Biochimica et Biophysica Acta 1829 (2013) 937–945

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Review

Transcriptional Control by NF-κB: Elongation in Focus

Gil Diamant, Rivka Dikstein ⁎Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

⁎ Corresponding author. Tel.: +972 8 9342117; fax: +E-mail address: rivka.dikstein@weizmann.ac.il (R. D

1874-9399/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbagrm.2013.04.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 January 2013Received in revised form 15 April 2013Accepted 17 April 2013Available online 25 April 2013

Keywords:NF-κBtranscription elongationP-TEFbBrd4DSIF

The NF-κB family of transcription factors governs the cellular reaction to a variety of extracellular signals.Following stimulation, NF-κB activates genes involved in inflammation, cell survival, cell cycle, immunecell homeostasis and more. This review focuses on studies of the past decade that uncover the transcriptionelongation process as a key regulatory stage in the activation pathway of NF-κB. Of interest are studies thatpoint to the elongation phase as central to the selectivity of target gene activation by NF-κB. Particularly,the cascade leading to phosphorylation and acetylation of the NF-κB subunit p65 on serine 276 and lysine310, respectively, was shown to mediate the recruitment of Brd4 and P-TEFb to many pro-inflammatorytarget genes, which in turn facilitate elongation and mRNA processing. On the other hand, some anti-inflammatory genes are refractory to this pathway and are dependent on the elongation factor DSIF forefficient elongation and mRNA processing. While these studies have advanced our knowledge of NF-κB tran-scriptional activity, they have also raised unresolved issues regarding the specific genomic and physiologicalcontexts by which NF-κB utilizes different mechanisms for activation.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The transcription elongation stage and regulatory factors

Transcription by RNA polymerase II (Pol II) is divided into threemechanistically distinct phases: initiation, elongation and termination.The initiation phase involves promoter recognition, assembly of thepre-initiation complex, formation of an openDNAduplex, and synthesisof the first few nucleotides. Elongation begins after Pol II clears thepromoter and enters into the productive synthesis of themRNA. Termi-nation occurs after recognition of the poly-A termination signal and isfollowed by the release of the nascent transcript and dissociation ofPol II from the DNA. An important regulatory module for all these tran-scriptional steps is the C-terminal domain (CTD) of the largest subunitof Pol II. This domain is composed of tandem heptad repeats enrichedwith phosphate-acceptor amino acids with the consensus sequenceYSPTSPS. The Pol II CTD is conserved amongall eukaryotes andundergoesextensive phosphorylation [1]. The CTD not only controls transcriptionbut also transcription-coupledmRNAprocessing [2]. During transcriptionthe CTD undergoes dynamic changes in phosphorylation [3]. The hypo-phosphorylated formof Pol II CTD is preferentially recruited to promoterswhere it is subsequently phosphorylated on serine 5 by the TFIIH-associated kinase CDK7, potentiating the transition from initiation toelongation (Fig. 1). As Pol II elongates, serine 2 is increasingly phosphor-ylated by P-TEFb,while serine 5 phosphorylation is gradually removedbyphosphatases (Fig. 1). Thus the serine 5 phosphorylation is usually seen

972 8 9344118.ikstein).

rights reserved.

around the transcription start site (TSS), and serine 2 phosphorylationis usually detected in the middle and the end of actively transcribedgenes (for review, see [4,5]). However, as discussed below, the situationis more complex than this generalized model and there are severalexceptions to this regulatory mode, in particular within the NF-κB path-way. In addition P-TEFb itself is highly regulated and exists in twodistinctforms: the active one which consists of the protein kinase Cdk9 andregulatory subunit cyclin T (T1, T2a or T2b); and the catalytically inactiveform which is bound and inhibited by the 7SK snRNP complex(consisting of 7SK snRNA, HEXIM1 and additional proteins) [6].

