Brd4 Coactivates Transcriptional Activation of NF- B via Specific Binding to Acetylated RelA

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1 MOLECULAR AND CELLULAR BIOLOGY, Mar. 2009, p Vol. 29, No /09/$ doi: /mcb Copyright 2009, American Society for Microbiology. All Rights Reserved. Brd4 Coactivates Transcriptional Activation of NF- B via Specific Binding to Acetylated RelA Bo Huang, 1 Xiao-Dong Yang, 1 Ming-Ming Zhou, 2 Keiko Ozato, 3 and Lin-Feng Chen 1 * Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois ; Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York ; and Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland Received 27 August 2008/Returned for modification 29 September 2008/Accepted 19 November 2008 Acetylation of the RelA subunit of NF- B, especially at lysine-310, is critical for the transcriptional activation of NF- B and the expression of inflammatory genes. In this study, we demonstrate that bromodomains of Brd4 bind to acetylated lysine-310. Brd4 enhances transcriptional activation of NF- B and the expression of a subset of NF- B-responsive inflammatory genes in an acetylated lysine-310-dependent manner. Bromodomains of Brd4 and acetylated lysine-310 of RelA are both required for the mutual interaction and coactivation function of Brd4. Finally, we demonstrate that Brd4 further recruits CDK9 to phosphorylate C-terminal domain of RNA polymerase II and facilitate the transcription of NF- B-dependent inflammatory genes. Our results identify Brd4 as a novel coactivator of NF- B through specifically binding to acetylated lysine-310 of RelA. In addition, these studies reveal a mechanism by which acetylated RelA stimulates the transcriptional activity of NF- B and the NF- B-dependent inflammatory response. The eukaryotic transcription factor NF- B/Rel family proteins regulate a wide range of host genes that govern the inflammatory and immune responses in mammals and play a key role in controlling programmed cell death, cell proliferation, and differentiation (18). The prototypical NF- B is a heterodimer of p50 and RelA and is sequestered in the cytoplasm by its association with the inhibitor protein I B in unstimulated cells. Stimulation of the cells with various stimuli leads to the activation of IKKs, phosphorylation and degradation of I B, and the nuclear translocation and the transcriptional activation of NF- B (17, 21). Transcriptional activation of NF- B involves the association of NF- B with various cofactors, including histone acetyltransferase (HAT) p300/cbp (16, 39, 42), and the nuclear receptor coactivators SRC-1/N-CoA-1, TIF2/GRIP-1, and SRC-3/Rac3 (42). These cofactors are thought to promote the rapid formation of the preinitiation and reinitiation complexes by bridging the sequence-specific activators to the basal transcription machinery, thereby facilitating multiple rounds of transcription (19). How these various cofactors are recruited to the promoter regions of NF- B target genes is not very clear. Posttranscriptional modifications of NF- B including phosphorylation and acetylation might play a role in the recruitment of these various cofactors. In support of this, phosphorylation of RelA at serines 276 and 536 has been demonstrated to facilitate the recruitment of p300/cbp and the subsequent acetylation of RelA (10, 22). Emerging evidence has demonstrated that reversible acetylation of RelA is important in modulating the nuclear action of * Corresponding author. Mailing address: Department of Biochemistry, College of Medicine, MC-714, University of Illinois at Urbana- Champaign, Urbana, IL Phone: (217) Fax: (217) lfchen@life.uiuc.edu. Published ahead of print on 22 December NF- B (6, 8, 9, 29, 49), as well as the inflammatory responses (20, 23, 24, 45). The RelA subunit of NF- B is acetylated by p300/cbp in a stimulus-coupled manner on different lysines (5, 9, 29). Modification of each of these lysines affects different functions of NF- B. For example, acetylation of lysine-221 enhances the DNA-binding properties of NF- B and, in conjunction with the acetylation of lysine-218, impairs the assembly of RelA with I B. Acetylation of lysine-310 is important for the transcriptional activity of RelA but does not affect its DNA binding or its assembly with I B (9). Abolishing lysine- 310 acetylation, either by mutating lysine-310 to arginine or by histone deacetylases, significantly inhibits the transactivation of NF- B and the expression of inflammatory cytokines (9, 10, 49). Acetylation of RelA is important for the NF- B-dependent inflammatory response. High levels of oxidative stress in chronic obstructive pulmonary disease enhance NF- B acetylation and the expression of inflammatory genes (24). Cigarette smoke promotes the acetylation of RelA, resulting in increased levels of proinflammatory cytokines in macrophages, as well as in rat lungs (45). Acetylation of RelA is also involved in Haemophilus influenzae (NTHi)- and DC-SIGN-induced NF- B activation and inflammation (20, 23). These data highlight the importance of acetylation of NF- B in the transcriptional activation of NF- B and NF- B-dependent inflammatory responses. However, the precise mechanism by which acetylation of RelA activates NF- B and contributes to the proinflammatory functions of NF- B remains elusive. Acetylation generates specific docking sites for bromodomain proteins, and acetylated lysine may regulate protein function in vivo through a signaling partnership with the bromodomain (35, 41, 46). For example, the bromodomains of Gcn5, PCAF, and CBP recognize acetylated lysines in histones, human immunodeficiency virus Tat, and p53, respectively (13, 33, 34, 43). Acetylation of p53 at lysine-382 is critical for the recruitment 1375

2 1376 HUANG ET AL. MOL. CELL. BIOL. of CBP, and acetylation at lysines 373 and 382 is critical for the recruitment of TAF1 to the promoter of the p21 gene (30, 34). The approximately 110-amino-acid bromodomain is a functional module that helps to decipher the histone code through its interactions with acetylated histones (46, 51). Many transcription and chromatin regulators, including HATs, chromatin remodeling factors, and basal transcription factors, contain one or two bromodomains, indicating its function in the regulation of chromatin structure and transcription (46, 51). We hypothesized that acetylated RelA might also recruit one of these bromodomain-containing factors to stimulate the transcriptional activation of NF- B. Brd4 belongs to the conserved BET family of proteins that contain two tandem bromodomains and an extra terminal domain (27, 44). Brd4 exerts its multiple functions by its association with various proteins. Brd4 binds to euchromatin through acetylated histones H3 and H4 (11). Brd4 has been isolated in complex with the replication factor C and the transcriptional mediator complexes (28, 32). Brd4 also binds to papillomavirus E2 proteins and tethers the viral DNA to host mitotic chromosomes for segregation of their genomes into daughter cells (50). Recent studies suggest that Brd4 functions as a positive regulatory component of P-TEFb (complex of cyclin T1 and CDK9). Brd4 