Mapping of the CBP–TAZ1 Binding Site on RelA–TAD

The RelA–TAD:CBP–TAZ1 interaction was previously mapped to RelA(477–504) region [20] (Figure 1A). We found only a weak interaction between this RelA fragment and TAZ1 as compared to the whole RelA–TAD [RelA(425–549)] and therefore scanned the entire TAD for TAZ1 binding using GST pull-down assays (Figure S1). A RelA construct spanning residues Lys425–Pro490 (in the TA2 region) emerged as the minimal fragment with maximal affinity to TAZ1. This fragment (henceforth referred to as RelA–TA2) was also optimal for structural analysis by NMR spectroscopy (Figure S1B,C). This highly acidic RelA–TA2 fragment is unstructured in its free state, as observed from the narrow dispersion of peaks in the [¹⁵N-¹H]–HSQC spectrum, but exhibits a well-dispersed spectrum indicative of a folded structure when in complex with TAZ1 (Figure 1C).

Structure of CBP–TAZ1 in the RelA–TA2:CBP–TAZ1 Complex

The structure of the complex between RelA–TA2 (residues 425–490) and CBP–TAZ1 (residues 340–439) was determined using distance and dihedral angle restraints derived from heteronuclear NMR experiments (see Table 1 for NMR statistics). TAZ1 is a scaffolding domain that interacts with intrinsically disordered TADs of various transcription factors, which fold upon complex formation [21]–[23]. The overall structure of TAZ1 in the complex (Figure 2) resembles that of TAZ1 in its free state (RMSD 1.6 Å) and in complex with other protein targets like HIF-1α (RMSD 1.8 Å), CITED2 (1.4 Å), and STAT2 (RMSD 1.3 Å) [21],[23]–[25]. As observed in our structure, the TAZ1 fold comprises four α-helices (α₁, α₂, α₃, and α₄) arranged in a roughly tetrahedral shape and is stabilized by three zinc atoms, each of which is bound to three Cys residues and one His residue. A prominent surface feature of the TAZ1 domain is a series of interlinked hydrophobic grooves that bind intrinsically disordered TADs (Figure S2).

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Figure 2. NMR structure of the RelA–TA2:CBP–TAZ1 complex.

RelA–TA2 is shown in green and TAZ1 in red. The three Zn²⁺ ligands are depicted in blue. The ordered region of the RelA fragment (Leu434–Val481) and TAZ1 (Ala345–Asp437) is depicted unless otherwise mentioned. (A) Twenty superimposed lowest energy NMR structures of RelA–TA2:CBP–TAZ1 complex. (B) Cartoon depiction of the lowest energy model of RelA–TA2:CBP–TAZ1 complex. (C) Electrostatic potential of solvent accessible surface of TAZ1 in complex with RelA–TA2. The positive potential is shown in blue and the negative in red. RelA–TA2 is shown as green ribbon with its acidic residues shown as red ball-and-sticks. RelA fragment (Gly430–Met483) is depicted in this figure.

https://doi.org/10.1371/journal.pbio.1001647.g002

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Table 1. NMR and refinement statistics for protein structures.

https://doi.org/10.1371/journal.pbio.1001647.t001

Structure of RelA–TA2 in the RelA–TA2:CBP–TAZ1 Complex

In the RelA–TA2:TAZ1 structure, the N- and C-terminal regions of the RelA–TA2 fragment used for structural analysis (Lys425–Thr433 and Ser482–Pro490, respectively) are dynamically disordered, with zero or negative [¹H]-¹⁵N heteronuclear NOEs (Figure S3A), and hence are omitted from the structures shown in Figure 2A,B. Our structure shows that RelA–TA2, essentially the Leu434–Val481 region, entirely wraps around TAZ1 in a predominantly extended conformation by docking itself through its well-conserved bulky hydrophobic residues into the interlinked hydrophobic grooves of TAZ1 (Figure 2 and Figure S4A). The C-terminal region (Glu471–Asn477) of RelA–TA2 folds into one short helix α_C that is anchored to the hydrophobic pocket formed by packing of α₁, α₂, and α₃ helices of TAZ1 (Figure 2B). Likewise, the N-terminal region (Leu434–Leu439) of RelA–TA2 also forms a short helix α_N, which is docked into the shallow hydrophobic groove formed at the junction of α₁ and α₄ of TAZ1. The α_N helix is dynamically disordered with only about 30% of the helical population, as calculated from the magnitude of the C-alpha and carbonyl secondary chemical shifts [26] (Figure S3B), and the small value of the [¹H]-¹⁵N heteronuclear NOE (∼0.3–0.5 for the α_N residues) confirms that this region is highly flexible on the nanosecond time scale (Figure S3A). RelA–TA2 also forms two additional helical turns, Leu449–Leu452 and Leu465–Val468, which make hydrophobic contacts in the α₁–α₂–α₃ and α₁–α₃ interfaces of TAZ1, respectively. The hydrophobic interactions are complemented by electrostatic interactions between the highly acidic RelA–TA2 region and the strongly electropositive surface of TAZ1. The region from Asp444–Asp448 in RelA–TA2 is particularly acidic, with four out of five residues being negatively charged, and passes through a deep cleft in the TAZ1 surface that is lined with basic residues (Lys366, Arg368, Arg369, Lys419, Lys438, Arg439) (Figure 2C). Our structural analysis was further corroborated by ITC experiments carried out at two different salt concentrations to study the electrostatic contribution of the binding event. Increasing the NaCl concentration from 50 to 150 mM weakened RelA–TA2:TAZ1 binding by 4-fold, consistent with a significant electrostatic contribution to binding from the complementary charges on TAZ1 and the RelA–TA2 region (Table 2).

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Table 2. Summary of dissociation constants and thermodynamic parameters obtained from ITC experiments.

https://doi.org/10.1371/journal.pbio.1001647.t002

RelA–TA2 Anchors on CBP–TAZ1 Through Multiple Hydrophobic Residues

The structure of the RelA–TA2:TAZ1 complex revealed that several hydrophobic residues in RelA–TA2 mediate binding within the exposed hydrophobic grooves of TAZ1. The RelA–TA2 amino acid residues, which extensively interact with the hydrophobic pockets of TAZ1, belong to two ψXXψψ motifs and a ψψXXψXXψ sequence (Figure 3A), where ψ is a bulky hydrophobic residue (Leu, Val or Phe in RelA–TA2) and X can be any residue (Figure 1B). The ψXXψψ motif is a generalization of the LXXLL motifs that are known to mediate protein–protein interactions [27]. Furthermore, Val481, Phe443, and the Leu residues belonging to the α_N helix of RelA–TA2 also contribute to complex formation as observed from the intermolecular NOEs (Figure S4B).