The transcription elongation factors, DRB sensitivity-inducing factor(DSIF, a heterodimer composed of Spt5 and Spt4 subunits) and negativeelongation factor (NELF) can function either together with P-TEFb or ina manner independent of it. In many genes, the two factors associatewith Pol II shortly after transcription initiation and pause the elongationdownstream from the TSS [7,8]. The release of Pol II frompausing occurswhen P-TEFb phosphorylates DSIF, NELF and the Pol II CTD leading todissociation of NELF from the paused Pol II [9–11]. Interestingly, a signif-icant portion of all genes seems to have a paused Pol II downstreamfrom their TSS [12–15]. Pausing is thought to constitute a mechanismto obtain rapid and coordinated transcription during developmentand in response to external stimuli [16–18].

NF-κB transcription factors

NF-κB was first discovered in 1986 in the lab of David Baltimore asa transcription factor that regulates immunoglobulin kappa lightchain in B cells [19,20]. Since then NF-κB has emerged as a family oftranscription factors that is activated in response to a variety of

Fig. 1. Pol II CTD phosphorylation cycle. During transcription the C-terminal domain of Rbp1, the RNA Polymerase II large subunit, undergoes dynamic changes in the pattern of itsphosphorylation. The hypo-phosphorylated form of Pol II CTD is preferentially recruited to promoters where it is subsequently phosphorylated on Serine 5 by the TFIIH-associatedkinase CDK7, correlating with the transition from initiation to elongation. When paused near the promoter, Pol II is predominately phosphorylated on Serine 5. As Pol II elongates,Serine 2 is increasingly phosphorylated by P-TEFb, while Serine 5 phosphorylation is gradually removed by phosphatases, such as downstream of the polyadenylation site onlySerine 2 phosphorylation is detected. These phosphorylations promote co-transcriptional recruitment of mRNA processing factors (such as splicing, export and termination factors)to the CTD.

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extracellular signals such as inflammatory cytokines, infections andmultiple stress situations. Activated NF-κB induces the expression ofgenes affecting diverse biological processes including development, im-munity, tissue homeostasis, inflammatory and stress responses, cell sur-vival and proliferation [21–25]. A subset of genes activated by NF-κBencodes for its own negative regulators thereby forming negative feed-back loops. Consequently, the activation of the NF-κB pathway is gener-ally a transient process, lasting up to several hours. However, in certaincancer and inflammatory diseases NF-κB activity becomes abnormallypersistent and directly contributes to the pathology [26].

The family of NF-κB is composed of structural homologs that inmammals include NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, andc-Rel. These proteins share a highly conserved DNA-binding and dimer-ization domain called Rel homology domain (RHD). NF-κB proteins bindto specific DNA sequences called κB sites. All Rel proteins can formhomodimers or heterodimers, except for RelB, which can only formheterodimers. This combinatorial diversity contributes to the regulationof distinct, but overlapping sets of genes since individual dimers displayvariable affinity towards a collection of related κB sites. Moreover, therelative abundance of different NF-κB proteins varies between differentcell types with the exception of p50-RelA heterodimer, which is ubiqui-tous and the most readily detected complex in most cells [27].

The activity of different NF-κB dimers is primarily regulated byinteraction with inhibitory proteins that hold these complexes in thecytoplasm in an inactive form. These inhibitors include p105, p100and IκB α, β, γ and proteins, which interact with NF-κB through anankyrin repeat domain.When a cell receives an NF-κB activating signal,the interaction between IκB and NF-κB is disrupted enabling NF-κB torapidly enter the nucleus [28]. Most of the NF-κB activating signalsconverge on the activation of a high molecular weight complex thatcontains an IκB kinase (IKK) which stimulates NF-κB in two distinctpathways, the canonical and non-canonical [29]. In the canonical path-way, the signal-activated IKK complex phosphorylates two specific

serine residues at IκBα N-terminus, which then trigger IκBαubiquitination and degradation by the 26S proteasome. In thenon-canonical pathway, the p100-RelB complex is activated by phos-phorylation of the C-terminal region of p100, which also leads toubiquitination and degradation of the p100 IκB-like C-terminal se-quences to generate p52-RelB. In bothof these pathwaysNF-κB complexis released from IκB proteins and translocates into the nucleus to acti-vate target gene transcription [30].