is necessary to form the transcriptionally active P-TEFb, recruits P-TEFb to a promoter, and stimulates RNA polymerase II (RNAPII)-dependent transcription (4, 26, 48) (40). P-TEFb has been shown to interact with NF- B and stimulate RNA polymerase II-dependent transcriptional elongation of a subset of NF- B target genes (1, 2, 31, 37). These findings raise an intriguing possibility that Brd4 might be also involved in the expression of NF- B target genes. However, it is not clear whether or how Brd4 regulates the expression of P-TEFb-dependent NF- B target genes. In an effort to understand the role of acetylation of lysine- 310 of RelA and the function of Brd4 in the transcriptional activation of NF- B, we found that acetylated lysine-310 is specifically recognized by two bromodomains of Brd4. Binding of Brd4 to acetylated lysine-310 is essential for the recruitment of Brd4 to the promoters of NF- B target genes and to coactivate NF- B. The bromodomains of Brd4 and the acetylation of lysine-310 of RelA are all indispensably required for the mutual interaction and the coactivation function of Brd4. Finally, we demonstrate that Brd4 further recruits CDK9 to phosphorylate RNAPII to activate the transcriptional activity of NF- B. Our results reveal the mechanism by which acetylated RelA activates the transcription of NF- B target genes and identify a novel function of Brd4 as a transcriptional coactivator of NF- B. MATERIALS AND METHODS Cell lines, recombinant proteins, and plasmids. Human A549 lung carcinoma cells, HEK293T and mouse embryonic fibroblast (MEF) cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Human THP-1 monocytic leukemia cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. RelA-deficient mouse embryonic fibroblast cells reconstituted with either wild-type (WT) RelA or RelA-K310R mutant have been described (10). Recombinant RelA and recombinant acetylated RelA proteins (with more than 70 to 80% acetylated RelA) were prepared as described previously (7). The glutathione S-transferase (GST) fusion proteins of GSTbromodomain 1 (BD1), GST-BD2, and GST-BD1/BD2 were expressed in Escherichia coli BL-21 and purified according to the manufacturer s instructions (GE Healthcare). Expression vectors for GST-BD1, -BD2, or -BD1/BD2 were generated by cloning the PCR fragments of BD1 (amino acids 55 to 168) or BD2 (amino acids 347 to 464) or fragments containing both BD1 and BD2 (amino acids 55 to 464) to the pgex-4t-1 vector. Expression vectors for Brd4 and its deletion mutants, T7-RelA, and T7-RelA-K310R have been previously described (9, 26). HA-p300 and the HAT deletion mutant ( ) of p300 (HA-p300 [-HAT]) expression vectors were kindly provided by R. Goodman (The Vollum Institute). 5,6-Dichloro-1- -D-ribofuranosylbenzimidazole (DRB; D1916) was purchased from Sigma. Antibodies. Rabbit normal immunoglobulin G (IgG), antibodies against RelA (sc-109), Brd4 (H-250), p300 (sc-584), CDK9 (H-169), RNA polymerase II (sc-899), and GST (sc-138) were from Santa Cruz Biotechnology, Inc. Anti-ser- 2-RNA polymerase II (H5) and anti-t7 antibodies were from Covance. Antiacetylated lysine-310 antibodies were kindly provided by W.C. Greene (Gladstone Institutes) (10). In vitro protein-protein interaction. Purified GST-bromodomains of p300, PCAF, and Brd4 were incubated with a biotinylated RelA acetylated-k310 peptide [biotin-rkrtyetfk(ac)simkkspfsgpt] bound to streptavidin-agarose beads. Binding of the bromodomain to the peptide was detected by immunoblotting with anti-gst antibody. For in vitro GST pull-down assay, 25 l of glutathione-sepharose beads conjugated to GST-BD1, GST-BD2, GST-BD1/ BD2, or GST were incubated with 0.5 g of acetylated recombinant RelA diluted in 500 l of GST pull-down buffer, followed by rotation at 4 C for 2 h. The GST-bromodomain-associated proteins were analyzed by immunoblotting with anti-ac-k310 RelA or anti-rela antibody. For the in vitro binding of Brd4 and RelA, 20 l of resuspended FLAG-Brd4 immunoprecipitation beads (prepared as previously described [10]) were incubated with 0.5 g of nonacetylated or acetylated recombinant RelA diluted in 500 l of lysis buffer, followed by rotation at 4 C for 2 h. The Brd4-associated proteins were analyzed by immunoblotting with anti-rela or anti-ac-k310-rela antibody. Transient-transfection and luciferase reporter assay. Transient-transfection and luciferase reporter assays were performed as previously described (10). In each experiment, cells were also cotransfected with EF1-Renilla luciferase reporter plasmid which was used as an internal control. The results represent the average of three independent experiments the standard deviation (SD). Immunoprecipitation and immunoblotting analysis and ChIP. Immunoprecipitation, immunoblotting analysis, and chromatin immunoprecipitation (ChIP) assay were performed as previously described (10). The sequence of primers in ChIP will be provided upon request. The ChIP data were quantified by densitometric analysis using ImageJ 1.40g image processing software (National Institutes of Health). Establishment of cell lines stably expressing Brd4 shrna. Brd4-specific short hairpin RNA oligomer (5 -AGCAAAGCCAAGGAACCTC-3 ) was subcloned into the psuperretro-neo vector (Oligoengine). The vector was transfected into Phoenix-Ampho packaging cells, and the supernatants were collected 48 h after transfection and used to infect A549 cells in the presence of 10 g of Polybrene (Sigma)/ml for 24 h. After the medium was changed, the cells were cultured in 10% fetal calf serum-dulbecco modified Eagle medium for 16 h and then selected with 1 mg of G418 (Sigma)/ml for 7 days. The knockdown efficiency was assessed by immunoblotting. Quantitative real-time PCR analysis. A549 stable cell lines expressing control shrna and Brd4 shrna were stimulated with TNF- for various times, and the total RNA was extracted by using an RNeasy minikit (Qiagen) according to the manufacturer s instructions. cdna was synthesized with an Omniscript RT kit (Qiagen) according to the manufacturer s protocol. Quantitative real-time PCR was performed by using a Qiagen SYBR green PCR kit by 7300 real-time PCR system (ABI). PCR primers for human E-selectin interleukin-8 (IL-8), A20, actin, and tumor necrosis factor alpha (TNF- ) were purchased from Qiagen. RNA interference. The predesigned and validated small interfering RNA (sirna) were purchased from Ambion. sirna (60 nm) was used for transfection using Lipofectamine 2000 (Invitrogen) according to the manufacturer s instructions. For sibrd4 in THP-1 cells, 48 h after transfection, the cells were stimulated with lipopolysaccharide (LPS; 1 g/ l) for indicated times, and quantitative real-time PCR were performed as described above. For sicdk9 in A549 cells, at 30 h after transfection the cells were transfected with reporter DNA and Brd4 expression constructs as indicated in the figure legends. After 24 h, cells were stimulated with TNF- (20 ng/ml) for 5 h, and then the luciferase activity was measured. The efficiency of the sirna knockdown was determined at the end of each experiment by immunoblotting with corresponding specific antibodies.