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Figure 3. RelA–TA2 docks itself onto a series of interlinked hydrophobic grooves of TAZ1 through a number of hydrophobic residues, which serve as anchor points.

Disruption of any of the anchor points leads to the destabilization of the whole binding architecture of the complex. ∼2,800 Ų of RelA–TA2 surface area are buried upon formation of RelA–TA2:TAZ1 complex. (A) Leu449 (left panel), Leu465 (middle panel), and Phe473 (right panel) residues of RelA–TA2 (shown as green spheres) buried into each face of the interlinked hydrophobic grooves of TAZ1. Residues of TAZ1 making contacts with the above mentioned hydrophobic residues of RelA are shown as red sticks. The other hydrophobic residues of RelA–TA2—namely, Leu452, Val468, Leu476, and Leu477—making vital contacts with TAZ1 are depicted as green sticks. (B) GST-pulldown experiments performed on the RelA–TA2 mutants binding to TAZ1. RelA fragment (Lys425–Pro490) with N-terminal GST-tag was used as the wild-type protein. All the mutations were made using this construct as a template. (C) Vertical bar chart of the association constants (left vertical axis, black bars) and enthalpy change (right vertical axis, grey bars) obtained from the ITC binding isotherms of the RelA–TA2:TAZ1 interaction corresponding to the respective RelA–TA2 wt and mutants (see Figure S5). The Leu449Ala and Leu465Ala mutants in RelA–TA2 lead to diminished association constants accompanied with decreased negative enthalpy change. The Phe473Ala mutant showed no binding (NB).

https://doi.org/10.1371/journal.pbio.1001647.g003

To investigate the role of the ψXXψψ motifs, we substituted alanine for Leu449 and Phe473 of RelA, each of which represents the first ψ residue of the motif. Both Leu449 and Phe473 are deeply buried in the molecular interface and participate in extensive hydrophobic interactions (Figures 1B and 3A). We also replaced the third ψ residue of ψψXXψXXψ sequence with Ala–Leu465Ala (Figure 3A, middle panel and Figure S4A). All three RelA mutants showed diminished to completely abrogated TAZ1 binding in the GST-pulldown assays (Figure 3B) and isothermal calorimetric (ITC) experiments (Figure S5, Table 2, Figure 3C), confirming the critical role of hydrophobic interactions in stabilizing the complex. The Leu449Ala and Leu465Ala substitutions each led to a 7-fold decrease in the TAZ1 binding affinity (Figure 3C and Table 2). As seen in the structure, the side chain of Leu465 is fully buried into a deep narrow hydrophobic pocket formed by Ile353, Leu357, Gln413, Ile414, His417, Trp418, Cys426, and Val428 of TAZ1 and Val468 of RelA (Figure 3A, middle panel and Figure S4A). This interaction contributes significantly to enthalpic stabilization of the complex, since the Leu465Ala substitution greatly decreases the enthalpic contribution to TAZ1 binding (Figure 3C). Leu449 binds in a shallower hydrophobic pocket, and accordingly substitution by alanine causes far less enthalpic destabilization (Figure 3A, left panel). The Phe473Ala mutation led to a complete loss of TAZ1 binding. In the structure, the side chains of Phe473, Leu476, and Leu477 (all the ψ residues of this ψXXψψ motif) are accommodated in the hydrophobic groove of TAZ1 (Figure 3A, right panel and Figure S4A). This arrangement is destabilized in the Phe473Ala mutant, leading to complete loss of TAZ1 binding. We speculate that a similar effect would be observed for Leu476Ala and Leu477Ala mutants.

Apart from the above-mentioned residues of RelA–TA2, the contacts between TAZ1 and the disordered α_N helix of RelA–TA2 contribute slightly to formation of the RelA–TA2:TAZ1 complex (Figure S4B). N-terminal truncation to remove residues Lys425–His440, thereby eliminating the entire α_N helix, decreases the affinity by only 1.6-fold (Figure 3C and Table 2). Likewise, deletion of residues Ser486–Pro490 from the disordered C-terminal end of RelA had no effect on TAZ1 binding (Figure S1D). However, further truncation to remove Val481–His485 greatly impaired binding by eliminating hydrophobic contacts between Val481 of RelA(TA2) and the side chains of Leu359, Leu381, and Pro382 in TAZ1. Replacement of Met483 by alanine had no effect on binding (Figure 3B), confirming that RelA residues beyond Val481 do not contribute to the interaction.

In summary, our structure shows that RelA–TA2 spirals through the exposed hydrophobic pockets of TAZ1 and anchors itself on TAZ1 at a number of points. As observed from the mutation studies, disruption of any of these anchoring residues of RelA–TA2 leads to the destabilization of the entire binding architecture of the entire complex.

Possible Role of Phosphorylation in RelA–TA2:CBP–TAZ1 Binding

The RelA–TA2 region also contains a well-conserved Ser467 (Ser468 in human RelA), which is a known phosphorylation site. Depending upon the stimulus, this site can be phosphorylated by GSK3β, IKKε, or IKKβ and plays a critical role in transcriptional regulation of NF-κB dependent genes [28]–[31]. Ser467 resides in the ψψXXψXXψ sequence of RelA–TA2 (Figure 1B), and in the complex it is close to the cluster of basic residues at the N-terminus of helix α₁ of TAZ1. Our ITC experiments show that a phosphomimetic mutant, RelA(Ser467Asp), binds TAZ1 with about 1.4-fold higher affinity than wild type RelA–TA2 and exhibits the same dependence on ionic strength, suggesting an enhanced but still nonspecific electrostatic interaction (Table 2 and Figure 3C). Thus, the increased negative charge of phosphoserine would likely enhance TAZ1 binding even further over nonphosphorylated RelA.

Comparative Binding Analysis of RelA–RHR:CBP–KIX and RelA–TA2:CBP–TAZ1 Interactions

In addition to the RelA–TA2:TAZ1 interaction, the RelA–RHR interacts with CBP–KIX via p-Ser276–RelA [20]. We compared the binding of RHR to KIX and TA2 to TAZ1 by GST-pulldown assay using nuclear extracts (NEs) of wild-type 3T3 cells stimulated with TNFα for 30 min. KIX and TAZ1 were purified as GST-fusion proteins and used to pull down nuclear-RelA (nRelA). As seen in Figure 4A, significantly lower amounts of nRelA were pulled down by GST–KIX compared to GST–TAZ1, which can be due to either of the two possibilities or both: first, a lower binding affinity of RelA–RHR:KIX compared to RelA–TA2:TAZ1 complex, and second, presence of only a small fraction of p-Ser276–RelA in the total pool of nRelA. In this context, we compared the binding affinities reported in the literature for KIX with other target proteins [32],[33] and found the reported binding affinities for KIX complexes typically 10–100-fold lower than those observed for various TAZ1 complexes [32],[33].