2. NF-κB-mediated transcription activation

Among the NF-κB family only the RelA, c-Rel and RelB subunitscontain transcription activation domains (TAD) in their C-termini,which are conserved among different species but less conservedbetween family members [31]. The two other NF-κB proteins p50and p52 are processed from precursor inhibitory proteins p105 andp100, respectively, to shorter proteins containing the RHD but lackingtranscriptional activity of their own [32]. However p50 and p52 canform transcriptionally active heterodimers in association with p65,c-Rel, and RelB. Moreover, p52 can activate transcription as a complexwith Bcl-3, an IκB-like molecule with coactivator functions [33]. TheTADs of NF-κB proteins are responsible for the recruitment of variousco-regulators to their target genes such as co-activators, chromatinmodifying factors and components of the basal transcriptional ma-chinery [34] (Fig. 2).

The most abundant and well-characterized NF-κB dimer consists ofthe ubiquitously expressed p65/RelA and p50. Since p65/RelA is respon-sible for the transcriptional activity of this complex, the mechanismunderlying its transcriptional activity has been explored in great details.p65/RelA has a composite TAD within its C-terminal 120 amino acidswhich is responsible for transcriptional activation. p65/RelA TAD isdivided into two distinct transactivation sub-domains, TA1 and TA2(Fig. 3), both of which are required for the full transcriptional activity

Fig. 2. Activation of transcription by NF-κB. The various stages in which NF-κB is known to influence the transcriptional process are indicated by arrows.

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[35,36]. These TADs were shown to mediate interactions with a varietyof transcriptional co-regulators in vitro and in cellular contexts such asbasal transcription factors, the coactivators CBP and p300 and manymore [37–43].

While nuclear translocation is the primary regulatory stage of NF-κB,phosphorylation of the RelA TADs at several key residues, in particularS468, T505, S529, S535, and S536, were found to be important for mod-ulation of its transcriptional activity and target gene activation [43–48].These signal-induced p65/RelA phosphorylations usually augment thetranscriptional activity of nuclear NF-κB rather than promoting itsnuclear translocation.

3. Regulation of NF-κB target genes by P-TEFb

The first study that brought into focus the ability of NF-κB to regulatetranscription elongationwas published in 2000. This study demonstratedthat the Glococorticoid Receptor (GR) mediated inhibition of certainNF-κB pro-inflammatory targets such as IL-8 and ICAM-1, involves directprotein–protein interaction with the RelA dimerization domain withoutdisrupting NF-κB-DNA interaction [49]. In addition, GR interfered withphosphorylation of Pol II CTD on serine 2 on these genes. These effectswere not apparent on the IκBα gene. As P-TEFb had already been charac-terized as one of the major CTD serine 2 kinases, a follow-up studyestablished that NF-κB activation of the IL-8 gene and an HIV reporterrequires P-TEFb to effectively promote elongation in response tothe cytokine TNFα, a well-known inducer of NF-κB [50]. This paperalso showed that inhibiting P-TEFb with DRB (5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole) sensitized cells to TNFα-induced apoptosis,suggesting an effect on a subset of anti-apoptotic genes normally acti-vated byNF-κB. Subsequently it was shown that NF-κB directly interactswith P-TEFb, implicating it in recruitment of P-TEFb to IL-8 but not toIκBα following activation [51]. Importantly, this study reported that GRactivation diminishes P-TEFb recruitment to the IL-8 gene. Interestingly,sequences of κB elements in promoters of IL-8 and IκBα differ by a singlenucleotide. It has been shown that reporters under the IL-8 and IκBαpromoters recapitulate the responses of the endogenous genes toTNFα and GR, and when κB sequences are switched between reportersboth exhibit increased levels of activation [52]. Strikingly, the IκBαmutant reporter is now also inhibited by GR. This finding points to a

