-p65 (C-20; Santa Cruz Biotechnology) and 0.8 μg of anti-p-RNAP II (CTD4H8; Upstate) antibodies were used. Monoclonal anti-p-RNAP II antibody was coupled to 2 μg of rabbit anti-mouse IgG (Jackson ImmunoResearch) before capturing with protein A-Sepharose. A list of primer sequences and targeting κB sites can be found in supplemental Table 2. The important conclusion that inhibition of p65 phosphorylation at serines 205, 276, 281, and 311 leads to impaired NF-κB activation after TNF stimulation is derived from the expression analysis of a limited number of genes (10Duran A. Diaz-Meco M.T. Moscat J. Essential role of RelA Ser-311 phosphorylation by ζPKC in NF-κB transcriptional activation.EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (269) Google Scholar, 19Hochrainer K. Racchumi G. Anrather J. Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis.FEBS Lett. 2007; 581: 5493-5499Crossref PubMed Scopus (15) Google Scholar, 23Dong J. Jimi E. Zhong H. Hayden M.S. Ghosh S. Repression of gene expression by unphosphorylated NF-κB p65 through epigenetic mechanisms.Genes Dev. 2008; 22: 1159-1173Crossref PubMed Scopus (114) Google Scholar). Here, we investigate the dependence of NF-κB-driven gene expression on these p65 phosphorylation sites on a broader level by constitutively knocking down endogenous WT p65 in the murine endothelial cell line bEND.3 and reconstituting the resulting cell pool with a control vector, human p65 WT or S205A, S276A, S281A, and S311A. p65 knockdown as well as equal protein expression of the introduced p65 forms was verified by Western blotting (Fig. 1A). Reconstituted p65 WT followed the characteristic wave of nuclear import and export after TNF stimulation (Fig. 1B), indicating its successful functional integration into the NF-κB pathway. p65 phospho-mutant nuclear translocation after 30 min of TNF stimulation was similar to p65 WT; however, as also seen in fibroblasts (19Hochrainer K. Racchumi G. Anrather J. Hypo-phosphorylation leads to nuclear retention of NF-κB p65 due to impaired IκBα gene synthesis.FEBS Lett. 2007; 581: 5493-5499Crossref PubMed Scopus (15) Google Scholar), basal nuclear p65 levels were higher in S205A-, S276A-, and S281A-expressing cells, and after 60 min of TNF stimulation, p65 S205A, S276A, and S281A were still nuclear, whereas p65 WT and the S311A mutant relocated to the cytoplasm (Fig. 1, B and C). To examine global NF-κB activity in these cells, RNA was prepared, and expression of 37 NF-κB-dependent genes, meeting our selection criteria (see “Experimental Procedures”), was analyzed in resting and 3-h TNF-treated cells. As a general finding, the transcriptional activity of p65 205, 276, and 281 serine to alanine mutants was impaired, whereas p65 S311A was inducing gene expression to equal levels as p65 WT on almost all genes tested (Fig. 2). Depending on whether their expression was or was not affected by p65 phosphorylation deficiency, we subdivided the tested genes into three regulatory groups. The first group includes 11 genes, which failed to be induced by all p65 Ser-205, Ser-276, and Ser-281 mutants. All but one (Cxcl5) of these genes were equally induced by p65 WT and S311A. Group II consists of 13 genes that were efficiently induced by p65 WT, S311A and S205A mutants. Thirteen group III genes were equally induced by all p65 variants. Supporting these results, we found that protein expression of selected NF-κB-dependent genes matched the mRNA levels in p65 WT and mutant cells (Fig. 3). p65 knockdown resulted in substantial but not complete repression of endogenous p65 protein (Fig. 1A). To minimize the probability that any of our observed effects were caused by residual endogenous p65 levels, we compared mRNA expression of representative genes for each regulatory group with those from p65−/− fibroblasts reconstituted with p65 WT and mutants. As shown in supplemental Fig. 1, expression profiles of all genes tested were similar in bEND.3 and 3T3 cells, except for minor variations for Cxcl10 and H2-k1, which could be cell type-specific.FIGURE 3Protein expression levels of three selected genes as observed by FACS analysis after 3 h of TNF treatment. Untreated or 3-h TNF-treated retrovirally transduced p65 WT and mutant bEND.3 cells were processed for FACS analysis as described under “Experimental Procedures.” Data represent mean ± S.E. (n = 3), calculated relative to the mean of unstimulated, pooled controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using isolated κB consensus sites to drive reporter gene expression, we have previously established that p65 phosphorylation-deficient forms are driving transcription in a cis-acting element-specific context (22Anrather J. Racchumi G. Iadecola