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Figure 4. Separating the components of the bipartite RelA:CBP interaction to delineate their individual roles.

(A, Upper panel) GST-pulldown assay using in-vitro purified GST–KIX/GST–TAZ1 to pull down nRelA from NEs of wild-type 3T3 cells stimulated with TNFα for 30 min. EDTA was used to remove the Zn²⁺ from TAZ1, thereby disrupting the TAZ1 structure. (Lower panel) Coomassie-stained SDS-PAGE gel showing the inputs for the GST-tagged proteins. (B) GST-pulldown assay using in vitro purified GST–TAZ1 to pull down nRelA from the NEs of RelA(wt/mutants) reconstituted rela⁻/⁻ cells stimulated with TNFα for 30 min. RelA(Leu449Ala+Phe473Ala) and RelA(Leu449Ala+Phe473Ala+Ser467Ala) mutants have slower mobility on SDS-PAGE gels due to unknown reasons. The expression level of the RelA(Ser276Ala) mutant in the reconstituted cell line was lower than that for the RelA(wt) (see Figure S6). Hence, a 3-fold excess of NEs was used for the RelA(Ser276Ala) mutant in this experiment. (C) Interaction of endogenous CBP with nRelA from the NEs of RelA(wt/mutants) reconstituted rela⁻/⁻ cells stimulated with TNFα for 30 min studied by co-immunoprecipitation assay of CBP/RelA followed by immunoblotting by RelA/CBP. The amount of NEs used for RelA(Ser276Ala) mutants was three times that of RelA(wt/TA2) mutants due to its lower expression levels. The RelA(Ser276Ala+Leu449Ala+Phe473Ala) mutant, which could potentially abolish the total RelA:CBP interaction, showed low and inconsistent expression and hence was not used in this study. * denotes IgG heavy chain. (D) The RelA mutants defective in RelA:CBP interaction also are defective binding to p300. The co-IP experiments for p300 was performed similarly to those in panel (C) of this figure. (E) RelA phosphorylated at Ser467 has higher CBP/p300 binding potential than the nonphosphorylated form. The immunoprecipitation assay was performed with α-RelA on CE (left column panels) and NE (right column panels) of rela⁻/⁻ cells reconstituted with RelA(wt) or RelA(Ser467Ala) mutant at three different time intervals after being stimulated with TNFα (5 ng/ml) in addition to the unstimulated cells. Co-immunoprecipitation assays were performed with α-CBP and α-p300 on NE in exactly the same manner as for the IP experiments above. The recruitment of nRelA by CBP/p300 is similar at 15 min relative to 30 min but diminishes at 45 min after TNFα stimulation despite the concentration of total RelA in the nucleus being significantly lower at 15 min after stimulation. Identical co-IP experiments with the RelA(Ser467Ala) mutant shows a direct proportionality in CBP/p300 binding with the concentrations of total nRelA at the different time points after stimulation. This indicates that the exclusively nuclear p-Ser467–RelA whose concentration peaks at about 10 to 15 min post-TNFα stimulation possesses a higher binding affinity for CBP/p300 compared to the nonphosphorylated form.

https://doi.org/10.1371/journal.pbio.1001647.g004

Overall, in either possible scenarios the total amount of RelA–TA2 bound to TAZ1 is significantly higher than that of RelA–RHR bound KIX, which should be reflected in a subset of RelA-activated genes that is regulated through RelA–TA2:TAZ1 interaction and is independent of p-Ser276–RelA:KIX binding.

RelA–TA2 Mutants Selectively Abrogate the RelA:TAZ1 Component of the Bipartite RelA:CBP/p300 Interaction

Next, we studied the role of the critical hydrophobic residues of RelA–TA2 (Leu449, Leu465, and Phe473) on TAZ1 binding in the context of full-length RelA. We used in vitro purified GST-tagged TAZ1 to pull down full-length wild-type (wt) or mutant RelA (Figure 4B) from the NEs of TNFα treated rela⁻/⁻ cells (see Materials and Methods) reconstituted with RelA(wt/mutants). We found that the RelA mutants, namely RelA(Leu449Ala+Phe473Ala) and RelA(Leu449Ala+Phe473Ala+Ser467Ala), were completely defective in binding to TAZ1 (Figure 4B). As expected, the Ser276Ala mutation had no effect on TAZ1 binding. Interestingly, the RelA(Leu449Ala) mutant did not show any noticeable decrease in binding affinity, contrary to the impaired TAZ1 binding observed in our in vitro GST pulldown and ITC experiments. A possible explanation is that in vivo posttranslational modifications like the phosphorylation of the well-conserved Ser467 could potentially mitigate the effects of the Leu449 mutation, thus increasing binding affinity of the RelA–TA2 to TAZ1, as observed by ITC experiments (Figure 3C and Table 2). Additionally, in the context of full-length RelA, the other domains of RelA could further influence the RelA–TA2:TAZ1 binding.

The above experiments established the minimal subset of mutations required in RelA to selectively prevent its interaction with the TAZ1 domain of CBP. These RelA mutants along with the RelA(Ser276Ala) were further tested for their interaction with endogenous CBP by coimmunoprecipitation (co-IP). Binding of all three RelA mutants [RelA(Leu449Ala+Phe473Ala), RelA(Leu449Ala+Ser467Ala+Phe473Ala), and RelA(Ser276Ala)] to endogenous CBP was impaired relative to RelA(wt) (Figure 4C). However, the disruption of either component of the RelA:CBP interaction—RHR:KIX or TA2:TAZ1—diminished but could not completely abrogate the entire RelA:CBP interaction. The RelA(Leu449Ala+Phe473Ala) and RelA(Ser276Ala) mutants were equally defective in binding to p300 (Figure 4D), whose TAZ1 domains share high sequence identity with CBP [22].