Fig. 3. Schematic structure of the major NF-κB subunit p65/RelA. The N-terminus of p65 coserves for DNA binding, dimerization and IκB binding. The C-terminus has a composite transmediate interactions with a variety of transcriptional co-regulators. p65 undergoes multiplemethylations, which regulate its specificity, kinetics and intensity. Of relevance to elongatio

connection between κB sequence and NF-κB's regulation of elongation.The importance of variation κB sequence is now becoming more evi-dent, as studies have shown that differences in NF-κB dimers and κB se-quences affect the recruitment of chromatin repressing complexes[52,53].

In parallel to these studies the Bromodomain 4 protein (Brd4)emerged as amajor recruiter of P-TEFb to active genes. Brd4 is amemberof the BET family of bromodomain-containing proteins. These domainswere first noted for their ability to bind acetylated lysine residues ofhistones [54,55]. Brd4 was initially reported to be a cell cycle regulatorand a player in translocation induced carcinoma [56,57]. Biochemically,it has the ability to interact with acetylated lysines on histones and onP-TEFb through its bromodomains. The latter interaction was shown torecruit P-TEFb to promoters where it phosphorylates the Pol II CTD tostimulate elongation [58,59]. Recent studies provided additional insightinto the role of Brd4 as a P-TEFb cofactor, demonstrating that it competeswith the HEXIM1-7SK complex for P-TEFb binding and recruitment tolysine-acetylated nucleosomes [60,61].

Considering the central role of P-TEFb in inflammatory gene activa-tion several studies have established Brd4 as a mediator of NF-κB stim-ulated elongation. Sharma et al. presented evidence that RelA recruitsthe histone acetyl transferases (HAT) p300 and PCAF. These cofactorsacetylate promoter bound histones, forming the necessary anchor forBrd4 and P-TEFb [62]. RelA had been shown to be phosphorylated atmultiple sites, including on Serine 276 by PKAc and MSK-1 in responseto TNFα [63–65]. Subsequently it was shown that S276 phosphorylatedform of RelA is specifically recruited to a subset of NF-κB-dependentgenes and is required for P-TEFb recruitment [65–67]. The genes infocus were again inflammatory response genes IL-8 and GRO-β. OtherNF-κB activated genes such asNaf1, NFκB2, and TRAF1 showed retardedkinetics of phospho-S276-RelA binding. Interestingly, in the same stud-ies the expression of IκBα was shown to be independent on this RelAmodification as well as on P-TEFb [66,67]. These findings connect withother studies which found that RelA phosphorylations on serines 276and 536 promote interaction with CBP/p300, which in turn acetylatesRelA lysine 310 and facilitates Brd4 and P-TEFb recruitment and tran-scriptional activity [68–71]. Recruitment of these factors can also beachieved through acetylated histones [72]. Another study examinedthe S276A mutation in mice and cell lines, and added an additional

ntains a Rel homology domain (RHD) that is shared by all other NF-κB members and-activation domain divided into two distinct sub-domains, TA1 and TA2. These domainspost translational modifications including phosphorylations (P), acetylations (Ac) andn control are S276 and S536 phosphorylations and K310 acetylation.

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layer of complexity by demonstrating that the defective CBP/p300recruitmentwas accompanied by a failure to displace repressive histonedeacetylases (HDAC) [73]. P-TEFb-mediated regulation of NF-κB targetwas also suggested to be facilitated through Brd4 that is associatedwith acquired H4K5/8/12 acetylations at promoters. It was shown thatselectively blocking NF-κB in LPS stimulated cells led to loss of theseH4K acetylations on NF-κB target promoters and consequently of Brd4and P-TEFb recruitment [74].

