IκB Kinase α and p65/RelA Contribute to Optimal Epidermal Growth Factor-induced c-fos Gene Expression Independent of IκBα Degradation

Mitogenic activation of expression of immediate-early genes, such as c-fos, is controlled through signal-induced phosphorylation of constitutively bound transcription factors that is correlated with a nucleosomal response that involves inducible chromatin modifications, such as histone phosphorylation and acetylation. Here we have explored a potential role for the transcription factor NF-κB and its associated signaling components in mediating induction of c-fos gene expression downstream of epidermal growth factor (EGF)-dependent signaling. Here we show that EGF treatment of quiescent fibroblast does not induce the classical pathway of NF-κB activation through IκB kinase (IKK)-directed IκBα phosphorylation. Interestingly, efficient induction of c-fos transcription requires IKKα, one of the subunits of the IκB kinase complex. The NF-κB subunit, p65/RelA, is found constitutively associated with the c-fos promoter, and knock-out of this transcription factor significantly reduces c-fos gene expression. Importantly, EGF induces the recruitment of IKKα to the c-fos promoter to regulate promoter-specific histone H3 Ser10 phosphorylation in a manner that is independent of p65/RelA. Collectively, our data demonstrate that IKKα and p65/RelA contribute significantly to EGF-induced c-fos gene expression in a manner independent of the classical, IκBα degradation, p65/RelA nuclear accumulation response pathway.

ubiquitination and subsequent proteasome-dependent degradation. This process frees NF-B to accumulate in the nucleus and to associate with specific gene regulatory regions to control gene expression (1)(2)(3)(4)(5). The IKK complex contains two highly conserved catalytic subunits, namely IKK␣ and IKK␤, of which IKK␤ appears to be the dominant kinase controlling IB phosphorylation downstream of cytokine-induced signaling (1,3). However little to no information exists regarding direct roles for IKK or NF-B in controlling growth factor-induced gene expression.
EGF family members interact with receptors belonging to the EGF receptor family, including EGF receptor, epidermal growth factor receptor family protein (ErbB)2, ErbB3, and ErbB4 (6). Interaction of these signaling molecules with the receptors initiates intracellular signaling cascades that lead to activation of a number of transcription factors such as AP-1 and STATs (7). It has previously been reported that EGF induces NF-B nuclear levels in cell types such as A431 cells and in several breast cancer cell lines that overexpress EGF receptor (8). However, it remains inconclusive whether EGF signaling to NF-B occurs in EGF receptor-normalized cell lines such as fibroblasts, and relevant gene targets are unknown.
The immediate early genes (including c-fos, c-jun, and c-myc) are characterized by their rapid inducibility in response to a variety of signals, including mitogenic stimulation (9). c-fos has been the most extensively studied of this group of genes relative to induction of gene expression, with evidence demonstrating the involvement of sequence-specific transcription factors and associated co-factors controlling transcription induction in a manner dependent on the activation of upstream signal transduction cascades (10 -12). A key regulatory element in the c-fos promoter is the serum response element (SRE), which interacts in a constitutive manner with the serum response ternary complex containing SRF, Elk-1, and the transcriptional co-activator CBP/p300 (10,11,13,14). It has been demonstrated that phosphorylation of Elk-1 by MAPKs leads to the activation of the ternary complex-associated CBP/p300 histone acetyltransferase co-activator (14). Additionally, the c-fos promoter undergoes inducible histone H3 phosphorylation and acetylation following mitogenic stimulation that correlates with immediate-early gene induction (15)(16)(17). The kinase that controls histone H3 phosphorylation downstream of mitogeninduced signaling has been proposed to be either MSK1/2 or ribosomal S6 Kinase 2 (18,19). More recent evidence supports the role of MSK1/2 in controlling this response (18). Recently we and others (20,21) described IKK␣ as a critical kinase involved in controlling TNF-induced H3 Ser 10 phosphorylation at certain NF-B-dependent promoters in a manner dependent on the recruitment of RelA/p65 to the promoters. Here we have analyzed the potential involvement of the p65/RelA subunit of NF-B and IKK␣ in controlling EGF-induced c-fos gene expression.