3 VOL. 29, 2009 Brd4 BINDS TO ACETYLATED RelA TO COACTIVATE NF- B 1377 FIG. 1. Brd4 binds to acetylated RelA in vitro and in vivo. (A) Bromodomains of Brd4 bind to acetylated lysine-310 peptide. Purified GST-bromodomains (1 g) of p300, PCAF, and Brd4 were incubated with a biotinylated RelA acetylated K310 peptide (21 amino acids) bound to streptavidin-agarose beads. Binding of the bromodomain to the peptide was detected by immunoblotting with anti-gst-antibody (upper panel). Levels of input for each reaction are shown in the lower panel. (B) Bromodomains of Brd4 bind to acetylated full-length RelA. Glutathione- Sepharose beads conjugated to GST, GST-BD1, GST-BD2, or GST fusion proteins containing BD1 and BD2 were incubated with the in vitro acetylated recombinant RelA. GST or GST fusion proteins associated proteins were immunoblotted with anti-ac-k310 RelA or anti-rela antibodies. The levels of input are shown in the right two panels. The levels of various GST fusion proteins are shown in the lower panel and indicated by asterisks. (C) Full-length Brd4 binds to acetylated RelA in vitro. FLAG-Brd4 immunoprecipitates from transfected HEK293T cells were incubated with nonacetylated or acetylated recombinant RelA. Brd4-associated proteins were immunoblotted with anti-ac-k310 RelA or anti-rela (upper two panels, pulldown). The levels of input are shown in the lower two panels. (D) Full-length Brd4 binds to acetylated RelA in vivo. HEK293T cells were transfected with the indicated combination of vectors expressing FLAG-Brd4, T7-RelA, HA-p300, or HA-p300 ( HAT). Brd4 immunoprecipitates were immunoblotted with anti-ac-k310 RelA or anti-t7 antibodies (upper two panels). The levels of Brd4, RelA, acetylated RelA, and p300 are shown in the lower four panels. (E) TNF- stimulates the interaction of Brd4 and RelA. A549 cells were stimulated with TNF- (20 ng/ml) for 60 min, cytoplasmic (Cyto) and nuclear (Nuc) extracts from stimulated and unstimulated cells were immunoprecipitated with anti-rela antibodies, and coimmunoprecipitated Brd4 was detected by anti-brd4 antibodies (upper panels). The levels of RelA, Brd4, HDAC1, and tubulin are shown in the lower three panels. RESULTS Brd4 binds to acetylated RelA in vitro and in vivo. To test the possibility that acetylated lysine-310 of RelA interacts with a bromodomain-containing protein to activate the transcriptional activity of NF- B, we tested whether acetylated lysine binds to the bromodomain of p300, PCAF or Brd4. Both p300 and PCAF acetylate RelA and coactivate NF- B; Brd4 is the positive regulator of the P-TEFb complex, which is also involved in the activation of NF- B (3, 31); therefore, we hypothesized that bromodomains from these proteins might interact with acetylated lysine-310. Using purified recombinant GST fusion proteins of the bromodomains of p300, PCAF, and Brd4, an in vitro peptide-binding assay was performed to test the binding ability of different bromodomains to biotin-labeled acetylated lysine-310 peptides. From the four bromodomains tested, both BD1 and BD2 of Brd4 bound to acetylated lysine-310 peptides (Fig. 1A). Surprisingly, bromodomains of p300 and PCAF barely bound to the acetylated lysine-310 peptides (Fig. 1A). These in vitro peptide binding data suggest that acetylated lysine-310 peptides bind to the BD1 and BD2 of Brd4 but likely not to the bromodomains of p300 and PCAF. To further confirm the binding of BD1 and BD2 to the acetylated lysine-310, we examined the binding of either bromodomain to acetylated

4 1378 HUANG ET AL. MOL. CELL. BIOL. full-length RelA in a GST pull-down experiment. GST-BD1 or GST-BD2 alone only associated with a small amount of acetylated RelA (Fig. 1B, lanes 2 and 3). Interestingly, GST fusion proteins containing both BD1 and BD2 pulled down a significantly enhanced amount of acetylated RelA (Fig. 1B, lane 4). These data indicate that although BD1 or BD2 alone is able to bind to acetylated RelA, these two bromodomains might function cooperatively for the effective binding to the acetylated full-length RelA. We next investigated whether acetylated full-length RelA associated with full-length Brd4. When full-length recombinant RelA was incubated with FLAG-tagged Brd4 immunoprecipitates and the Brd4-associated acetylated RelA was detected by anti-acetylated lysine-310 RelA antibodies, we found that without acetylation RelA was barely pulled down by Brd4 (Fig. 1C, lane 1). However, when RelA was acetylated by p300 in vitro, a significant amount of acetylated RelA was pulled down by Brd4 (Fig. 1C, lane 2), suggesting a direct association of acetylated RelA with Brd4. Furthermore, when the interaction of Brd4 and RelA was examined in vivo by cotransfecting HEK293T cells with the expression vectors for Brd4 and RelA, we found that without coexpression of p300, which mediates the acetylation of RelA, Brd4 barely coimmunoprecipitated RelA (Fig. 1D, lane 3). However, Brd4 coimmunoprecipitated acetylated RelA in the presence of p300, when RelA was acetylated (Fig. 1D, lane 4). Conversely, when the HAT domain deletion mutant of p300 was used, no RelA was acetylated, and the interaction between RelA and Brd4 was significantly reduced (Fig. 1D, lane 5), indicating that interaction of RelA with Brd4 depends on the acetylation status of RelA rather than on the possible bridging effect of the cotransfected p300. Finally, we investigated the interaction of endogenous RelA and Brd4 upon TNF- stimulation. When nuclear extracts from TNF- -treated lung epithelial A549 cells were immunoprecipitated with anti-rela antibodies, we found that Brd4 coimmunoprecipitated with RelA (Fig. 1E), indicating a stimulus-dependent interaction between endogenous RelA and Brd4 in the nucleus. However, in an attempt to examine whether endogenous Brd4 associates with acetylated RelA, we could not detect acetylated RelA in the Brd4 coimmunoprecipitates (data not shown). This is probably due to the relatively low abundance of acetylated endogenous RelA, since Brd4-associated acetylated RelA could be detected in vitro and in vivo when the amount of acetylated RelA was increased by p300 (Fig. 1C and D). Brd4 coactivates NF- B in cooperation with p300. We next assessed the possibility that Brd4 might regulate the transcriptional activity of NF- B since acetylation of RelA enhances the transcriptional activation of NF- B (9) and Brd4 positively regulates the activity of P-TEFb, which is involved in the transcriptional activation of NF- B target genes (3). We first examined the effect of Brd4 on the transcriptional activity of NF- B using a 5X B-luciferase reporter containing five B binding sites. In this transient-transfection reporter assay, transcriptional activation of RelA was enhanced