Fig. 4. Modes of regulation of NF-κB target gene expression by P-TEFb. Upon activation of cesignaling pathway converges on the IKK complex which phosphorylates IκBα, targeting itranscription. IKK also phosphorylates certain residues on NF-κB subunits themselves. TNFR aphosphorylation leads to downstream acetylation of p65 on K310 which promotes recruitmespecific manner. Various isoforms of p65 and p50 also differentially recruit HATs and HDAnucleosomes, thus alsomodulating Brd4-P-TEFb recruitment and transcript elongation. Finally, ainhibiting expression of a subset of NF-κB targets genes.

Combined, these findings outline a pathway (Fig. 4) in which TNFαor LPS signaling leads to phosphorylation events of RelA which recruitHATs such as CPB/p300 and PCAF. These HATs acetylate K310 of RelAas well as several histones, which are then recognized by Brd4 throughits bromodomains. In this way Brd4 recruits P-TEFb to promoters of asubset of NF-κB-responsive inflammatory genes . This pathway istargeted, at least in part, by glucocorticoids (Fig. 4). The understandingof Brd4 regulation over a specific subset of NF-κB targets led to studies

ll surface receptors (such as TNFR, TLR's and other inflammatory cytokine receptors) at to degradation and releasing NF-κB to translocate to the nucleus and activate genelso activates a cascade resulting p65 phosphorylation on S276 by PKAc and/or S536. Thisnt of Brd4 and P-TEFb by p65 to a subset of promoters, controlling elongation in a geneCs to different promoters, leading to acetylation and deacetylation of promoter boundctivatedGlococorticoid Receptor (GR) inhibits P-TEFb binding byNF-κB dimers, selectively

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that apply Brd4 inhibition to achieve modulation of the inflammatoryresponse [72,75].

4. Regulation of NF-κB target genes by DSIF and other factors

As mentioned above, regulation by the CBP/p300-Brd4-P-TEFbpathway occurs in most, but not all NF-κB target genes. In particular,NF-κB targets andnegative feedback regulators IκBα,A20 and TRAF1wereshown to be refractory to P-TEFb and Brd4 inhibition [51,67,69,76]and recruitments of Ac-K310-RelA, p300 and Brd4 were not detected[69]. A20 is an ubiquitinase-deubiquitinase enzyme which is a well-characterized inhibitor of the TNF receptor and a negative feedback com-ponent of the NF-κB signaling [77–80]. This gene, like IκBα, is essentiallyanti-inflammatory, and like the TRAF1 and NFκB2 genes mentionedabove, is part of the NF-κB pathway that does not show requirement forS276 phosphorylation of RelA.

Initial studies aimed to elucidate the mechanistic basis of NF-κB-mediated rapid transcriptional induction of A20 and IκBα genes showedthat before stimulation the promoters of these genes are already boundby the transcriptionmachinery, indicating that NF-κB is not required forPIC formation. However, NF-κB was found to enhance the re-initiationrate [81]. For A20 gene, DSIF was shown to promote promoter proximalpausing under basal conditions, and this regulation is dependent on aspecific promoter element [16,82]. Occupancy of DSIF on the A20 andIκBα promoters and gene bodies was further enhanced upon NF-κBactivation. Interestingly on some NF-κB target gene regulation byP-TEFb and DSIF is mutually exclusive [76]. This differential regulationwas found to be dependent on NF-κB itself, the core promoter typeand the initiation complex. P-TEFb controls NF-κB target genes drivenby TATA-containing promoters which are independent of TFIID andDSIF regulatesweakTATAandTATA-less genes that are TFIID dependent(Fig. 5). In otherwords,while TBP is always required, the requirement ofcertain TAFs varies among NF-κB target genes. Using the A20 promoteras a model it was shown that converting its core promoter fromTATA-less to a TATA was sufficient to convert the regulation from DSIFto P-TEFb, and from TFIID dependency to TFIID independency [76].This mode of regulation may not be restricted to the NF-κB pathwayas another study reported that regulation of the c-myc gene by P-TEFbis also dependent on the TATA-box [83].