Chromatin Immunoprecipitation Assay-ChIP analysis was performed following a protocol provided by Upstate Biotechnology, Inc. under modified conditions and as previously described (20). The following promoter-specific primers were used; primer pair CACGGCCGGTC-CCTGTTGTTC and GTCGCGGTTGGAGTAGTAGGCG was used to amplify the proximal region of the c-fos promoter. The primers for ␤-globin are CAGCATGTGCTGAGGACTTGG and ACTGCCTTCA-GAGAATCGCCC. Quantitative real time PCR was performed in triplicate to determine the association of phospho-Ser 10 -H3, acetyl-Lys 9 -H3, and acetyl-Lys 14 with the c-fos promoter by using the above oligonucleotide primers and input DNA standards diluted in 3-fold increment from 10 to 0.01% with SYBR Green Master Mix and the ABI Prism 7700 sequence detection system.
Quantitative Real Time PCR-Total RNA was prepared from MEF, IKK␣ Ϫ/Ϫ , IKK␣ ϩ/ϩ , IKK␤ Ϫ/Ϫ , or p65 Ϫ/Ϫ cells using TRIzol (Invitrogen) as recommended by the manufacturer. For real time PCR, the cDNA was prepared with random primers (Invitrogen) and analyzed in triplicate with the SYBR Green Master Mix (Qiagen) for 15 min at 95°C for initial denaturing, followed by 40 cycles of 95°C for 30 s and 63°C for 30 s in the ABI sequence detection system. The oligonucleotide primers used to analyzed IB␣ transcripts contained the sequences 5Ј-GATC-CGCCAGGTGAAGGG-3Ј and 5Ј-GCAATTTCTGGCTGGTTGG-3Ј. Primers used to analyze the c-fos and 18 S ribosomal RNAs were obtained using an Ambion gene-specific relative reverse transcription-PCR kit with the suggested conditions.
Western Blot Analysis-Western blot analysis was performed after preparing nuclear, cytoplasmic, or whole cell extracts and separating proteins by SDS-PAGE followed by immunoblotting with antibodies as indicated. Extractions of acid-soluble proteins were done according to the protocol described by Upstate Biotechnology, Inc. and resolved on 10 -20% Tris-Tricine SDS-PAGE gels.

Analysis of the Classical NF-B Activation Pathway in
Response to EGF Stimulation of MEFs-It has previously been reported that EGF induces NF-B nuclear levels in cell types that overexpress EGF receptor, such as A431 cells and certain breast cancer cell lines (8). We sought to determine whether EGF signaling activates the classical NF-B pathway in cells that do not express high levels of the EGF receptor. Thus, EGF-treated mouse embryonic fibroblasts were analyzed for the characteristic degradation of IB␣ and nuclear accumulation of the p65 NF-B subunit typically associated with the TNF response. MEFs were stimulated with EGF and assayed for degradation of IB␣ through Western blot analysis. Interestingly, EGF did not result in any significant level of IB␣ degradation (Fig. 1A). Additionally, analysis of nuclear and cytoplasmic extracts from EGFtreated MEFs did not reveal detectable nuclear translocation of p65 (Fig. 1B), although similar analysis using TNF results in strong IB␣ degradation and p65 nuclear accumulation (Ref. 20 and data not shown). This is consistent with previous reports indicating that maximal nuclear accumulation of p65 is largely dependent on IKK-directed IB␣ phosphorylation. We previously reported that the subcellular localization of IKK␣ in unstimulated MEFs is both nuclear and cytoplasmic and that IKK␣ undergoes a nuclear translocation event in response to TNF␣ stimulation (20). To determine whether EGF induces a similar response, IKK␣ levels were examined in nuclear and cytoplasmic extracts by Western analysis. Similar to p65, nuclear levels of IKK␣ remained unchanged in response to EGF (Fig. 1B). NF-B DNA binding potential, analyzed by electrophoretic mobility shift assay, did not show any appreciable increase in response to EGF (data not shown). Additionally, real time PCR analysis of wild-type MEFs demonstrated only a modest induction of expression of the NF-Bdependent IB␣ gene in response to EGF in contrast to the stronger inducible profile observed for induction of c-fos gene expression (Fig. 1C). In contrast, TNF strongly activated the IB␣ gene and only weakly induced c-fos gene expression (Fig. 1D), as previously reported (22). Taken together, these results demonstrate that EGF does not activate the classical NF-B activation pathway in mouse embryonic fibroblasts.