with cotransfected Brd4 in a dose-dependent manner (Fig. 2A, left panel). To rule out the possibility that these results reflect a selective effect to activate the 5X B reporter, we tested two other reporter plasmids containing the B enhancer from E-selectin and IL-8 gene promoters. Brd4 coactivates RelA-mediated induction of luciferase activity in both E-selectin-Luc and IL-8-Luc constructs (Fig. 2A, middle and right panels), indicating that Brd4 functions as a coactivator for NF- B. To further confirm that this coactivation is a direct effect on NF- B, we compared the ability of Brd4 to coactivate NF- B in a luciferase reporter plasmid containing minimum CD28 responsive element of IL-2 promoter or the element with the mutated NF- B binding sites in the presence of cotransfected RelA and Brd4. Similar to the results from other NF- B reporters (Fig. 2A), dose-dependent coactivation of NF- B by Brd4 was observed in the WT IL-2 promoter reporter, whereas no activation was seen in the mutated reporter even when Brd4 was cotransfected (Fig. 2B), indicating that binding of NF- B to the promoter is required for the coactivation function of Brd4 and Brd4 directly targets on NF- B. Similar to the coactivation effect observed for RelA, coexpression of Brd4 enhanced TNF- -induced activation of endogenous NF- B in a dose-dependent manner in three different reporter constructs (Fig. 2C). Since p300 acetylates RelA and coactivates NF- B through acetylation of RelA (9), and Brd4 binds to acetylated RelA and coactivates NF- B (Fig. 1 and 2A to C), we next evaluated whether Brd4 and p300 act in cooperation. In the E-selectin- B luciferase reporter assay, expression of p300 or Brd4 individually or in combination had no effect on the basal transcriptional activity of the reporter. Expression of p300 or Brd4 individually with RelA resulted in a slight increase in transcriptional activation compared to RelA alone. Interestingly, coexpression of both Brd4 and p300 with RelA further enhanced RelA-mediated transcriptional activation (Fig. 2D). This cooperative activation is dependent on the HAT activity of p300, since the synergistic effect of Brd4 and p300 on transcription was abolished when the HAT-deficient mutant of p300 was used (Fig. 2D). To further confirm this observation, the synergistic effect of Brd4 and p300 on the transcriptional activation of endogenous NF- B in response to TNF- stimulation was evaluated. Similar cooperation between p300 and Brd4 was observed, and the synergistic activation also depends on the HAT activity of p300 (Fig. 2E). Collectively, these data reveal a potential cooperative function of Brd4 and p300. Brd4 coactivates transcriptional activation of NF- B, and this coactivation appears to be dependent on p300-mediated acetylation of RelA. RNA interference-mediated depletion of Brd4 impairs the transcriptional activation of NF- B. To further demonstrate that Brd4 is a coactivator for NF- B, we generated an A549 cell line stably expressing an shrna against Brd4 and evaluated TNF- -induced activation of NF- B target genes in these Brd4 knockdown cells. Examining the TNF- -induced expression of two NF- B target genes, the E-selectin and TNF- genes, in these cells (with more than 50% knockdown efficiency [Fig. 3A, right panel]) by quantitative real-time PCR, we found that depletion of Brd4 by shrna compromised TNF- -induced transcription of both E-selectin and TNF- compared to control cells, although the expression patterns of the two genes varied (Fig. 3A, left and middle panels). Interestingly, when the expression of another NF- B target gene, the A20 gene, was examined, we found that depletion of Brd4 barely affected TNF- -induced A20 expression (Fig. 3A, second to right panel), indicating that coactivation of NF- B by

5 VOL. 29, 2009 Brd4 BINDS TO ACETYLATED RelA TO COACTIVATE NF- B 1379 Downloaded from FIG. 2. Brd4 coactivates NF- B. (A) Brd4 coactivates RelA in a dose-dependent manner. 5X B-luciferase (5X B-Luc), E-selectin-luciferase (E-selectin-Luc), or IL-8-luciferase (IL-8-Luc) reporter plasmids (0.1 g) were cotransfected with RelA expression vector (1 ng) alone or in combination with increasing amounts of Brd4 (250 and 500 ng) into HEK293T cells. The luciferase activity was measured 30 h after transfection. The results represent the average of three independent experiments the standard deviation. (B) HEK293T cells were cotransfected with IL-2-luc or IL-2m-luc reporter plasmid (0.1 g) combined with increasing amounts of Brd4 expression vector (250 and 500 ng). The luciferase activity was measured as described for panel A. (C) Brd4 coactivates TNF- -mediated activation of NF- B. HEK293T cells were cotransfected with 5X B-luc, E-selectin-Luc, or IL-8-Luc reporter plasmid (0.1 g) alone or in combination with increasing amounts of Brd4 expression vector (250 and 500 ng). At 24 h after transfection, cells were treated with TNF- (20 ng/ml) for 5 h, and the luciferase activity was measured as described in panel A. (D and E) Brd4 coactivates NF- B in cooperation with p300. HEK293T cells were cotransfected with E-selectin-Luc reporter plasmid (0.1 g) in the presence of various combinations of expression vectors for RelA (1 ng), Brd4 (50 ng), p300 (50 ng), and the p300 ( HAT) catalytic mutant (50 ng) as indicated. In panel D, the luciferase activity was measured 30 h after transfection. In panel E, at 24 h after transfection the cells were treated with TNF- (20 ng/ml) for 5 h, and then the luciferase activity was measured. on August 17, 2018 by guest Brd4 might be promoter specific and that Brd4 is only involved in the transcriptional activation of a subset of NF- B targets. To exclude a possible off-target effect of the shrna in A549 cells, we used sirna targeting a different Brd4 sequence to knock down Brd4 in human monocytic leukemia THP-1 cells and examined LPS-induced expression of NF- B target genes. Consistent with the results in A549 cells, depletion of Brd4 in THP-1 cells by sirna impairs LPS-induced expression of IL-8 and TNF- mrna but not the expression of A20 (Fig. 3B). Together, these results indicate that Brd4 is essential for the expression of a subset of NF- B target genes induced by different stimuli in different cell types. Brd4 is recruited to the promoters of a subset of NF- B target genes. Having identified Brd4 as a coactivator of NF- B, we next used ChIP assays to examine whether Brd4 is recruited to the promoters of NF- B target genes, an essential step for the function of a coactivator. We stimulated A549 cells with TNF- for 0, 30, or 60 min and examined the binding of RelA, acetylated lysine-310 RelA, p300, and Brd4 to the promoter of E-selectin, the expression of which is regulated by Brd4 (Fig.