Another study that addressed selective NF-κB target activationshowed that in resting macrophages, the TNFα gene has a NELF-associated Pol II with serine 5 phosphorylation paused near its pro-moter, whereas the CXCL10 gene does not [84]. Upon LPS inductionNELF is dissociated and releases the polymerase to transcribe the TNFαgene, whereas CXCL10 transcription rate was chiefly dependent on PolII recruitment. This results in significantly different kinetics of transcrip-tional peaks in spite of both genes not being CpG rich and containing aTATA box. It is of course true that LPS and polyIC are both TLR agonists

Fig. 5. Regulation of NF-κB target genes by DSIF. On promoters with a weak or no TATA-bremains associated with Pol II. DSIF subsequently aids to promote mRNA processing even i

that activate not only NF-κB but also additional pathways. Still this high-lights a reality where in addition to promoter CpG content and relatedchromatin structure, other mechanisms also regulate the selective acti-vation of inflammatory genes andmore specifically— differentially reg-ulate the elongation of NF-κB targets. In addition, some NF-κB targetsshowdelayed kinetics of expression and require de-novo protein synthe-sis and chromatin remodeling [85]. In such genes, promoter proximalpausing is not likely to be a major regulatory step.

IKKα, a major component of the NF-κB pathway, has also beenimplicated in elongation control. This kinase complex is also found inthe nucleus and is recruited alongwith RelA via CBP to NF-κB regulatedgenes where it phosphorylates histone H3 on S10/S31, which triggershistone lysine acetylations [86,87]. Once recruited to active genes byRelA, IKKα continues into the gene body in association with Pol II viainteraction with CTD phosphor-serine 2 in an elongation dependentmanner and further regulates elongation via interaction with HP1γand chromatin remodeling [88].

5. Coupling of elongation andmRNAprocessing in theNF-κBpathway

What could be the significance of elongation control for NF-κB targetgenes? The Pol II CTD serine 2 phosphorylation by P-TEFb has long beenknown to facilitate co-transcriptional mRNA processing [89–94]. It wastherefore suggested that P-TEFb acts to promote efficient processingof NF-κB-induced transcripts as well [74,95]. In this respect, it wasreported that in resting macrophages full length but incompletelyspliced transcripts were detected in association with the chromatin ofpro-inflammatory genes, along with Pol II CTD serine 5 but not serine2 phosphorylation [74,96]. Only upon inductionwas serine 2 phosphor-ylation acquired andmRNAproduction evident. In addition itwas foundthat promoters with low CpG correlate with low basal levels and highinduction folds while CpG-rich promoters correlate with more promis-cuous expression andweaker induction [96]. In this context it should benoted that TATA-box containing promoters typically have low CpGcontent [97] and that this type of promoter is highly enriched amongNF-κB targets [76]. Importantly, Bhatt and colleagues [96] noted thattheir data did not support a model for inducible CpG-rich promoters,in which efficient elongation and processing of existing pre-mRNA arethe central steps towards mRNA production. Rather, their data suggestthat the described stimulation of efficient elongation and processingsupports expression of genes in which induction has increased theinitiation and release rates [74,96].

With regards to genes such as A20 and IκBα in which P-TEFb isreleased and the elongating form of Pol II is hypo-phosphorylated[82,98], a recent study addressed the interplay between elongationand mRNA processing in these genes. It appears that in these genesDSIF substitutes for P-TEFb-mediated phosphorylation in promotingmRNA processing (Fig. 5). This was evident from DSIF knocked-down

ox a TFIID dependent PIC assembles, and upon induction P-TEFb is released and DSIFn the absence Pol II CTD phosphorylation.

