Fig. 1. p300 acetylates RelA in vivo. (A) The HAT activity of p300 is required for acetylation of RelA. 293T cells were co-transfected with the indicated amounts of T7-RelA and wild-type p300 or p300 (HAT-) mutant expression vector DNA. Acetylation was detected by immunoblotting of anti-T7 immunoprecipitates with anti-acetylated lysine antibodies (Cell Signaling) (middle panel). Levels of p300, p300 (HAT-) and T7-RelA are shown in the upper and lower panels, respectively. (B) p300 (HAT-) inhibits the acetylation of RelA. COS-7 cells were co-transfected with expression plasmid DNA encoding T7-RelA alone (5 µg) or in combination with p300 (HAT-) (10 µg) or adenovirus E1A (10 µg). Acetylation levels of RelA (upper panel) were assessed by [³H]sodium acetate labeling as described previously (Chen,L. et al., 2001). Levels of T7-RelA expression in each sample are shown in the lower panel.

Fig. 3. RelA K221R, K310R and RelA-KR display impaired transcriptional activation properties. (A) 293T cells were transfected with E-selectin–luciferase reporter plasmid DNA (0.1 µg) and the various lysine-to-arginine substitution mutants of RelA (1 ng). Luciferase activity was measured as described previously (Chen,L. et al., 2001). Results represent the average of three independent experiments ± SD. (B) The HAT-deficient mutant of p300 inhibits RelA-mediated transactivation of RelA. 293T cells were transfected with E-selectin–luciferase reporter plasmid DNA (0.1 µg) and expression plasmid DNA encoding RelA (5 ng) alone or with graded amounts of p300 (HAT-) expression plasmid DNA (100 ng, 200 ng and 400 ng). Luciferase activity was measured as in (A). (C) Lack of cooperative transactivation of RelA-KR and p300/CBP. 293T cells were co-transfected with E-selectin–luciferase reporter plasmid DNA (0.1 µg) and either wild-type RelA or RelA-KR expression plasmid DNA (1 ng), alone or in combination with increasing amounts of p300 expression vector DNA (100 ng and 200 ng). Luciferase activity was measured as in (A).

Fig. 5. Acetylation of lysine 221 in RelA plays a key role in regulating assembly with IκBα. (A) p300 inhibits the interaction of RelA with IκBα in a mammalian one-hybrid assay. Expression plasmids encoding wild-type RelA (0.1 µg) were co-transfected into 293T cells with plasmids encoding the GAL4 DNA binding domain fused to IκBα (pFA-IκBα) (1 ng) together with a GAL4 enhancer–luciferase reporter (0.1 µg) (pFR-luc) (Stratagene) in the presence of increasing amounts of either p300 or p300 (HAT-) mutant expression plasmids (100 ng and 200 ng, respectively). Luciferase activity was measured as in Figure 3A. (B) p300 prevents the cytoplasmic sequestration of wild-type RelA but not RelA-KR induced by co-expression of IκBα. HeLa cells were co-transfected with expression vectors encoding GFP–RelA (0.2 µg) or GFP–RelA-KR and IκBα (50 ng) together with wild-type p300 expression vector (1 µg). Note that in the presence of p300, the majority of GFP–RelA localizes in the nucleus while GFP–RelA-KR localizes predominantly in the cytoplasm. The percentage of cells displaying the depicted phenotype derived from inspection of at least 500 cells present in multiple fields in two independent experiments is presented in the bottom right corner of each panel. (C) The RelA-KR mutant displays a stronger interaction with IκBα in vivo. Interaction of IκBα with wild-type RelA or the lysine-to-arginine substitution mutants was tested in the mammalian one-hybrid assay system as in (A). (D) Whole-cell extracts from 293T cells co-transfected with expression vectors encoding wild-type RelA or the various RelA substitution mutants as indicated together with expression vectors encoding p50 and p300 were prepared and tested in EMSA. Binding of the nuclear complexes to DNA in the presence (lower panel) or absence (upper panel) of added recombinant of GST–IκBα (50 ng) is shown.