6 1380 HUANG ET AL. MOL. CELL. BIOL. Downloaded from FIG. 3. Brd4 is required for the activation of NF- B and is recruited to the promoters of NF- B target genes. (A) Depletion of Brd4 by shrna impairs TNF- -induced expression of NF- B target genes. A549 cells stably expressing a Brd4 shrna or a control (ctr) shrna were stimulated with TNF- (20 ng/ml) for the indicated time points. Quantitative reverse transcription-pcr was performed to analyze the TNF- -induced expression of three NF- B target genes (for E-selectin, TNF-, and A20) in response to Brd4 knockdown (left three panels). The efficiency of the knockdown of Brd4 was analyzed by immunoblotting nuclear extracts with anti-brd4 antibodies. HDAC1 is a nuclear protein control (right panel). (B) Depletion of Brd4 by sirna impairs LPS-induced expression of NF- B target genes. THP-1 cells were transiently transfected with Brd4 sirna or a control (ctr) sirna, and after 48 h the cells were stimulated with LPS (1 g/ml) for the indicated time points. Quantitative reverse transcription-pcr was performed to analyze the LPS-induced expression of three NF- B target genes (IL-8, TNF-, and A20) in response to Brd4 knockdown (left three panels). The efficiency of the knockdown of Brd4 was analyzed by immunoblotting nuclear extracts with anti-brd4 antibodies. HDAC1 is a nuclear protein control (right panel). (C) Brd4 is recruited to the promoters of a subset of NF- B target genes. A549 cells were treated with TNF- (20 ng/ml) for the indicated time periods. ChIP assays were performed with the indicated antibodies or rabbit normal IgG as a control and probed for the E-selectin, TNF-, or A20 promoter sequences spanning the B binding sites or for nonspecific -actin DNA (right panels). The data are representative of three independent experiments. (D) The quantified results, normalized to the input, were derived from three independent experiments. on August 17, 2018 by guest 3A). In the absence of stimulation, the binding of Brd4, p300, acetylated RelA, or RelA to the E-selectin promoter was barely detected (Fig. 3C, left panel, lane 1). However, after stimulation with TNF-, RelA, p300, and acetylated lysine-310 RelA were recruited to the promoter of E-selectin (Fig. 3C). More importantly, Brd4 was also recruited to the promoter of E-selectin after stimulation with TNF-. In contrast, no recruitment of RelA, acetylated RelA, p300, or Brd4 to the -actin DNA was observed, demonstrating the specificity of the recruitment. Similar recruitment of Brd4 to the promoter

7 VOL. 29, 2009 Brd4 BINDS TO ACETYLATED RelA TO COACTIVATE NF- B 1381 Downloaded from FIG. 4. Brd4 coactivates NF- B through specific binding to acetylated lysine-310. (A) Acetylation of lysine-310 is required for the interaction of RelA with Brd4 in vivo. HEK293T cells were transfected with the indicated combination of plasmids expressing FLAG-Brd4, p300, and T7-RelA, as well as its mutant form, T7-RelA-K310R. Brd4 was immunoprecipitated with anti-flag antibody, and the associated RelA was detected by immunoblotting with anti-t7 antibody (upper panel). The levels of Brd4, RelA, and acetylated RelA are shown in the lower three panels. (B) Acetylated lysine-310 is required for the recruitment of Brd4 to the promoters of NF- B target genes. RelA-deficient MEFs reconstituted with either WT RelA or RelA-K310R mutant were stimulated with TNF- (20 ng/ml) for the indicated periods. ChIP assays were performed with indicated antibodies or rabbit normal IgG as a control and probed for the E-selectin promoter sequences spanning the B binding sites. The data are representative of three independent experiments. The quantified results, normalized to the input, derived from three independent experiments are shown on the right. (C) Acetylated lysine-310 is important for the coactivation function of Brd4. HEK293T cells were cotransfected with 5X B luciferase reporter plasmid (0.1 g) and either WT RelA or RelA-K310R mutant expression plasmid (1 ng), alone or in combination with increasing amounts of Brd4 expression vector (250 and 500 ng). The luciferase activity was measured 30 h after transfection. was also observed for another NF- B target gene, TNF- (Fig. 3C). However, when we examined the promoter of A20, whose expression was not regulated by Brd4 (Fig. 3A), we found that Brd4 was not recruited to the promoter, although RelA was still recruited to the promoter after TNF- stimulation (Fig. 3C). This is probably due to the lack of recruitment of p300 and p300-dependent acetylated lysine-310 RelA to the promoter (Fig. 3C). Quantitation of these ChIP assays further confirms the stimulus-dependent recruitment of Brd4 to the promoters of E-selectin and TNF- but not to that of A20. Similar to the recruitment of acetylated lysine-310 to the promoters, more promoter-bound Brd4 was detected at 60 min than at 30 min after TNF- stimulation for E-selectin and TNF- promoters (Fig. 3D). These ChIP data suggest that Brd4 is recruited to the promoters of a subset of NF- B target genes during the activation of NF- B. Brd4 coactivates NF- B through specific binding to acetylated lysine-310. The observations that the bromodomains of Brd4 bind to acetylated lysine-310 peptides and that Brd4 coactivates NF- B (Fig. 1A and Fig. 2) prompted us to examine whether Brd4 coactivates NF- B through binding to acetylated lysine-310. We first examined whether acetylation of lysine-310 was required for the interaction of RelA with Brd4. WT RelA interacted with Brd4 in the presence of p300; however, when the acetylation of lysine-310 was blocked by mutation of lysine to arginine, the interaction between Brd4 and RelA was completely abolished (Fig. 4A), although WT RelA and RelA-K310R were expressed at comparable levels. These on August 17, 2018 by guest