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experiments, which revealed that a significant portion of A20 and IκBαmRNA was uncapped, unspliced, and most notably-failed to beexported from the nucleus. As a consequence, much less IκBα andA20 proteins were synthesized and NF-κB activity was abnormallyprolonged [98]. The C-terminal region (CTR) of DSIF large subunitSpt5 is itself a known target for phosphorylation by P-TEFb. The phos-phorylation of this domain by P-TEFb was proposed to be a switchfrom negative to positive effects on elongation [99]. Interestingly, theonly Spt5 domain that is dispensable for regulation of A20 and IκBα byDSIF is the CTR, while all other domains are essential. This is distinctfrom many other DSIF-regulated genes in which the phosphorylationof this domain by P-TEFb is critical for its function [99–101]. This is inline with the observation that P-TEFb is released from these genesupon their activation, and accordingly their induced transcription isresistant to DRB. Thus the investigation of the negative feedback branchof the NF-κB pathway has uncovered an alternative pathway for tran-scription coupledmRNA processing, whichmay operate in other genes.

6. NF-κB-mediated transcription elongation and HIV

The NF-κB pathway has a significant impact on the HIV life cycle,contributing in various ways to HIV latency through positive and neg-ative regulations of the viral genome itself and genes that affect HIVlatency [102]. Among the viral encoded proteins, the Tat has longbeen established as the major activator of HIV transcription by power-fully stimulating transcription elongation. Tat interacts with a viralinhibitory RNA element called TAR as well as with P-TEFb to promoteCTD phosphorylation of Pol II that is paused near the viral promoterand to facilitate productive elongation. This, along with multiple regula-torymodifications of Tat, brings about an intense activation of expressionof viral genes [103]. Within this frame NF-κB has been shown to impact

Table 1Summary of major papers addressing transcription regulation of the NF-κB pathway at the leare noted.

Elongation modulator Target genes Cell type Stimulu

CTD Ser2 kinase, GR IL-8, ICAM-1 CV-1, A549 TNFα

P-TEFb IL-8, HIV reporter CV-1, A549 TNFαMSK-1 IL-6, IL-8, E-selectin L929sA, HEK293,

primary fibroblastsTNFα

DSIF, NELF A20, IκBα Jurkat, 293 T TNFα, p

P-TEFb, GR IL-8 A549 TNFαP-TEFb, p300 Murine CD80 FO Anti- CD

DSIF, P-TEFb A20, IP-10, RANTES, IκBα Jurkat, 293T TNFα, p

P-TEFb, PKAc IL-8, Gro-β U937, A549, 293T TNFαDSIF A20 293T TNFα, p

NELF, P-TEFb TNFα, IP-10, RANTES,TTP

RAW264.7, BMMΦ LPS

MSK-1 IL-8, Gro-β A549 RSV infeBrd4 IL-8, E-selectin TNFα A549, 293 T, MEF, THP-1 TNFαP-TEFb, Brd4, HACs(PCAF, GCN5, CBP/P300)

