TNF is a pleiotropic cytokine that mediates the pulmonary cytokine cascade, the hepatic acute-phase response, and the regulator of leukocyte activation and apoptosis. Upon its ligation to cell surface receptors, TNF induces protein recruitment to cytoplasmic death domains, assembling a signaling complex composed of TRADD, FADD, and TRAF2 (and others) that activates divergent intracellular signals, including the Jun N-terminal protein kinase-AP-1 and the IKK-NF-κB pathways to induce genomic responses in the target cell (23
). Although the canonical IKK-NF-κB pathway is critical for inducing tissue inflammation and preventing TNF-induced programmed cell death, surprisingly little is known about its mechanism for activating downstream gene targets. We have used high-density DNA microarrays of Tet-regulated dominant-negative cell lines to systematically identify the network of genes downstream of the NF-κB transcription factor (54
). Surprisingly, these studies indicate that genes under direct NF-κB control are expressed in temporally distinct waves, with each group affecting a different biological process (54
). In this study, we have exploited our recent developments in a quantitative two-step ChIP assay (39
), an assay sensitive to binding and dissociation of NF-κB, to probe the diverse mechanisms governing NF-κB-inducible gene expression.
NF-κB subunits are nuclear phosphoproteins responsive to diverse signaling pathways (35
). Specifically, the potent RelA-transactivating subunit can be phosphorylated at multiple sites, including Ser276
by PKAc (26
) and mitogen and stress kinase 1 (57
) and Ser536
by IKKβ, NK-κB-inducing kinase, and c-Src (28
). These phosphorylation events lead to an increase in RelA nuclear translocation, DNA binding, and/or transcriptional activity, through the mechanisms not completely understood. Of relevance to this study is the fact that RelA Ser276
phosphorylation occurs upon the activation of the canonical NF-κB pathway and cytoplasmic release from IκBα. Although the relative roles of PKAc and mitogen and stress kinase 1 have yet to be fully understood, we have recently observed that PKAc mediates TNF-induced phospho-Ser276
RelA formation via an intracellular ROS signal (26
). This second ROS-PKAc pathway is completely independent of that controlling RelA translocation and is necessary for the acquisition of full transcriptional activity (26
). The consequence of Ser276
phosphorylation is thought to be a destabilization of the intermolecular association of the NH2
- and COOH-terminal ends of RelA (60
), producing a conformational change that may produce a more stable association with CBP/p300 and other coactivators (60
). As a result, phospho-Ser276
RelA becomes a substrate for acetylation, resulting in enhanced transcriptional activity (12
Our findings extend the range of protein interactions involving activated RelA, as we have observed that Ser276
phosphorylation is also required for interaction with the P-TEFb complex. This finding is important because it suggests that phospho-Ser276
RelA binding can mediate transcriptional elongation by recruiting P-TEFb to target genes as an additional mechanism for inducible gene expression over that mediated by p300/CBP recruitment. Importantly, the requirement for P-TEFb recruitment is not a uniform feature of NF-κB-dependent gene activation. Rather, our data suggest that phospho-Ser276
RelA plays only a restricted role in the activation of two NF-κB-dependent targets, the IL-8 and Gro-β genes (but not the IκBα gene). Our ChIP studies indicate that the IL-8 and Gro-β genes are highly inducible through a mechanism involving Pol II recruitment, whereas the IκBα gene is one whose magnitude of expression is not as highly inducible and is stably engaged with Pol II in the absence of TNF stimulation (Fig. ). Previously, using genomic footprinting of the IL-8 gene by ligation-mediated PCR, we found that NF-κB activation produced dramatic modifications of the proteins binding the TATA box and transcriptional initiation site, suggesting that NF-κB activated this gene by a promoter recruitment mechanism (9
). Here, we extend this mechanism to include both Pol II and P-TEFb recruitment. In response to CDK-9-mediated CTD phosphorylation, recruited Pol II is able to produce full-length transcripts. In the case of the IκBα gene, others have shown that the gene is preloaded with RNA polymerase, making it able to rapidly respond to NF-κB binding without needing time-dependent formation of a preinitiation complex (1
). Together, these studies indicate that NF-κB activates its downstream gene targets through pleiotropic mechanisms.
By comparing the kinetics of bulk RelA binding and those of phospho-Ser276
RelA binding to genomic targets, this study represents the first demonstration that there is a dissociation in the timing between phospho- and non-phospho-RelA recruitment. Specifically, our data show that phospho-Ser276
RelA binding is the functionally important complex in Gro-β and IL-8 gene expression because (i) phospho-Ser276
RelA binding temporally coincides with peak transcriptional activation and peak steady-state mRNA abundance, (ii) inhibition of phospho-Ser276
formation by antagonism of the ROS pathway selectively blocks phospho-RelA (but not bulk RelA) binding and transcriptional activation of IL-8/Gro-β, and (iii) a phosphorylation-deficient Ser-to-Ala site mutation at residue 276 is unable to efficiently transactivate Gro-β in a RelA-deficient MEFs. Others have that shown Ser276
phosphorylation was necessary for a strong response to the NF-κB-inducing stimuli TNF and IL-1 (2
). It is possible that the size or shape of this multiprotein complex or the nature of chromatin where the NF-κB binding site is found may restrict the access of the phospho-Ser276
RelA complex to certain genes. It will be interesting to compare protein complexes formed by phospho-Ser276
RelA and nonphosphorylated RelA.