8 1382 HUANG ET AL. MOL. CELL. BIOL. data highlight the importance of acetylated lysine-310 in the interaction of RelA with Brd4. To further demonstrate that Brd4 interacts with RelA through acetylated lysine-310, we used ChIP assays in the RelA-deficient MEF cells reconstituted with WT RelA or RelA-K310R. Similar to A549 cells, in reconstituted WT RelA cells, TNF- stimulated the recruitment of Brd4, as well as RelA, p300, and acetylated lysine-310 RelA, to the promoter of E-selectin (Fig. 4B, lanes 2 and 3). Conversely, although RelA was still recruited to the promoter after TNF- stimulation in reconstituted RelA-K310R MEFs, acetylated lysine-310 RelA was not recruited to the promoter at all (Fig. 4B). Importantly, TNF- failed to stimulate the binding of Brd4 to the E-selectin promoter in RelA-K310R reconstituted cells (Fig. 4B, lanes 5 and 6). Consistent with the in vitro peptide binding assay, acetylated lysine-310 appears to not be involved in the binding of the bromodomain of p300 and its recruitment to the promoter, since p300 is still recruited to the E-selectin promoter in RelA-K310R mutant cells. Altogether, these results further confirm the recruitment of Brd4 to the promoter of E-selectin after TNF- stimulation and suggest an acetylated lysine-310-dependent recruitment. These results demonstrated the critical role of acetylated lysine-310 in the interaction of RelA with Brd4. We next examined whether acetylation of lysine-310 is required for the coactivation function of Brd4. We compared the coactivation effect of Brd4 on WT RelA and RelA-K310R in a B-luciferase reporter assay. Brd4 coactivated WT RelA in a dose-dependent manner (Fig. 4C). Consistent with previous findings, RelA-K310R retained a significantly lowered degree of transcriptional activity compared to WT RelA (9). However, in contrast to its effect on the WT RelA, coexpression of Brd4 did not further enhance the transcriptional activity of RelA- K310R. It appears that Brd4 coactivates NF- B through binding to acetylated lysine-310. Bromodomains are required for the interaction of Brd4 with RelA and the coactivation function of Brd4. We next investigated whether the bromodomains of Brd4 are required for its physical interaction with RelA and its transcriptional coactivation function. In order to explore the role of bromodomains in the coactivation function of Brd4, we used bromodomain deletion mutants of Brd4 (26) and examined their abilities to associate with RelA and coactivate NF- B. We first examined the interaction in a pull-down experiment using in vitro acetylated full-length recombinant RelA and immunoprecipitates of WT Brd4 and its bromodomain deletion mutants from transfected HEK293T cells. In this in vitro interaction assay, fulllength Brd4 pulled down a significant amount of RelA when RelA was acetylated (Fig. 5A, left panels). However, deletion of either BD1, BD2, or both BD1 and BD2 significantly compromised Brd4 s interaction with RelA (Fig. 5A, left panels), indicating that both bromodomains are required for the physical interaction with RelA. Next, we examined the physical interaction in vivo by immunoprecipitation assay. Consistent with the in vitro pull-down data, deletion of either BD1 or BD2 impaired Brd4 s interaction with RelA even in the presence of p300 when RelA is acetylated (Fig. 5B). Deletion of both BD1 and BD2 completely abolished the interaction between Brd4 and RelA. Collectively, these in vitro and in vivo results demonstrate that both bromodomains of Brd4 are required for the interaction with RelA. We next assessed whether the bromodomains of Brd4 are required for the transcriptional coactivation of NF- B. When the coactivation capabilities of WT Brd4 and its various bromodomain deletion mutants were tested in the B-luciferase reporter assay, we found that WT Brd4 coactivated RelA. However, deletion of BD1 or BD2 or both BD1 and BD2 sharply impaired the coactivation function of Brd4 (Fig. 5C, left panel). In addition, when Brd4 and its various mutants were examined for their abilities to coactivate TNF- -induced activation of NF- B, deletion of either bromodomain compromised its ability to coactivate NF- B (Fig. 5C, right panels). The coactivation potential of Brd4 and its bromodomain deletion mutants correlated closely with their abilities to bind to RelA (Fig. 5A and B). These results further underscore the importance of the bromodomains of Brd4 in the coactivation of NF- B. CDK9 is required for the coactivation function of Brd4. Brd4 is a positive regulator of the P-TEFb complex; it binds to P-TEFb and activates CDK9 and polymerase II-dependent transcription (26, 48). We therefore examined whether Brd4 further recruits P-TEFb to activate the transcription of NF- B target genes. We first used ChIP assays in the RelA-deficient MEF cells reconstituted with WT and RelA-K310R. In reconstituted WT RelA cells, TNF- stimulated the binding of RelA, acetylated RelA, p300, and Brd4 to the promoter of E-selectin (Fig. 6A, upper four panels). TNF- also stimulated the binding of CDK9, RNAPII and the phosphorylated form of RNAPII to the promoter (Fig. 6A). However, in reconstituted RelA-K310R cells, Brd4 was not recruited to the promoter, and neither were CDK9 or the phosphorylated form of RNAPII, although RNAPII was still recruited to the promoter, probably through RelA. These data suggest that acetylation of lysine-310 is critical for the recruitment of Brd4 and CDK9 and that recruitment of CDK9 is required for Brd4 to coactivate NF- B. To determine whether CDK9 is involved in the coactivation function of Brd4, we examined the transcriptional coactivation function of Brd4 in the presence of the CDK9 inhibitor DRB (15). Brd4 barely enhanced the transcription activity of NF- B when the activity of CDK9 was inhibited by DRB at the two different doses tested (Fig. 6B). These data suggest that the enhancement of NF- B activation by Brd4 requires the activity of CDK9. To further confirm this, we used sirna to reduce the expression of endogenous CDK9 in A549 cells and assessed the coactivation function of Brd4. sirna against CDK9 effectively depleted the expression of CDK9 (Fig. 6C, upper panel). Akin to the effect of DRB, depletion of CDK9 abolished the ability of Brd4 to coactivate TNF- -induced activation of NF- B (Fig. 6C). Altogether, these data support the notion that CDK9 is required for Brd4 to activate the expression of NF- B target genes. DISCUSSION Growing evidence from in vivo studies demonstrates that stimulus-induced acetylation of RelA plays an important role in the transcriptional activation of NF- B and the induction of inflammatory responses (6, 8, 9, 20, 23, 24, 29, 45, 49). Al-