C. cis-Acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB.J. Biol. Chem. 2005; 280: 244-252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Here, we investigated whether the κB site preference of hypo-phosphorylated p65 mutants was also reflected in binding to endogenous gene promoters. Because the location and DNA sequence of functional κB sites has not been established for the majority of genes analyzed in this study, we restricted our analysis to selected genes of each induction group whose κB sites were functionally characterized and compared their annotated DNA sequences (Fig. 4). We found that the group affiliation of genes was largely dictated by the composition of the 5′ half-sites of the κB consensus sequence. The more guanines located in the N terminus of the κB site, the less the expression of the tested gene was susceptible to p65 Ser-to-Ala mutations. Whereas κB-binding sites in group I had preference for thymidine or maximal two guanines at the 5′ end, group II and III consensus sites were characterized by three or more guanine residues. The highest conservation was observed in group III with all but one κB site featuring four to five guanine residues at the 5′ end. In addition, all 5′ half-sites of group III genes were entirely composed of purines, a feature not as prevalent in κB sites of group I and II genes. The 3′ half-site showed higher homology spanning all groups with sites featuring two cytosines at the 3′ end preceded by two pyrimidines. In general, group I κB elements were found to be more diverse, whereas group II sites were similar, differing only in maximal two-base substitutions. Group III showed the highest homology with Cxcl10 site 2 and H2-k1, as well as H2-k1 and Nfkbie κB elements being identical except for a one-base shift. In summary, we confirm that differential p65 phosphorylation directs NF-κB activity to particular subsets of genes. The transcriptional specificity of NF-κB p65 phospho-mutants is thereby, as suggested previously (22Anrather J. Racchumi G. Iadecola C. cis-Acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB.J. Biol. Chem. 2005; 280: 244-252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), dictated by the structure of the κB element in the promoter of respective genes. Next, we investigated whether differential DNA-binding properties could account for differential transcriptional activities of p65 mutants by performing ChIP with a p65-specific antibody from resting, 0.5, and 3 h TNF-stimulated p65 knockdown, WT, or mutant-expressing bEND.3 cells. Maximal p65 binding was achieved after 0.5 h TNF stimulation for all p65 variants regardless of the expression profile of the corresponding gene (Fig. 5, left panel). p65 binding to gene promoters in group I was apparent for p65 WT but was markedly reduced for all p65 mutants (Fig. 5, left panel). This is in concordance with the observed gene expression patterns for p65 Ser-205, Ser-276, and Ser-281 mutants but is surprising for p65 S311A, which showed transcriptional activity comparable with the WT protein. Within group II, p65 S205A, S276A, and S311A were similarly recruited to the promoter as p65 WT, whereas p65 S281A DNA binding was impaired (Fig. 5, left panel). This suggests that reduced expression of group II genes in p65 S276A cells is not solely mediated by reduced p65 DNA binding, while the inability of the S281A mutant to be efficiently recruited to the promoter correlated well with its reduced transcriptional activity. In group III, promoter binding profiles of all p65 proteins were equal (Fig. 5, left panel), reflecting the observed mRNA expression patterns of genes belonging to this group. Because mRNA expression patterns of some genes could not be explained by p65 DNA binding properties alone, we next examined the levels of promoter-bound p-RNAP II, thought to be indicative for transcriptionally active genes (Fig. 5, right panel) (32O'Brien T. Hardin S. Greenleaf A. Lis J.T. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation.Nature. 1994; 370: 75-77Crossref PubMed Scopus (285) Google Scholar). Similarly to p65, p-RNAP II reached maximum binding at 0.5 h after TNF addition on most promoters, but recruitment was sustained at Vcam1, Cxcl10, H2-k1, and Nfkbie promoters. We" @default.
• W2056512813 created "2016-06-24" @default.
• W2056512813 creator A5008243101 @default.
• W2056512813 creator A5058899441 @default.
• W2056512813 creator A5063306805 @default.
• W2056512813 date "2013-01-01" @default.
• W2056512813 modified "2023-10-14" @default.
• W2056512813 title "Site-specific Phosphorylation of the p65 Protein Subunit Mediates Selective Gene Expression by Differential NF-κB and RNA Polymerase II Promoter Recruitment" @default.
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