Although the Ser276
RelA complex plays a key functional role in IL-8 and Gro-β gene expression, it is not required for IκBα expression, even though our ChIP assays show that phospho-Ser276
RelA rapidly and inducibly binds to this promoter. This conclusion is made based on the ability of hypo-Ser276
-phosphorylated RelA to transactivate IκBα after the inhibition of the ROS-PKAc pathway, the ability of the RelA Ser276
Ala mutant to transactivate IκBα in RelA−/−
MEFs, and the lack of effect of CDK-9 downregulation on IκBα expression. Therefore, although we observed an induction of phospho-Ser276
RelA binding to IκBα in the ChIP assay, this RelA modification and P-TEFb recruitment were not required to activate productive transcription. In this regard, we note earlier studies that have shown phospho-defective RelA at residues Ser205
, and Ser281
are unable to mediate a subset of NF-κB-dependent genes (the ICAM-1, VCAM-1, and MIP-2 genes) in response to lipopolysaccharide and gamma interferon, whereas induction of two other NF-κB-dependent genes, the major histocompatibility complex class I and Mn superoxide genes, are essentially unaffected (2
). These data suggest to us that a phosphorylation code controls NF-κB's ability to activate subnetworks of target genes, where distinct groups are induced by particular forms of phospho-RelA and the protein complexes that they form. Elucidation of the complete spectrum of genes under phospho-Ser276
RelA-dependent control will require further investigation.
CDK-9, along with Ccn T1, are core constituents of P-TEFb, a complex that functions as a transcriptional elongation factor mediating human immunodeficiency virus TAT-dependent transactivation (5
) and the activation of heat shock genes in Drosophila melanogaster
). P-TEFb mediates transcriptional elongation by its ability to phosphorylate several targets in the paused Pol II-dependent promoter, including serine 2 in the heptad repeat in the Pol II CTD, as well as inhibitory factors such as negative elongation factor (NELF) and DRB sensitivity-inducing factor (DRIF) (41
). Our studies extend the understanding of inducible phospho-RelA protein interactions and suggest for the first time that phospho-Ser276
RelA is required for complex formation with the P-TEFb kinase. Our findings using a highly potent and specific CDK inhibitor and siRNA-mediated knockdown both suggest that IL-8 and Gro-β are activated by a mechanism involving P-TEFb recruitment. That CDK kinase activity mediates Pol II CTD phosphorylation is indicated by the reduction in CTD phosphorylation in response to FP (Fig. ). Further studies are needed to more precisely identify the spectrum of proteins modified by CDK-9 on NF-κB-dependent promoters, particularly NELF and DRIF.
Our fluorescence colocalization studies suggest that a significant fraction of RelA colocalizes with CDK-9 upon nuclear entry. Although spatial colocalization is subject to random error, especially in this case, where the concentration of one interacting protein is increasing in the cellular compartment of interest, we have analyzed these data using a formal statistical randomization algorithm which distinguishes random color overlap due to compartmentalization from that arising due to specific colocalization (13
). Using this technique, we find that a significant fraction of RelA associated with CDK-9 in the nucleus. Because much of the nuclear fraction of RelA is not associated with chromatin, we believe that RelA-CDK-9 interaction occurs prior to promoter binding. To this end, we can coimmunoprecipitate NF-κB/RelA-CDK-9 from the nuclei of TNF-stimulated cells.
Recent work using fluorescence microscopy (photobleaching and fluorescence lifetime measurements) has shown that NF-κB transiently binds its chromatin targets with an exchange rate of seconds (8
), resulting in the appreciation that chromatin-bound NF-κB is in dynamic exchange with its non-DNA-associated nucleoplasmic pool. This finding suggests that concentration changes in phospho-NF-κB isoforms entering the nuclear compartment can rapidly exchange with promoter-associated NF-κB. This phenomenon has been experimentally verified by us using Ang II, a ligand that activates phospho-Ser536
RelA formation without affecting total RelA nuclear abundance. In this case, an increase in the fraction of nuclear phospho-Ser536
RelA can rapidly exchange with promoter-bound hypophosphorylated RelA, producing a transition in gene expression from an inactive state to an activated one (14
). Our ChIP analysis of phospho-Ser276
RelA binding, however, indicates that this isoform does not rapidly exchange with the hypophosphorylated RelA bound to the Naf1 gene. We suspect that phospho-Ser276
RelA complexed with p300/CBP and P-TEFb is a macromolecular complex whose promoter accessibility is dependent on the architecture and topology of the target promoters.
The findings of this study indicate that NF-κB controls downstream genes through at least two distinct mechanisms, schematically illustrated in Fig. . The first mechanism, exemplified by IL-8 and Gro-β, controls genes that are not significantly engaged with RNA Pol II in the absence of stimulation and require phospho-Ser276 RelA binding and P-TEFb recruitment for inducible Pol II recruitment and activation of transcriptional elongation. The second mechanism, exemplified by IκBα, binds with RNA Pol II in the absence of stimulation. Although IκBα inducibly binds phospho-Ser276 RelA and P-TEFb, this complex is not required for promoter activation.
FIG. 9. Model of NF-κB-dependent initiation of transcription. This schematic represents the steps involved in the two mechanistically distinct pathways involved in the transcriptional initiation of NF-κB-dependent gene expression mediated by inducible (more ...)
Epithelial cells play important roles in the initiation and maintenance of innate immunity. For example, in the airway, epithelial cells respond to a variety of viral, environmental, and hormonal (cytokine) stimuli to activate NF-κB-dependent cytokine cascades important in the genesis and maintenance of airway inflammation (42
). Our delineation of distinct mechanisms by which NF-κB activates target genes has important implications for targeted anti-inflammatory therapy at epithelial surfaces, where distinct biological responses could be modified. For example, inhibition of the ROS-dependent phospho-Ser276
RelA-P-TEFb pathway may be useful to control the cytokine cascade without influencing other NF-κB-mediated antiapoptotic responses.