Role of RelA(Ser467) Phosphorylation in RelA:CBP/p300 Interaction

To further elucidate the role of Ser467 in RelA:CBP/p300 binding, we first studied the RelA(Ser467) phosphorylation event both spatially and temporally following TNFα stimulation. As shown in Figure S6A, the amount of p-Ser467–RelA is negligible in the unstimulated cells. Phosphorylation increases within 5 min of TNFα stimulation, reaching its maximum at 10 to 15 min followed by gradual dephosphorylation starting at 20 min after stimulation. We confirmed the presence of p-Ser467–RelA exclusively in the nucleus [34] (Figure 4E). The timing of RelA nuclear entry coincides with the p-Ser467–RelA maximum, indicating a possible correlation between the Ser467 phosphorylation event and an enhanced early RelA:CBP/p300 interaction. This correlation holds true only if Ser467 phosphorylation contributes towards increasing the affinity. To confirm this, co-IP experiments with α-CBP and α-p300 using NEs of RelA(wt) and RelA(Ser467Ala) mutants at three different time points after TNFα stimulation (Figure 4E) in addition to unstimulated condition were performed. Interestingly, in the RelA(wt) reconstituted cells, despite lower amounts of nRelA at 15 min than at 30 min of TNFα treatment, equivalent amounts of RelA co-immunoprecipitated with CBP/p300. The amount of nRelA bound to CBP/p300 at 45 min after TNFα stimulation diminished significantly. The nonlinearity of the amounts of nRelA co-immunoprecipitated with CBP/p300 with respect to the nRelA concentration is attributed to the enhanced binding affinity of p-Ser467–RelA for CBP/p300, as this nonlinearity was not observed for the identical experiment performed using RelA(Ser467Ala) mutant reconstituted rela⁻/⁻ cells (Figure 4E, Figure S6B).

Despite the role of p-Ser467–RelA in RelA:CBP/p300 binding, RelA(Leu449Ala+Phe473Ala) mutant [henceforth known as the RelA(TA2) mutant] was selected for further gene expression experiments due to two reasons: First, it had the minimal number of mutations required to impair the RelA–TA2:TAZ1 interaction. Second, RelA(Ser467) is a key determining factor for various other processes in the NF-κB pathway [28],[35],[36], which could further complicate and lead to misinterpretation of our experimental observations. Therefore, the RelA(Leu449Ala+Ser467Ala+Phe473Ala) mutant was not used further in this study.

RelA–TA2:TAZ1 Drives the Transcription of a Subset of NF-κB Target Genes Independent of RelA(Ser276) Phosphorylation

Next we investigated the effect of RelA:CBP/p300 interaction on the inducible expression profile of NF-κB target genes by qRT-PCR. Expression of the target genes was tested in the rela⁻/⁻ cells reconstituted with RelA(wt/mutants) following TNFα stimulation. Fibroblast cells were used because in this cell type the NF-κB-driven transcription is mainly carried out by the RelA subunit [16],[37]. rela⁻/⁻ cells reconstituted with an empty vector were used as a control, which also ensured that the genes tested were RelA dependent. The set of nine RelA-dependent genes are well-studied early TNFα responsive genes (Figure 5A) [16],[19],[38]. In this gene set, ptgs2 and nfkbia are NF-κB target genes believed to be independent of RelA:CBP/p300 interaction [19].

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Figure 5. A subset of RelA:CBP/p300 dependent genes is independent of RelA(Ser276) phosphorylation.

(A) Total RNA was isolated and purified from unstimulated cells at 30, 60, and 120 min of TNFα stimulation and prepared for analysis by RT-PCR. GAPDH was used as a reference and the 0 min time point for each cell line was used as the calibrator. The respective protein products of the genes (with names other than the gene name) are in parentheses. The rela⁻/⁻ cell line reconstituted with empty vector was included to ensure that RelA regulates the genes under study. The genes with their respective gene accession numbers (mouse) are as follows: csf2 (NM_009969), cxcl2 (NM_009140), icam1 (NM_010493), nfkbia (NM_010907), ptgs2 (NM_011198), tnf (NM_013693), tnfaip3 variant 1 (NM_009397), tnfaip3 variant 2 (NM_001166402), tnfsf9 (NM_009404), and vcam1 (NM_011693). Note that tnfaip3 has two variants. (B) The RelA–TA2:TAZ1 interaction activates a set of RelA target genes independent of p-Ser276RelA. The expression phenotype was determined for 99 genes that undergo at least 2-fold activation at 1 h of TNFα stimulation as compared to unstimulated RelA(wt) reconstituted rela⁻/⁻ cells. These TNFα-activated genes were grouped into four groups, A–D, based on the observed expression defect (with 95% confidence) with respect to RelA(wt), in the two distinct RelA:CBP/p300 interaction defective mutant cells (see main text). The gene list was sorted according to the differences between the RelA(wt) and RelA(TA2) mutant reconstituted cells at 1 h of TNFα treatment. The genes that switch to other groups upon relaxation of the confidence limit to 67% are labeled with their prospective group names. The genes that were tested by qPCR are marked with a blue asterisk. vcam1 did not satisfy the stringent criteria of at least 2-fold activation at 1 h of TNFα treatment and hence could not enter the list.

https://doi.org/10.1371/journal.pbio.1001647.g005

Our qRT-PCR data demonstrated inducible expression for all of the genes in the chosen set following TNFα stimulation in the RelA(wt) reconstituted rela⁻/⁻ cells. However, the expression of all the nine genes was significantly diminished in the cells reconstituted with RelA(TA2) mutant (Figure 5), suggesting that genes like ptgs2 and nfkbia, which are believed to be independent of RelA:CBP/p300 interaction, are actually dependent on RelA:CBP/p300 binding through the RelA–TA2:TAZ1 component (Figure 5A).

On the other hand, for the RelA(Ser276Ala) reconstituted cells, only seven of the nine genes displayed reduced gene expression. The relative mRNA levels of the remaining two genes, namely vcam1 (NM_011693) and ptgs2, were not affected by the RelA(Ser276Ala) mutation following TNFα stimulation (Figure 5). In our study, expression of nfkbia, which is believed to be independent of Ser276 phosphorylation, appeared to be partially dependent on it. The lower levels of nfkbia mRNA in the RelA(Ser276Ala) reconstituted cell line following TNFα stimulation as compared to those observed for RelA(wt) reconstituted cells could possibly be due to the lower expression of RelA(Ser276Ala) mutant in the cell relative to RelA(wt) and RelA(TA2) mutant (Figure S7).