9 VOL. 29, 2009 Brd4 BINDS TO ACETYLATED RelA TO COACTIVATE NF- B 1383 FIG. 5. Bromodomains are required for the interaction of Brd4 with RelA and the coactivation function of Brd4. (A) Both BD1 and BD2 of Brd4 are required for the in vitro interaction of Brd4 with acetylated RelA. Expression vectors for the full-length Brd4 (FL) or its deletion mutants ( BD1, BD2, and BD1&2) were transfected in HEK293T cells, and the anti-flag immunoprecipitates were incubated with nonacetylated (U) or acetylated (Ac) recombinant RelA. Proteins associated with Brd4 and its deletion mutants were analyzed by immunoblotting with anti-rela antibody (left panel). The levels of input of nonacetylated or acetylated recombinant RelA are shown in the right panel. (B) Bromodomains of Brd4 are important for the in vivo interaction of Brd4 with acetylated RelA. HEK293T cells were transfected with the indicated combination of vectors expressing full-length Brd4 (FL) or its deletion mutants ( BD1, BD2, and BD1&2), T7-RelA, and p300. Brd4 was immunoprecipitated with anti-flag antibody, and the associated RelA was detected by immunoblotting with anti-t7 antibody (upper panel). The levels of Brd4, RelA, and acetylated RelA are shown in the lower three panels. (C) Both BD1 and BD2 of Brd4 are important for the coactivation function of Brd4. HEK293T cells were cotransfected with E-selectin-luciferase reporter plasmid (0.1 g) in the presence of various combinations of expression vectors for RelA (1 ng) and Brd4, as well as its deletion mutants (250 and 500 ng) as indicated. In the left diagram, the luciferase activity was measured 30 h after transfection. In the right diagram, at 24 h after transfection, the cells were first treated with TNF- (20 ng/ml) for 5 h, and then the luciferase activity was measured. though acetylation of lysine-310 is responsible for the optimized transcriptional potential of NF- B, the precise mechanism underlying the specific effect of this single lysine acetylation remains elusive. In the present study, we demonstrated that acetylated RelA contributes to the transcriptional activation of NF- B and the expression of inflammatory genes by specific recruitment of transcriptional coactivator Brd4 via an interaction between the acetylated lysine-310 and the bromodomains of Brd4 (Fig. 6D). Brd4 further recruits CDK9, which phosphorylates CTD of RNAPII to stimulate the transcription of a subset of NF- B genes (Fig. 6D). The interaction of the bromodomains of Brd4 with acetylated lysine-310 underlines the role of Brd4 in NF- B-dependent expression of inflammatory genes and demonstrates a potential chromatinindependent property of Brd4 in transcriptional activation. These studies also provide a better understanding of the molecular mechanism by which acetylation of RelA contributes to the full transcriptional activity of NF- B and the NF- B-dependent inflammatory response. Bromodomains that specifically recognize acetylated lysine and the binding of bromodomain-containing proteins to acetylated histones help to interpret the histone code that is embedded within chromatin to signify regions of distinct nuclear activities such as heterochromatin formation or transcriptional activation (35, 51). HATs, including p300/cbp and PCAF, play an important role in acetylating lysines, as well as in recognizing those same acetylated lysines (12). p300/cbp and PCAF both acetylate RelA and function as coactivators for NF- B (6, 29, 39, 42). Interestingly, bromodomains of Brd4, but not bromodomains of p300 and PCAF, bound to acetylated lysine-310 in our in vitro peptide-binding assay (Fig. 1A). The specific binding is further demonstrated in the in vivo ChIP experiments, where the recruitment of p300 but not Brd4 can be found in the RelA-K310R reconstituted cells (Fig. 4B). This specific binding of the bromodomains of Brd4 to acetylated lysine-310 indicates that various mechanisms might be involved in the recruitment of different cofactors to NF- B. Indeed, phosphorylation provides such a mechanism for the recruitment of p300/cbp. Phosphorylation of RelA at serines 276 and 536 facilitates the recruitment of p300/cbp and enhances the acetylation of RelA (10, 22). Thus, phosphorylation and acetylation seem to form a combined regulatory mechanism and create a signaling cascade that dictates the order of recruitment of p300 and Brd4 to the promoter to activate the transcription. RelA, p300, and Brd4 cooperatively activate NF- B-depen-

10 1384 HUANG ET AL. MOL. CELL. BIOL. Downloaded from FIG. 6. CDK9 binds to Brd4 and is required for its coactivation function for NF- B. (A) CDK9 is not recruited to the promoter of E-selectin in RelA-K310R reconstituted cells. RelA-deficient MEFs reconstituted with either WT RelA or RelA-K310R mutant were stimulated with TNF- (20 ng/ml) for the indicated periods. ChIP assays were performed with indicated antibodies or rabbit normal IgG as a control and probed for the E-selectin promoter sequences as in Fig. 4B. The data are representative of two independent experiments. The quantified results, normalized to the input, derived from two independent experiments are shown on the right. (B) CDK9 inhibitor DRB impaired the coactivation function of Brd4. A549 cells were cotransfected with E-selectin-luciferase reporter plasmid (0.1 g) with increasing amounts of Brd4 (250 or 500 ng). At 24 h after transfection, cells were first treated with TNF- (20 ng/ml) alone or with TNF- (20 ng/ml) combined with DRB (10 M or20 M) for 5 h, and then the luciferase activity was measured. (C) Depletion of CDK9 impaired the ability of Brd4 to coactivate NF- B. A549 cells were transfected with CDK9 sirna or control sirna; at 30 h after transfection, E-selectin-Luc reporter was cotransfected with increasing amounts of Brd4. At 24 h after transfection, the cells were first treated with TNF- (20 ng/ml) for 5 h, and then the luciferase activity was measured. A representative immunoblot showing CDK9 depletion in transfected cells is shown on the right. (D) Schematic model for the binding of Brd4 to acetylated lysine-310 of RelA and the role of this interaction in the transcriptional activation of NF- B. Stimulus-dependent acetylation of RelA at lysine-310 by p300 triggers the recruitment of Brd4 to the promoter via its bromodomains. Brd4 further activates CDK9 to phosphorylate the CTD of RNAPII and facilitate RNAPII-mediated transcription of NF- B target genes. on August 17, 2018 by guest dent transcription (Fig. 2D and E). The cooperative effect appears to derive from the sequential binding of p300 and Brd4 to RelA, since synergy depends on the HAT activity of p300 and the acetylation of lysine-310. Acetylation enhances the transcriptional activity of the transcription factor (3); how the acetylation of transcription factors is directly involved in the transcriptional activation is not clear. It has been suggested that acetylation determines the recruitment of factors involved in the regulation of transcription, including basal transcription factors (3, 47). For example, acetylation of p53 at lysines 373 and 382 promotes recruitment of the TFIID subunit TAF1 to the p21 promoter through interaction with the bromodomains of TAF1 (30). Our data suggest that acetylation of lysine-310 specifically recruits Brd4, a positive regulator of P-TEFb, thus