IKK␣ Is Required for Optimal EGF-induced c-fos Gene
Expression-Previously, we had found that IKK␣ contributes to cytokine-induced gene expression. Thus, we addressed whether the loss of IKK␣ has an observable affect on c-fos gene expression in response to EGF. Analysis of c-fos gene expression was measured by reverse transcription-PCR ( Fig. 2A) and independently by quantitative real time PCR analysis (Fig. 2B) in wild-type MEFs and MEFs deficient for either IKK␣ or IKK␤. MEFs were maintained in low serum (0.5% fetal bovine serum) for 48 h followed by EGF stimulation for the indicated times to induce c-fos gene expression. As expected, wild-type MEFs exhibited rapid induction of c-fos mRNA with peak levels occurring 15 min after EGF stimulation, which decreased by the 1-h time point (Fig. 2). Interestingly, induction of c-fos in IKK␣ Ϫ/Ϫ MEFs was considerably reduced and more transient following EGF stimulation (Fig. 2). Although induction of c-fos was reduced at the 15-min time point in IKK␤ Ϫ/Ϫ cells, the levels were more comparable with wild-type cells following 30 min of EGF stimulation (Fig. 2). Restoration of IKK␣-deficient cells with wild-type IKK␣ (IKK␣ ϩ/ϩ ) resulted in strong c-fos expression following EGF treatment (Fig. 2B). The enhanced level of c-fos gene expression in the IKK␣ ϩ/ϩ MEFs is likely due to the relatively high expression of reconstituted IKK␣ in these cells (data not shown). Additionally, a similar requirement for IKK␣ in the inducible regulation of two other immediate-early genes, c-myc and Fra-2, was observed (data not shown). These data provide evidence that IKK␣ is required for optimal EGFinduced c-fos gene expression.
The Loss of IKK␣ Does Not Affect EGF-induced Signaling Pathways-One possible mechanism for the reduction of c-fos gene expression observed in the IKK␣ Ϫ/Ϫ MEFs may involve inhibition of EGF-induced signaling pathways. Therefore, we investigated the effect of EGF on MAPK activation in IKK␣deficient cells by Western blot analysis using phospho-specific antibodies. Treatment of wild-type MEFs with EGF resulted in the induction of MEK1/2 phosphorylation within 10 min and decreased after 30 min (Fig. 3A). Induction of MEK1/2 phosphorylation in IKK␣ Ϫ/Ϫ MEFs was similar to wild-type MEFs with slight kinetic differences in activation (Fig. 3A). MAPKdirected phosphorylation of Elk-1 was analyzed by immunoblotting with a phospho-specific Elk-1 antibody. Inducible and transient levels of Elk-1 phosphorylation were detected in both wild-type and IKK␣-deficient MEFs (Fig. 3B). These results indicate no significant defect in MAPK activation in IKK␣ Ϫ/Ϫ cells and suggest that the altered induction in c-fos gene expression in IKK␣ Ϫ/Ϫ cells is downstream of MAPK activation, specifically at the level of c-fos gene regulation.
Recruitment of IKK␣ to the Endogenous c-fos Promoter Is Enhanced in Response to EGF-Prior studies have correlated enhanced H3 Ser 10 phosphorylation with transcriptional induction of immediate-early genes, such as c-fos and c-myc, upon mitogenic treatment (15)(16)(17)19). However, the promoter association of potential H3 Ser 10 kinases has not been explored. Thus, we investigated whether IKK␣ is recruited to the c-fos promoter in response to EGF in a manner associated with specific histone modifications. Specifically, MEFs were maintained for 48 h in 0.5% serum. Quiescent cells were treated with EGF (50 ng/ml) for various times, DNA and protein was cross-linked with formaldehyde, and DNA was sonicated to shear chromatin into average fragments of ϳ1 kb. Subsequently, protein-DNA complexes were immunoprecipitated using antibodies that specifically recognize phospho-Ser 10 histone H3, acetyl-Lys 9 -H3, or acetyl-Lys 14 -H3 in parallel with an IKK␣-specific antibody. DNA recovered from the antibodybound fractions as well as the DNA from input chromatin (input) were analyzed by semi-quantitative PCR using primers specific for the proximal region of the c-fos promoter. The specificity for all ChIP assays was demonstrated both by the absence of c-fos amplification in the mock samples immunoprecipitated with or without an irrelevant antibody, IgG, and by analysis of the transcriptionally inactive ␤-globin locus (data not shown).