Comparative Genome-Wide Expression Analysis Reveals That a Major Set of RelA Target Genes Is Regulated by the RelA–TA2:TAZ1 Interaction

To ascertain the effect of the RelA:CBP/p300 interaction on the RelA-regulated transcriptome, we carried out RNA-seq analysis of total mRNA isolated from RelA(wt/mutants) reconstituted cells prior to TNFα stimulation as well as at two different times points after TNFα stimulation (Figure 5B). The set of genes activated upon TNFα stimulation was classified into four groups: Group A contains genes that are dependent on both the interaction components of the RelA:CBP/p300 complex. Group B contains those dependent only on the RelA–TA2:TAZ1 interaction. Group C contains genes that are dependent only on p-Ser276–RelA, and Group D contains TNFα activated genes, which are RelA regulated but are altogether independent of RelA:CBP/p300 interaction, or the genes that are activated by transcription factors other than RelA. The RNA-seq data revealed that a majority of the RelA:CBP/p300 target genes belong to Group B (25 genes) followed by Group A (20 genes) and Group C (six genes). A sizable number of RelA target genes belong to RelA:CBP/p300 independent Group D (48 genes) (Figure 5B). Based on these experiments, it is clear that the RelA–TA2:TAZ1 interaction regulates a subset of RelA target genes independent of p-Ser276–RelA. Thus, the subset of RelA-activated genes dependent on RelA:CBP/p300 interaction is much larger than previously believed.

The gene expression analysis revealed nfkbia (IκBα) and tnfaip3 (A20), which control the negative feedback loop of the NF-κB pathway, are dependent on RelA:CBP/p300 interaction. IκBα essentially is the main NF-κB inhibitor, which strongly controls the negative feedback and is responsible for the fast turnoff of the NF-κB response after TNFα treatment in fibroblast cells [39]. Therefore, we asked whether reduced expression of nfkbia due to defective RelA:CBP/p300 interaction had any effect on the NF-κB pathway.

RelA:CBP/p300 Interaction Regulates the Temporal Control of IκBα and nRelA

To study the effect of the RelA:CBP/p300 interaction on nfkbia expression in detail, we investigated the temporal profile of the IκBα protein in rela⁻/⁻ cells reconstituted with RelA(wt/mutants) following TNFα stimulation. This study was also performed to ensure that the RelA mutations did not interfere with the upstream NF-κB pathway in the cytoplasm or with the DNA binding property of RelA. nfkbia is a prominent RelA target gene, which has a robust NF-κB responsive promoter. IκBα is a strong negative regulator of nuclear NF-κB (nNF-κB) and hence a key determinant of the nNF-κB temporal profile [39]. The canonical NF-κB pathway stimulated by TNFα has been extensively studied and focuses on the activation of the IKK-IκB-NF-κB signaling module. A mathematical model, which accounts for experimentally observed nNF-κB activity and IκB expression profiles, has been established [40],[41]. This model depends on the abundance and the transcriptional activation potential of NF-κB (RelA) [42],[43], among other parameters.

We observed that the normalized protein levels of RelA in the rela⁻/⁻ cells reconstituted with RelA-mutants varied relative to that in RelA(wt)-reconstituted cells (Figure 6A). This prompted us to simulate the effect of NF-κB abundance on the temporal profile of nNF-κB activity following TNFα stimulation (Figure 6B) before studying the transcriptional activation potential of the RelA-mutants. The simulations show that a decrease in the total NF-κB concentration leads to sustained nNF-κB activity and lower levels of IκBα synthesis following TNFα stimulation (Figure 6B).

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Figure 6. Impaired RelA–TA2:CBP–TAZ1 interaction disrupts the negative feedback loop of the NF-κB pathway.

(A) Expression of RelA mutants with respect to RelA(wt) in the RelA reconstituted rela⁻/⁻ cell lines. The protein expression levels of RelA(Ser276Ala) mutant was about three times lower than that for RelA(wt). For the quantitative estimation, the RelA to Actin signal ratio for each cell line was normalized with that for the RelA(wt) reconstituted rela⁻/⁻ cells (lower panel). (B) Model predictions for NF-κB (top) and total IκBα abundance (bottom) for different RelA expression levels (2×, 1.5×, 1×, 0.5×, and 0.2× relative to RelA(wt) values). In the nNF-κB curves, the peak is normalized to one for the highest level of nNF-κB activity and to zero for its levels in the resting cells (time = 0 min) for each individual expression level. Similarly, for the IκBα curves, the amount of IκBα in the resting cell (time = 0 min) is normalized to one and the minimum amount after degradation (basal levels) to zero following TNFα stimulation. (C) nRelA activity assay in response to TNFα stimulation as measured by EMSA using labeled κB probe and control NF-Y probe in the above-mentioned cells. The activity due to RelA binding to the κB probe was indicated by supershift of the EMSA band corresponding to the probe-bound NF-κB detected with anti-RelA antibody. 12 µg of NE was used for EMSA. (D) IκBα degradation and regeneration assay in RelA(wt/mutants) reconstituted rela⁻/⁻ cells following TNFα stimulation. IκBα protein levels after stimulation were monitored with respect to Actin. (E) Comparison of experimentally determined (symbols) and model predictions (solid line) based on adjusted RelA levels for NF-κB (left) and total IκBα abundance (right). Time courses for rela⁻/⁻ cells reconstituted with RelA(wt) (top), RelA(TA2) mutant (middle), and RelA(Ser276Ala) (bottom) stimulated with TNFα are shown. The experimental data were normalized as mentioned in panel (B) of this figure. The RMSD values correspond to the combined NF-κB and IκB datasets. (F) Comparison of experimentally determined (•) and model predictions (solid line) for NF-κB (left column) and total IκBα abundance (right column) for different degrees of suppression of IκBα mRNA production for RelA(TA2) mutant reconstituted rela⁻/⁻ cells.

https://doi.org/10.1371/journal.pbio.1001647.g006

We next compared the nuclear translocation of RelA and the temporal profile of IκBα protein following TNFα stimulation in rela⁻/⁻ cells reconstituted with wild-type or mutant RelA. To detect nNF-κB in NEs, the NF-κB DNA binding activity was measured by Electrophoretic Mobility Shift Assay (EMSA) (Figure 6C). nRelA levels were also monitored directly by immunoblotting (Figure S8). The time of entry of RelA(TA2) and RelA(Ser276Ala) mutants into the nucleus after TNFα stimulation is identical to that observed for RelA(wt). However, a prolonged nuclear residence time is observed for RelA(TA2) mutant relative to RelA(wt) reconstituted cells (Figure 6C and Figure S8). We also performed similar time course experiments using the whole cell extracts (WEs) of the respective cells and monitored the IκBα protein levels by immunoblot (Figure 6D). While the time of IκBα degradation and RelA entry into the nucleus are identical in the RelA(wt), RelA(Ser276Ala) and RelA(TA2) mutant reconstituted rela⁻/⁻ cells (Figure 6C,D), the temporal profile of IκBα regeneration and the nuclear residence time of RelA differed significantly (Figure 6C–E). For the RelA(TA2) mutant reconstituted cells, IκBα regeneration is delayed and does not reach its initial levels within the time course of the experiment. The RelA(Ser276Ala) mutant on the other hand did not show any significant delay in IκBα regeneration, although the IκBα level was reduced.