11 VOL. 29, 2009 Brd4 BINDS TO ACETYLATED RelA TO COACTIVATE NF- B 1385 providing a different mechanism for the acetylated transcription factor to activate the transcriptional machinery. Different acetylated transcription factors might recruit different transcriptional regulators within the transcriptional machinery. The ability of these acetylated factors to recruit different transcriptional regulators might be determined by the number of acetylated lysines, the flanking sequences around acetylated lysines, and the specific structure of the bromodomain proteins. It has been suggested that Brd4 is a general cofactor for most RNAPII-dependent transcription (40, 48). It will be interesting to determine whether other acetylated transcription factors similarly recruit Brd4 to activate RNAPII-dependent transcription and to identify a sequence motif that is specifically recognized by the bromodomains of Brd4. Brd4 recognizes diacetylated lysines-9/14 on histone H3 and lysines-5/12 on H4 via its two bromodomains (11). Similar to the binding to chromatin, both bromodomains are required for binding to acetylated lysine-310 (Fig. 5) and the two bromodomains function cooperatively as one functional unit to recognize acetylated lysine-310. Supporting this, in vitro GST pull-down experiments demonstrate that a single bromodomain was able to bind to a relatively small amount of fulllength acetylated RelA (Fig. 1B). However, the binding was significantly enhanced when both BD1 and BD2 were present (Fig. 1B). Consistent with these in vitro binding data, deletion of either of the domains abolished the interaction between Brd4 and RelA and the ability of Brd4 to coactivate NF- B (Fig. 5). For the acetylated RelA, both BD1 and BD2 interact with a single acetylated lysine-310. Although RelA can be acetylated at multiple sites, including lysines 218 and 221, these additional sites do not seem to contribute to the recruitment of Brd4, since mutation of lysines 218 or 221 does not affect the binding of Brd4 to acetylated RelA (data not shown). How do two bromodomains recognize a single acetylated lysine? Crystal structure analysis of the bromodomains of human Brd2 and TAF1 reveals that bromodomains form homo- or heterodimers to recognize acetylated lysines (25, 36). The homodimer of BD1 in Brd2 contains two acetyl-lysine-binding pockets and a negatively charged secondary binding pocket formed at the dimer interface (36). This secondary binding pocket, formed by homodimeric (e.g., BD1-BD1) or heterodimeric (e.g., BD1- BD2) bromodomains, likely helps determine the binding specificity between the bromodomains and the different acetylated lysines. Because the BD1 or BD2 of Brd4 and Brd2 exhibit high sequence similarity with 75% sequence identity (36), it is possible that BD1 and BD2 of Brd4 form a heterodimer to recognize two molecules of acetylated RelA. The additional secondary pocket might further recognize a histone or another transcription regulator such as the P-TEFb complex to activate transcription (26). In supporting this model, we found that BD1 associated with BD2 in vivo (B. Huang and L.-F. Chen, unpublished data). Brd4 is a positive regulatory component of P-TEFb, and it stimulates RNAPII-dependent transcription (14, 26, 48). P- TEFb is involved in the transcriptional elongation of a subset of NF- B genes. Based on the differential requirement for P-TEFb in NF- B transcription, NF- B target genes can be classed as P-TEFb-dependent or P-TEFb-independent genes (2, 31). Most genes mediating the physiological response of NF- B, including those for cytokines and chemokines, are P- TEFb dependent, whereas genes involved in the signaling pathway of NF- B, such as those for A20 and I B, are P- TEFb independent (1). Intriguingly, the E-selectin, TNF- and IL-8 genes, with downregulated expression in Brd4 knockdown cells (Fig. 3A and B), are P-TEFb-dependent genes, whereas the A20 and I B genes, whose expression is not affected by Brd4 knockdown (Fig. 3A and B and data not shown), are P-TEFb-independent genes. It appears that p300-mediated acetylation of lysine-310 and the subsequent recruitment of Brd4 to the promoters of NF- B target genes may play an important role in determining the differential requirement for P-TEFb for the transcription of NF- B target genes. p300, acetylated lysine-310 RelA, and Brd4 are recruited to the promoters of E-selectin and TNF- but not to the promoter of A20 after TNF- stimulation (Fig. 3C). It is not surprising that without p300, neither acetylated RelA nor Brd4 is recruited to the promoter (Fig. 3C). However, how p300 is differentially recruited to the promoters in response to TNF- stimulation is not clear and remains an interesting subject to be explored further. Previous studies reveal that P-TEFb promotes the transcription elongation of NF- B target genes by its association with RelA (2). However, the molecular determinants that regulate the assembly of RelA with P-TEFb are not known. Posttranslational modifications of RelA might play an important role in this regulation. Our findings here indicate that association of P-TEFb with RelA is mediated by Brd4, which is recruited to the promoters of NF- B target genes through acetylated lysine-310 (Fig. 4B). Brd4 functions as a bridging factor that binds to both acetylated lysine-310 and P-TEFb to activate P-TEFb-dependent NF- B target genes (Fig. 6D). Phosphorylation also regulates the association of P-TEFb with RelA. Phosphorylation of serine 276 is required for the binding of P-TEFb to RelA and for the transcription of a subset of NF- B target genes (37). It is worth noting that phosphorylation of serine 276 of RelA is required for the effective recruitment of p300 to RelA and the subsequent acetylation of lysine-310 (10). It is possible that enhanced binding of P-TEFb to phosphorylated RelA might derive directly from increased acetylation of lysine-310 triggered by phosphorylation. It is still possible that different posttranslational modifications differentially regulate the interaction between RelA and P-TEFb in different cell types in response to distinct stimuli. While Brd4 is recruited to the promoters of certain NF- B target genes via acetylated lysine-310 of RelA upon TNF- stimulation (Fig. 3C and 4B), other bromodomain-containing proteins might also be recruited to this acetylated lysine and regulate the function of NF- B in different physiological settings. Posttranslational modifications of NF- B have been proposed to construct a transcription factor code for NF- B and regulate the target gene specificity (8). NF- B target gene specificity could be dictated by the recruitment of different bromodomain-containing proteins to an acetylated lysine or to different acetylated lysines. Thus far, there are seven acetylated lysines that have been identified within RelA (5, 38); whether other acetylated lysines might similarly recruit different bromodomain proteins to regulate expression of various NF- B genes remains to be further explored. The identification of Brd4 as a NF- B coactivator provides a mechanism for the enhanced transcriptional activity of acety-