In wild-type MEFs, stimulation with EGF led to an increase of H3 Ser 10 phosphorylation at the c-fos promoter, with peak levels occurring at the 10-min time point that declined to unstimulated levels 1 h following EGF stimulation (Fig. 4A, left  panel). In contrast to H3 Ser 10 phosphorylation, acetylation of H3 at Lys 9 and Lys 14 was detected in unstimulated cells with a modest increase detected following EGF stimulation. Interestingly, association of IKK␣ with the c-fos promoter was detectable at low levels in unstimulated cells and increased transiently in response to EGF, with enhanced association occurring after 10 min of EGF treatment (Fig. 4A, left panel). This analysis demonstrates that IKK␣ association with the c-fos promoter correlates with induced H3 Ser 10 phosphorylation levels in quiescent MEFs stimulated with EGF. IKK␣ Is Required for EGF-induced H3 Ser 10 Phosphorylation at the c-fos Promoter-To determine whether IKK␣ is required for EGF-stimulated H3 Ser 10 phosphorylation at the c-fos promoter, ChIP assays were performed using quiescent IKK␣ Ϫ/Ϫ MEFs. In contrast to wild-type MEFs (Fig. 4A, left panel), enrichment for H3 Ser 10 phosphorylation at the c-fos promoter was undetected in IKK␣ Ϫ/Ϫ MEFs following EGF treatment (Fig. 4A, middle panel). Additionally, the kinetic profiles of Lys 9 -and Lys 14 -acetylated H3 were similar to wild-type cells; however, reduced levels of acetylated H3 at Lys 19 or Lys 14 were detected in IKK␣ Ϫ/Ϫ cells. Next, quantification of ChIP DNA by real time PCR was performed to measure the amount of c-fos genomic DNA that co-immunoprecipitated with the specific histone modifications in wild-type and IKK␣ Ϫ/Ϫ cells relative to total input chromatin. These results show that in IKK␣ Ϫ/Ϫ MEFs, H3 Ser 10 phosphorylation levels are quantitatively reduced most significantly after 10 min of EGF treatment with more comparable levels to wild type at later time points (Fig.  4B). Both Lys 9 -and Lys 14 -acetylated H3 show modest induction in response to EGF in wild-type MEFs. However, induction and overall levels of acetylated H3 are reduced in IKK␣ Ϫ/Ϫ MEFs (Figs. 4, C and D).
Next, to more directly address a potential requirement for IKK␣ in modulating H3 Ser 10 phosphorylation at the c-fos promoter, ChIP assays were performed using IKK␣-null MEFs where wild-type IKK␣ was restored via stable expression (indicated as IKK␣ ϩ/ϩ ). ChIP assays revealed that H3 Ser 10 phosphorylation levels were restored in response to EGF in IKK␣ ϩ/ϩ MEFs with slight differences in kinetics as compared with wild-type cells, with peak levels occurring after 15 min of EGF treatment (Fig. 4A, right panel). The kinetic profile of IKK␣ recruitment was delayed as compared with wild type with peak levels of promoter-associated IKK␣ detected after 15 min of EGF treatment. Additionally, the kinetic profiles for Lys 9 -and Lys 14 -acetylated H3 were comparable with wild-type MEFs. Overall, the recruitment of IKK␣ at the c-fos promoter coincided with and was required for promoter-associated H3 Ser 10 phosphorylation following EGF treatment.
IKK␣ Controls Global Levels of EGF-induced Phosphorylation of Histone H3 on Ser 10 -To address whether IKK␣ plays a broader role in regulating global levels of histone H3 phosphorylation, core histones were acid-extracted from EGF-treated wild-type MEFs or IKK␣-deficient cells (IKK␣ Ϫ/Ϫ ) and examined by Western blot analysis using an antibody specific to phospho-histone H3 (Ser 10 ). As expected, wild-type MEFs exhibit inducible H3 Ser 10 phosphorylation in response to EGF in a time-dependent manner (Fig. 4E). This histone modification was visible within 15 min after the addition of EGF with overall levels decreasing between 30 and 60 min postinduction. Although H3 Ser 10 phosphorylation levels in IKK␣ Ϫ/Ϫ MEFs were similar to wild type after 15 min of EGF treatment, overall levels were lost at the later time points (Fig. 4E). IKK␣ ϩ/ϩ MEFs, in which functional IKK␣ was stably expressed in IKK␣ Ϫ/Ϫ cells, result in restored levels of phosphorylated Ser 10 levels with similar kinetics as wildtype MEFs (Fig. 4E). Global levels of EGF-induced histone H3 acetylation at Lys 9 and Lys 14 were slightly reduced in IKK␣ Ϫ/Ϫ cells as compared with wild-type cells (Fig. 4E). These data support a role for IKK␣ in modulating global levels of histone H3 phosphorylation on Ser 10 in response to mitogenic (EGF) stimuli.