In order to address our observed results, we applied the available mathematical model of NF-κB regulation [40],[41]. We assumed that all parameters other than the RelA protein abundance and its transcriptional activation potential remained unchanged in all of the RelA reconstituted rela⁻/⁻ cell lines. Identical IκBα degradation and nuclear translocation of RelA for the three cell lines validated our assumption (through 30 min after TNFα stimulation in Figure 6C,D and Figure S8). Further, when the cell-line-specific RelA concentration estimates were incorporated into the model, the calculated profile for nNF-κB activity and IκBα abundance matched the experimental data of the RelA(Ser276Ala) mutant to a significant extent (Figure 6E, lowest panel), but did not resemble the observed profiles for the RelA(TA2) mutant (Figure 6E, middle panel). The root mean square deviation (RMSD) was used as a measure of agreement between calculated and the observed data. The results suggest that the observed partial expression of nfkbia in RelA(Ser276Ala) mutant reconstituted cells in our qRT-PCR data (Figure 5) was to a great extent due to lower expression of this RelA mutant in the cell line used here.

For the RelA(TA2) mutant, we investigated whether agreement between the experimentally observed and calculated data could be obtained by reducing the NF-κB-dependent IκBα mRNA production rate (k_mRNA) parameter to 25%, 12.5%, or 6.25% of its value for RelA(wt) (Figure 6F). We found that the lowest k_mRNA of 6.25% of RelA(wt) produced the best superimposition of the calculated and experimentally determined temporal profiles of the nNF-κB activity and IκBα regeneration as determined by the RMSD score (Figure 6F, top panel). It should be noted that the k_mRNA is directly proportional to the transcriptional activation potential of nNF-κB. Our study suggests that the Leu449Ala and Phe473Ala mutations in RelA lower the transcriptional activation of NF-κB by impairing the RelA–TA2:TAZ1 component of RelA:CBP/p300 interaction.

From the above results we conclude that while nfkbia is dependent on RelA–TA2:TAZ1 interaction, it is independent of RelA–RHR:KIX interaction. Thus, our data show that disrupting the RelA–TA2:TAZ1 component of the RelA:CBP/p300 interaction disturbs the temporal profile of the nuclear activity of RelA by interfering with the negative feedback loop of the NF-κB pathway. This could further deregulate the expression of the subset of NF-κB-dependent genes, which are independent of the overall RelA:CBP/p300 interaction.

RelA:CBP/p300 Interaction at the RelA Target Promoters

To further understand the nature of RelA:CBP/p300 interaction on the chromatin, ChIP-qPCR assay was performed to analyze the chromatin at six RelA target genes in unstimulated and TNFα-stimulated RelA(wt/mutants) reconstituted rela⁻/⁻ cells. We examined the chromatin for RelA occupancy on its target promoter set (Figures 7A and S9). As expected, we found that RelA(wt) was recruited to the promoters of all the six genes (although to a varied extent) at 30 min after TNFα treatment. This enrichment of RelA(wt) was diminished at 60 min after stimulation in congruence with its nuclear exit. The recruitment of RelA(TA2) and RelA(Ser276Ala) mutants to the promoter sites of highly expressed tnfaip3, nfkbia, and ptgs2 was significantly reduced at 30 min following TNFα treatment compared to RelA(wt), while for the mutants there was no significant RelA signal detected on the promoters with low mRNA expression levels (cxcl2, csf2, and tnf). In agreement with the prolonged nuclear retention of RelA(TA2) mutant due to a defective negative feedback loop, reduced amounts of the RelA(TA2) mutant remained bound to the tnfaip3, nfkbia, and ptgs2 promoters even at 60 min after stimulation. As indicated earlier, expression of tnfaip3 and nfkbia were reduced but not completely eliminated. Therefore, the mRNA levels of the RelA target genes correlate well with the amounts of RelA recruited to their respective promoters. These results clearly indicate defects in promoter recruitment of RelA mutants despite their uncompromised DNA binding potential (Figure 6C). This led us to hypothesize that CBP/p300 might be responsible for recruitment of RelA to its target gene promoters. If our hypothesis were true, then CBP/p300 should be constitutively loaded on the promoters and there should exist a direct correlation between the amounts of CBP/p300 present on the promoters to that of RelA recruited upon stimulation. To test our hypothesis, the chromatins were analyzed for CBP/p300 enrichment for our promoter set. The results show CBP/p300 enriched on all of the promoters to a varied extent, with no significant change upon TNFα stimulation (Figures 7B and S9). A comparison of CBP/p300 and RelA signals in this assay revealed a direct correlation between enrichment of RelA upon stimulation with that of CBP/p300 (Figure 7C). Similar RelA recruitment was observed for RelA(wt), RelA(TA2), and RelA(Ser276Ala) mutants on RelA target promoters independent of RelA:CBP/p300 interaction (Figure S10).

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Figure 7. The RelA:CBP/p300 interaction influences RelA recruitment to its target gene promoters.