Fig. 2. Lysines 218, 221 and 310 in RelA correspond to major sites of p300-mediated acetylation. (A) Left: schematic depiction of the various C-terminal deletion mutants of RelA tested and the position of each of the 18 lysines that form potential acetylation sites. Asterisks indicate the relative positions of lysines 218, 221 and 310. Right: p300-mediated acetylation of the RelA deletion mutants. 293T cells were co-transfected with expression vector DNA (0.5 µg) encoding either full-length RelA or the indicated deletion mutants with p300 expression plasmid DNA (2 µg). Acetylation levels of each RelA deletion mutant (upper panel) were detected as in Figure 1A. Levels of expression of each of the RelA deletion mutants are shown in the lower panel. All these deletion mutants retain the ability to bind to p300 at comparable levels (see Supplementary figure 1). (B) Schematic depiction of the domain structure of RelA and the protein sequence alignment of RelA (amino acids 198–320), c-Rel, RelB, p50/p105 and p52/p100. Note that lysines 218 and 221 are highly conserved while lysine 310 is uniquely present in RelA. (C) In vivo acetylation assay of K-to-R substitution mutations in the context of full-length RelA. Mutants corresponding to K218/221R, K310R and RelA-K218/221/310R (designated RelA-KR) were transfected into COS-7 cells. Acetylation levels of wild-type RelA and the various substitution mutants were assessed by [³H]sodium acetate labeling as in Figure 1B (upper panel). Levels of expression of wild-type RelA and the various substitution mutants are shown in the lower panel. (D) 293T cells were transfected with the various lysine-to-arginine substitution mutants of RelA (0.5 µg) together with expression vector encoding p300 (2 µg). The level of acetylation of wild-type RelA and the various mutants was tested by immunoprecipitation with an anti-acetylated lysine antibody (Cell Signaling) followed by immunoblotting with anti-T7 antibodies (levels of expression of wild-type RelA and each mutant are shown in the lower panel).

Fig. 4. The hypoacetylated RelA-KR mutant displays lower binding affinity for κB enhancer DNA. (A) Analyses of κB enhancer binding activity of wild-type RelA and the lysine-to-arginine mutants of RelA. Whole-cell extracts from 293T cells transfected with expression vectors encoding p300 and wild-type RelA or the indicated substitution mutants of RelA were prepared. EMSA was performed (upper panel) as described in Materials and methods. The levels of expression of RelA and each RelA mutant are shown in the lower panel (B) NF-κB heterodimers composed of p50 and wild-type RelA or the RelA-KR mutant display different sensitivity to unlabeled κB enhancer competition in EMSA. Whole-cell extracts from 293T cells co-transfected with expression vectors encoding wild-type RelA or the RelA-KR mutant together with p50 and p300 expression plasmids were isolated and tested in EMSA for binding to ³²P-radiolabeled κB enhancer probes in the presence of increasing amounts of unlabeled κB enhancer oligonucleotides (cold probe). (C) Analyses of the off-rate of RelA binding to the κB enhancer. The off-rate of binding of wild-type RelA and RelA-KR to the κB enhancer was tested by EMSA as described in the Materials and methods. The results for wild-type RelA (left panel) and the RelA-KR mutant (right panel) are shown. (D) Measurement of the off-rate of binding of wild-type RelA in the presence of p300 (HAT-) mutant and wild-type RelA and the collection of lysine-to-arginine substitution mutants in the presence of p300 and p50. Binding to ³²P-radiolabeled κB enhancer was measured as in (B) followed by assessment of the EMSA results by radiodensitometry. The results presented are an average of two independent experiments.

Fig. 6. GFP–RelA-KR and K221R mutants are principally localized in the cytoplasm of expressing cells. (A) HeLa cells were transfected with GFP–RelA (2 µg) (panels g, h and i), GFP–RelA-KR (2 µg) (panels d, e and f) or GFP–RleA-K221R (panels g, h and i) expression plasmid DNA. Selected cultures of these cells were treated with TSA (400 nM, 5 h) (panels b, e and h) or with leptomycin B (20 nM, 2 h) (panels c, f and i). (B) GFP–RelA-KR is principally expressed in the nucleus of IκBα^–/– MEFs. MEFs derived from IκBα^–/– mice were transfected with GFP–RelA-KR (1 µg) (panel a) expression vector DNA. The IκBα^–/– MEFs were also reconstituted with expression vectors encoding either wild-type IκBα (panel b) or an IκBα NES-deficient mutant (panel c). (C) A model summarizing how acetylation of different lysine residues regulates distinct functions of RelA. Acetylation of lysine 221 increases DNA binding affinity for the κB enhancer and prevents the association of RelA with IκBα; acetylation of lysine 310 likely controls the association of an unknown factor that is required for full transactivation by RelA.