p65/RelA Is Constitutively Associated with the c-fos Promoter and Contributes to the Induction of c-fos Gene Expression-
Transcriptional regulation of c-fos is controlled by cis-acting elements in the promoter regions, including the SRE and the 12-O-tetradecanoylphorbol-13-acetate response element (see Ref. 23 and references therein). Full activation of the c-fos SRE requires association with the ubiquitous transcription factor SRF and formation of a complex with ternary complex factors, including the Ets family of transcription factor Elk-1 (10). Additionally, transactivation by these nuclear factors is facilitated by CBP/p300 (14). In terms of NF-B-dependent promoter assembly, previous reports have demonstrated that IKK␣ interacts with CBP; however, recruitment of CBP to NF-B-dependent promoters is not dependent on IKK␣ (21). In addition, utilizing p65 Ϫ/Ϫ MEFs, the p65 subunit was shown to be required for IKK␣ association with NF-B-regulated promoters following TNF stimulation (20,21). To explore the mode of recruitment for IKK␣ to the c-fos promoter, ChIP assays were performed using antibodies against p65 and CBP. In wild-type MEFs, p65 was detected at the c-fos promoter in unstimulated cells, and this remained relatively unchanged following EGF stimulation (Fig. 5A, left panel). A similar profile of p65 association with the c-fos promoter was observed in IKK␣ Ϫ/Ϫ MEFs (Fig. 5A, right panel). Consistent with previous reports (see Ref. 14 and references therein); CBP is found constitutively associated with the c-fos promoter in wild-type MEFs (Fig. 5A, left panel). Additionally, CBP recruitment was unaltered in cells deficient for IKK␣ (Fig. 5A, right panel). p65 was not found associated with the ␤-globin promoter (data not shown), indicating the specificity of the ChIP assays. These data indicate that p65 is preassociated with the c-fos promoter in an IKK␣-independent manner.
To explore a potential role for p65 at the c-fos gene promoter, quantitative real time PCR analysis was used to measure c-fos gene expression in p65 Ϫ/Ϫ MEFs following EGF treatment. p65 Ϫ/Ϫ MEFs exhibited a marked defect in c-fos gene induction after EGF treatment in contrast to wild-type MEFs that show rapid induction of c-fos mRNA within 15 min of EGF stimulation (Fig. 5B). These results indicate that p65 may play a direct regulatory role in controlling c-fos gene expression or may facilitate promoter-associated IKK␣ or other factors to stimulate gene expression.