ChIP was performed to analyze the chromatin of six TNFα-induced RelA target genes in unstimulated and TNFα (5 ng/ml) stimulated for 30 and 60 min RelA(wt/mutants) reconstituted rela⁻/⁻ cells. Immunoprecipitation using antibodies against RelA, CBP, p300, histone H3, and H3K27ac were performed. The H3K27ac antibody showed higher signals compared to the total H3 antibody due to differences in their qualities. The results shown are the average of two independent replicates, with standard deviations shown as error bars. (A) Fold enrichment of RelA on the promoter of six RelA target genes after TNFα treatment relative to that in unstimulated cells. (B) CBP/p300 enrichment as depicted by the respective %input for six RelA target genes in unstimulated and TNFα-stimulated RelA(wt/mutants) reconstituted rela⁻/⁻ cells. (C) Comparison of RelA, CBP, p300, H3, and H3K27ac ChIP signals in unstimulated RelA(wt) reconstituted rela⁻/⁻ cells for six RelA target genes.

https://doi.org/10.1371/journal.pbio.1001647.g007

Since acetylation of histone-3 (H3) at Lys27 by CBP/p300 is a characteristic of active genes [44], we further examined the levels of H3 occupancy and compared them with that of acetylated histone H3 at Lys 27 (H3K27ac). We expected to observe increased H3K27ac signals on the promoters only after TNFα treatment as a mark of activated promoters as compared to those in unstimulated cells. To our surprise we found H3K27ac enriched in the unstimulated promoters, whose levels remained constant even after TNFα stimulation. Additionally, the H3K27ac levels were directly proportional to the amount of CBP/p300 present on the specific promoters (Figure 7C). This suggests that CBP/p300 bound to promoters of RelA targets is poised for transcription initiation. Upon nuclear entry RelA is recruited to its respective promoter targets by CBP/p300, which is preloaded on these sites, leading to transcription activation.

Upon TNFα stimulation in RelA(TA2) or RelA(Ser276Ala) mutant reconstituted rela⁻/⁻ cells, the mRNA expression levels of the highly expressed genes like tnfaip3 and nfkbia registered only partial reduction (Figure 5A). This is because of a reduced but significant amount of RelA recruited to their respective promoters (Figure 7A). Both the promoters also maintain high levels of CBP/p300, thereby leading to recruitment of the RelA(TA2) and RelA(Ser276Ala) mutants, which possess reduced but significant CBP/p300 binding affinity (Figure 4C,D). This further proves that the RelA-dependent gene expression is regulated by the total amount of RelA recruited on the promoter by CBP/p300.

Overall our study indirectly shows that CBP/p300, which marks the promoter/enhancers of genes, also aids in recruiting RelA to the promoters of its target genes.

Protein Expression and Purification

The GST tagged mouse RelA fragments were expressed in E. coli [BL21(DE3) cells] and purified by affinity chromatography using Glutathione sepharose beads (GE Life Sciences). The purified protein fragments were then dialyzed in the binding buffer of the respective experiments.

For NMR experiments, the mouse RelA fragments were expressed as fusion proteins with an N-terminal His6-tag followed by the 58 amino acid residue B1 domain of streptococcal protein G (GB1) as per previous reports [57],[58]. The protein was isotopically labeled by growth in M9 minimal media supplemented with ¹⁵N ammonium chloride and ¹³C-glucose. The fragments were purified under denaturing conditions on a Ni-NTA (SIGMA) column followed by dialysis against cleavage buffer [20 mM Tris (pH 7.5), 200 mM NaCl] at 4°C. The His6–GB1 peptide was cleaved by thrombin at room temperature for 18 h. The cleaved RelA peptides were further purified by reverse-phase HPLC (RPHPLC) using a C18 column on ÄKTA purifier system. The purified peptides were lyophilized immediately. For NMR and ITC experiments, the TAZ1 domain (residues 340–439 of mouse CBP) was expressed and refolded as described previously [24]. For ITC experiments, the His6-tag was cleaved from the GB1–RelA peptides by TEV protease (in house purified).

GST-Pulldown Assay

The GST-pulldown assay was carried out in binding buffer (20 mM Tris pH 7.5, 150 mM NaCl, and 0.5 mM DTT). GST-RelA fragments (20 µg) were mixed with purified TAZ1 (10 µg) and 20 µl of a 50% slurry of washed glutathione sepharose beads and incubated at 4°C for 30 min in a rotator. The beads were then washed 5 times with 500 µL of binding buffer containing 0.3% TritonX-100, followed by elution and analysis by 15% SDS-PAGE.

Isothermal Titration Calorimetry (ITC)

ITC experiments were performed at 25°C using a MicroCal Omega VP-ITC instrument. For ITC experiments, GB1-tagged RelA-TA2 fragments were used. The proteins were dialyzed overnight in the ITC buffer [20 mM Tris (pH 8.0), 50 mM or 150 mM NaCl and 1 mM DTT]. The protein concentrations were in the range of 10 to 18 µM of RelA–TA2 peptide in the cell and 169 to 185 µM TAZ1 in the syringe. Protein concentrations were determined by absorbance at 280 nm. The GB1-tag was used for accurate concentration determination of the RelA–TA2 peptides, which have a low extinction coefficient. The GB1-tag did not interact with TAZ1. A typical ITC experiment consisted of a total of 25 injections, using 5–6 µl of TAZ1 for the first injection followed by 24 injections of 11–12 µl of TAZ1 into the cell containing either blank or RelA peptide. Data were analyzed using a single-binding site model in the MicroCal Origin Software. The stoichiometry of binding ranged from 0.9 to 1.2. Errors for the K_d values were estimated from duplicate measurements. All the buffers used were made using water purged with nitrogen.

NMR Spectroscopy and Structure Calculations

NMR experiments were carried out at 25°C on Bruker DRX600 MHz and Avance 900 MHz spectrometers. The typical protein concentration for NMR experiments was 0.7–1.2 mM. All spectra were referenced to external DSS. Refolded TAZ1 and RelA fragments were mixed to form the complex followed by overnight dialysis in NMR buffer (20 mM Tris pH 6.5, 40 mM NaCl, and 2 mM DTT). We added 5% D₂O to the sample prior to NMR experiments. NMR data were processed using NMRPipe [59] and analyzed using CARA [60]. Backbone and side-chain resonances were assigned using standard triple resonance experiments [61]. Distance restraints were obtained from 3D ¹⁵N-edited NOESY-HSQC (τ_m = 100 ms) and ¹³C-edited NOESY-HSQC (τ_m = 100 ms) spectra. Intermolecular NOEs were derived from ¹²C-filtered-¹³C-edited NOESY-HSQC (τ_m = 200 ms) [62] experiments. Unambiguous intermolecular NOES were assigned manually and were used in the initial structure calculations, which were performed using CYANA [63] with CANDID [64]. Chemical shift-based torsion angles were obtained using TALOS+ [65]. Distance and torsion angle restraints were imposed to ensure tetrahedral geometry for the zinc atoms of TAZ1 [25]. A total of 200 structures were generated in CYANA and were further refined by restrained molecular dynamics simulated annealing using the AMBER 10 software package [66],[67].