Association of IKK␣ at the c-fos Promoter Occurs Independently of the p65 NF-B Subunit-The data described above raise the question of whether p65 is required for the association of IKK␣ with the c-fos promoter. To address this point, ChIP assays were performed in p65 Ϫ/Ϫ MEFs. Quiescent p65 Ϫ/Ϫ MEFs were left untreated or were treated with EGF along a time course followed by ChIP analysis with antibodies against IKK␣ or phospho-Ser 10 histone H3. Our data demonstrate inducible levels of H3 Ser 10 phosphorylation in response to EGF, with peak levels after 15 min of EGF treatment (Fig. 5C). Interestingly, peak levels of promoter-associated IKK␣ were detected after 10 min of EGF treatment, indicating that recruitment of IKK␣ at the c-fos promoter is p65-independent (Fig. 5C). The results were confirmed by quantification of ChIP DNA by real time PCR to measure the amount of c-fos genomic DNA that co-immunoprecipitated with the H3 Ser 10 phosphorylation levels in p65 Ϫ/Ϫ MEFs as well as wild-type MEFs relative to total input chromatin (data not shown). Collectively, FIG. 4. EGF-induced H3 Ser 10 phosphorylation at the c-fos promoter requires IKK␣ recruitment. A, chromatin was prepared from quiescent IKK wild-type (left panel), IKK␣ Ϫ/Ϫ (middle panel), or IKK␣ ϩ/ϩ , IKK␣ reconstituted MEFs (right panel) that were treated with EGF (50 ng/ml) at the indicated time points. ChIP assays were performed using anti-IKK␣, anti-phospho-Ser 10 (H3-pS10), anti-acetyl-Lys 9 (H3-AcK9), or anti-acetyl-Lys 14 (H3-AcK14) as indicated. c-fos promoter DNA sequences were detected by semi-quantitative PCR. B-D, chromatin was prepared from quiescent IKK wild-type (black bars) or IKK␣ Ϫ/Ϫ (gray bars) MEFs that were treated with EGF (50 ng/ml) at the indicated time points. ChIP assays were performed using anti-phospho-Ser 10 (B), anti-acetyl-Lys 14 (C), or anti-acetyl-Lys 9 (D) as indicated. c-fos promoter DNA sequences were detected by quantitative real time PCR. The data are presented as percentages of total input. E, acid-soluble proteins were extracted from quiescent IKK wild-type (WT), IKK␣ Ϫ/Ϫ , and IKK␣ ϩ/ϩ MEFs and immunoblotted with anti-phospho-Ser 10 , anti-acetyl-Lys 9 , or anti-acetyl-Lys 14 H3 antibodies. Coomassie staining of acidextracted histones was used to visualize equal loading.
these results indicate that EGF-induced recruitment of IKK␣ to the c-fos promoter occurs independently of p65/RelA. DISCUSSION The results presented in this report indicate that IKK␣ is a critical regulator of mitogenic-induced H3 Ser 10 phosphorylation, at least for the c-fos promoter. The data also support previously published work regarding the association between inducible phosphorylation and acetylation at certain promoters (17), whereby the loss of H3 Ser 10 phosphorylation reduces but does not eliminate inducible levels of acetylation of histone H3 in IKK␣ Ϫ/Ϫ MEFs. However, we do not exclude the possibility that these dynamic modifications occur independently as previously reported for the c-jun promoter (24). In addition to these dynamic modifications, interplay among other modifications, such as histone H3 methylation, may differentially contribute to c-fos gene regulation either positively or negatively (25,26). The experiments also provide an interesting regulatory model whereby NF-B controls gene expression in a manner that is independent of inducible promoter association of the NF-B subunit, p65/RelA.
Evidence that IKK␣ controls c-fos histone H3 phosphoryla-tion and inducible gene expression led us to address the potential association of the p65 NF-B subunit with the c-fos promoter, because it was previously found that IKK␣ association with certain cytokine-regulated promoters was dependent on the recruitment of p65 (20,21). Our current studies indicate that p65 is found constitutively associated with c-fos promoter. In this regard, the association of p65 with the c-fos promoter occurs in a manner that is similar to other c-fos regulatory factors (such as SRF) and that is different from the traditional mechanism of NF-B regulation. Although others have observed the classical NF-B activation response in cells that express high levels of EGF receptor (8), at least in quiescent MEFs, we did not observe any significant degradation of IB␣ or nuclear accumulation of p65 following treatment with EGF. Interestingly, p65 contributed to the induced levels of c-fos gene expression following EGF treatment of quiescent MEFs (Fig. 5B). Thus, the role of the p65 subunit in controlling EGF-induced gene expression is not dependent on the enhanced recruitment of this transcription factor with its gene target. However, the mode of p65 activation downstream of EGF and the role that p65 plays in regulating c-fos gene expression are presently unclear.
The basis for the association of p65 with the c-fos promoter is unknown. Previous studies have suggested that p65 might participate in regulating the transcriptional activity of SRF, and a physical interaction was reported between p65 and SRF in vitro (27). Thus, it is possible that p65 is constitutively associated with the c-fos promoter through interactions with SRF. Additionally, we identified a previously uncharacterized NF-B consensus site in the c-fos promoter (positions Ϫ214 to Ϫ223). The position of the region amplified with c-fos promoterspecific primer pairs used in the PCR step of the ChIP assays includes this putative NF-B site. Electrophoretic mobility shift assays did not detect NF-B DNA binding to this site in the c-fos promoter (data not shown), suggesting that this site is not a high affinity site for p65 in vitro and supports the possibility that p65 may function in concert with other nuclear factors at this promoter in vivo. Possibilities for promoterassociated p65 include cooperative interactions with SRF resulting in the formation of higher transcriptional complexes, as described above. In fact, antibodies to p65 (but not to p50) can partially supershift an SRF/SRE complex using a gel mobility shift assay (data not shown).
Although the importance of chromatin modification, including histone phosphorylation, in regulating gene expression is well recognized (28 -30), the regulation of and identity of factors that control these modifications often remain unclear. Recent evidence, using MSK1/2 double knock-out MEFs, indicate that MSK proteins control global levels of H3 Ser 10 phosphorylation as well as inducible H3 Ser 10 phosphorylation at the c-jun promoter in response to mitogenic or stress stimuli (19). Furthermore, our studies and those of Soloaga et al. (19) indicate that the loss of inducible H3 Ser 10 phosphorylation results in deficient c-fos gene expression levels. It is intriguing to speculate that this reduction may reflect the contribution of H3 Ser 10 phosphorylation to immediate early gene expression. However, MSK1/2 and IKK␣ may play other roles in controlling c-fos gene expression. For example, MSK1 may play a role in activating the function of p65 at the c-fos promoter, because it has been proposed that MSK1 controls the phosphorylation of p65 on Ser 276 (31), an event important in the interaction of p65 with co-activators, such as CBP (32). This concept could reflect a role for p65 and for MSK at the c-fos promoter in controlling expression of immediate-early genes. Longer term experimentation is directed toward understanding the complex regulatory network involving MSK1/2 and IKK␣ in controlling chromatin FIG. 5. Promoter-associated p65/RelA contributes to the induction of c-fos gene expression. A, chromatin was prepared from quiescent IKK wild-type (left panel) or IKK␣ Ϫ/Ϫ (right panel) MEFs that were treated with EGF (50 ng/ml) at the indicated time points. ChIP assays were performed using anti-p65 or anti-CBP as indicated. c-fos promoter DNA sequences were detected by semi-quantitative PCR. B, real time PCR analysis was performed to measure the endogenous levels of c-fos mRNA in EGF stimulated wild-type or p65 Ϫ/Ϫ MEFs. Quiescent cells were treated with EGF at the indicated time points, similar to A. The values are reported as molecules of c-fos per attomol of 18 S rRNA copies. C, EGF-induced recruitment of IKK␣ at the c-fos promoter occurs independently of p65. Chromatin was prepared from quiescent p65 Ϫ/Ϫ MEFs that were treated with EGF (50 ng/ml) at the indicated time points. ChIP assays were performed using anti-IKK␣ or anti-phospho-Ser 10 as indicated. c-fos promoter DNA sequences were detected by semi-quantitative PCR. modification and ultimately inducible gene expression.
The loss of p65 did not affect promoter-associated IKK␣ or EGF-induced H3 Ser 10 phosphorylation at the c-fos promoter (Fig. 5C). This result was surprising as compared with our previous findings and those by Yamamoto et al. (21) where recruitment of IKK␣ to cytokine-inducible promoters is required the presence of p65. Thus, the mechanism of IKK␣ recruitment to the c-fos promoter could be dependent on the presence of another NF-B subunit or via interactions with a distinct factor. It is important to note that IKK␣ interacts with the transcriptional co-activator CBP (21) and that this interaction may underlie promoter recruitment. In this regard, MAPK-directed phosphorylation of Elk-1 that stabilizes CBP/ p300 interactions with the SRE ternary complex (14) may also lead to enhanced association of IKK␣ with the c-fos promoter.
Collectively, our findings provide the first evidence that promoter association of IKK␣ can occur in a p65-independent manner and that both IKK␣ and p65 contribute to EGF-induced c-fos gene expression. In this regard, IKK␣ has been shown to control keratinocyte differentiation in a manner that is independent of NF-B (33). Additionally, IKK␣ knock-out mice exhibited skin and skeletal abnormalities (34), consistent with the effect on keratinocyte differentiation. Overall, our results indicate that both IKK␣ and p65 contribute toward maximal EGF-induced c-fos gene expression, potentially through distinct mechanisms. This is consistent with previous reports highlighting the complexity of c-fos gene regulation through multiple promoter-bound factors and/or specific signaling pathways (10).