These structures were subjected to 2,000 steps of energy minimization, followed by 20 ps of simulated annealing in vacuum and another 20 ps of simulated annealing using a generalized Born solvent model [68] and finally 2,000 steps of energy minimization. During the simulated annealing, the system was heated to 1,000 K for the first 2 ps, followed by 4 ps at constant temperature, and final cooling to 0 K for the remaining 14 ps. Force constants were 30 kcal mol⁻¹ Å⁻² for NOE restraints and 100 kcal mol⁻¹ rad⁻² for dihedral angle restraints. The 20 lowest energy structures were analyzed using PROCHECK-NMR [69].

Cell Culture and Reagents

Immortalized rela⁻/⁻ fibroblast cells [70] were reconstituted with retroviral vectors pBABE-puro with mouse RelA(wt/mutants) inserted or with empty vector controls. A final concentration of 5 ng/ml TNFα (Roche Diagnostics) was used for stimulation. Antibodies used for IB and IP in this study were from Santa Cruz: anti-RelA (sc-372g), anti-IκBα (sc-371), anti-CBP (sc-369), anti-Actin (sc-1615) and anti-USF2 (sc-861), anti-α-Tubulin (sc-5286), anti-p300 (sc-584 and sc-585), and Cell-Signaling Technology: p-Ser468-RelA (#3039). cOmplete, EDTA-free Protease Inhibitor cocktail was from Roche Diagnostics. Protein G-beads were from GE Life Sciences.

Preparation of Cytoplasmic, Nuclear, and WE

For the NE, 3T3 cells were cultured in 10 cm³ plates and harvested using PBS buffer. Cell pellets were prepared by 5 min centrifugation at 500 g at 4°C and then resuspended in CE buffer (0.32 M Sucrose, 10 mM Tris HCl pH 8.0, 3 mM CaCl₂, 2 mM MgOAc, 0.5% NP-40, 1 mM DTT, 0.5 mM PMSF) followed by centrifugation at 500 g for 5 min at 4°C. The supernatant (CE) was flash frozen and stored at −80°C. The nuclear pellet was washed twice with CE buffer without NP-40 followed by centrifugation as above. The washed pellet was resuspended in hypotonic buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 20 mM KCl, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF] followed by gradual addition of high salt buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 800 mM KCl, 25% glycerol, 1% NP-40, 0.5 mM DTT, 0.5 mM PMSF, and 1× complete EDTA-free protease inhibitor]. The samples were incubated with mild agitation for 45 min at 4°C followed by centrifugation at 12,000 g at 4°C for 15 min. The supernatant was collected as NE and was further subjected to quantification.

For WE, the cells were harvested as above followed by addition of RIPA lysis buffer containing protease inhibitor cocktail. The samples were centrifuged at 12,000 g at 4°C. The supernatant was collected as the WE. All of the buffers contained phosphatase inhibitor cocktail.

Immunoblotting and Co-Immunoprecipitation

For immunoblotting, WEs and NEs were resolved on SDS-PAGE and immunoblotted with respective antibodies. For co-immunoprecipitation (co-IP) assay, NEs were incubated with anti-RelA or anti-CBP antibodies for 1 h followed by addition of prewashed protein-G beads and incubation for 4 h. The beads were then washed thoroughly and resolved on SDS-PAGE and immunoblotted with anti-RelA and anti-CBP antibodies. For quantitative analysis of the immunoblots, the loading controls were analyzed from the same gel as for the protein monitored.

Gene Expression Analysis

RNA was extracted and purified as per the manufacturer's protocol using the RNeasy kit (QIAGEN). We reverse transcribed 1 µg of the quantified RNA using superscript II RT system (Invitrogen) and poly-dT primers. cDNA fragments generated were analyzed by qPCR using the KAPA system and Realplex thermocycler (EPPENDORF). PCR amplification conditions were 95°C (4 min) and 40 cycles of 95°C (15 s), 56°C (30 s), and 72°C (30 s). Primer pairs used were from previous studies [38],[71] and were tested by melt-curve analysis. Δ(ΔCt) method was used for data analysis with gapdh as normalization control to derive fold induction over basal levels. For each set of time-course experiment (0 min, 10 min, 60 min, and 120 min) RNA was collected from empty vector, RelA(wt), RelA(TA2), RelA(Ser276Ala) mutant reconstituted rela⁻/⁻ cells stimulated with 5 ng/ml TNFα. For qRT-PCR experiments, the data were collected for three set of biological replicates.

Next generation sequencing was performed at the UCSD Biogem core facility. Reads were obtained from Illumina HiSeq 2000 sequencing system. Sequencing experiments were performed on the total mRNA extract from RelA(wt), RelA(TA2), and RelA(Ser276Ala) reconstituted rela⁻/⁻ cells at two different time points after TNFα (5 ng/ml) treatment in addition to unstimulated cells. RNA-seq was performed on nine different experimental conditions in total. Two independent replicates were used for each experimental condition. Reads were mapped to the mm10 mouse genome using TopHat [72], and the transcriptome assembly was generated using Cufflinks [73] followed by detection of differentially expressed genes using Cuffdiff.

Computational Simulation

The ordinary differential equation (ODE)-based model for NF-κB regulation [40] was adapted (Table S1) by increasing IκB mRNA degradation rates by a factor of 1.8 in line with recent measurements [74] and replacing the terms describing transcription-factor-dependent mRNA production for IκBα and IκBε by those used in [41] (Model 3, additive Pol II recruitment). Model simulations were carried out with the “Stiffness Switching” method of the NDSolve function in the package Mathematica 8 (Wolfram Research, Champagne, IL) using a numerically defined IKK activity induced by exposure to TNFα Table S2 [40].

ChIP Experiments

Samples for ChIP experiments were prepared using SimpleChIP Plus Enzymatic chromatin IP kit (Cell Signaling Technology #9005) according to the manufacturer's protocol with some modifications. Primer pairs used were from previous studies [38],[71],[75] and were tested by melt-curve analysis. ChIP experiments were performed with anti-H3 (Cell Signaling #4620), anti-acetyl H3K27 (Cell Signaling #8173), anti-RNA Pol II (Santa Cruz sc-900), anti-p300 (Santa Cruz sc-585), anti-CBP (Santa Cruz sc-369), anti-RelA (Santa Cruz sc-372), and anti-IgG (Cell Signaling #2729)

Accession Numbers

Coordinates and structural restraints for the RelA–TA2:CBP–TAZ1 complex have been deposited in the Protein Data Bank under the accession number 2LWW. The chemical shifts have been deposited in the BioMagResBank, accession number 18650. The RNA-seq data discussed in this publication have been deposited in NCBI's Gene Expression omnibus [76] and are accessible through GEO Series accession number GSE46213. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46213).