Array of NF-κB targetgenes

C57BL/F10 mice, BMMΦand RAW macrophages,293 T, MEF

LPS, IL-1

Brd4 IL-6, TNFα Murine BMDM, mice invivo

LPS

P-TEFb, Brd4 CXCL1, CXCL2, IL-6,RANTES

A549, MEF RSV infe

Brd4 Array of NF-κB targetgenes

RTEC (kidney cells),HIV-1 transgenic mice

TNFα, H

IKKα TNFα RAW264.7 macrophage TNFα, L

DSIF A20, IκBα HeLa TNFα

viral gene expression mainly at the level of initiation and early stages ofviral expression [104]. However NF-κB can still impact the elongation ofHIV. West and colleagues compared total and full length transcriptsto show that HIV transcription in T cells can be highly induced byp65/RelA at the level of elongation in the absence of Tat [105]. Tat alonehad an impact on elongation to a greater extent; however with NF-κBelongation was activated synergistically. This work also showed thatp65/RelA elongation stimulation was actually less sensitive to knock-down of the P-TEFb subunit Cdk9, bringing the authors to postulatethat other CTD kinases may confer some of the RelA activity. Severalstudies have demonstrated inverse correlations between Brd4 levelsand viral gene expression [106,107]. Thus NF-κB may play an importantrole in elongation control during the initial stages of viral gene expres-sion, before Tat protein reaches the critical levels required for stimulationof Pol II elongation. When considering all the established links betweenNF-κB, Brd4 and HIV, it seems likely that the effect of NF-κB on HIV tran-scription elongation may be underestimated and needs to be furtherelucidated.

7. Future prospective

Over the past decade our understanding of the mechanisms bywhich NF-κB activates different target genes at various stages of thetranscription process has remarkably expanded (Fig. 2 and Table 1).In particular the findings that pro-inflammatory genes are so stronglydependent on the CBP/p300-Brd4-P-TEFb cascade for elongation andmRNA processing have been well established. On the other hand,some of the anti-inflammatory genes are refractory to this pathwayand aremore dependent onDSIF for efficient elongation andmRNApro-cessing. These observations have already yielded valuable informationthat was used to target the pro-inflammatory response in a selective

vel of elongation. For each paper, the elongation modulator in focus and central finding

s Finding Reference

GR inhibits NF-κB activation and Ser2 phosphorylationthrough protein–protein interaction

[49]

RelA recruits P-TEFb to genes to stimulate elongation [50]MSK-1 phosphorylates RelA S276 [63]

65 expression DSIF regulates promoter proximal pausing of A20 at basalconditions and of A20 and IκBα upon TNFα induction

[16]

GR competes with P-TEFb on target promoters [51]40 antibody RelA recruits acetyl transferases which acetylate the first

nucleosome downstream to TSS, facilitating Brd4-P-TEFbrecruitment

[62]

65 expression Differential regulation of NF-κB targets by DSIF or P-TEFbis determined by the core promoter

[76]

TNFα induces ROS-PKAc-RelA S276P-P-TEFb pathway [66,67]65 expression Interplay between E-box and NF-κB in regulation of A20

elongation under basal and NF-κB induced conditions[82]

Pol II promoter proximal pausing enables differentialinduction kinetics of NF-κB targets

[84]

ction RSV induces ROS-MSK1-RelA S276P pathway [65]Brd4 binds RelA K310Ac and recruits P-TEFb [69]

β NF-κB induces H4 acetylations and Brd4-P-TEFb recruit-ment, P-TEFb recruitment promotes both elongation andmRNA processing

[74]

Acetylated-histone mimicking compounds deplete Brdproteins from inflammatory cytokine gene promoters

[72]

ction, TNFα RelA K310Ac facilitates association with P-TEFb and Brd4to control inflammatory cytokine expression

[71]

IV infection Brd4 inhibition downregulates NF-κB transcriptionalactivity and kidney diseases associated inflammation

[75]

PS IKKα, recruited to NF-κB responsive genes, travels withPol II into gene body and phosphorylates histone 3,modulating elongation

[88]

DSIF coordinates elongation and mRNA processing ofA20 and IκBα, affecting NF-κB negative feedback

[98]

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manner by drugs that inhibit Brd4. However there are still a number ofopen questions that need to be addressed in future research.

An important question is the mechanistic basis underlying theregulation of the anti-inflammatory arm of NF-κB by DSIF. In analogyto the p65-Brd4-P-TEFb pathway, it is possible that specific post-transcriptional modifications either on p65/RelA and/or chromatinthat have yet to be discovered are involved in the selective regulationof IκBα and A20 genes by DSIF. If a mechanistic link will be found topathways that can be modified by drugs such as the control of post-translational modifications, it may possible to develop a new class ofdrugs to enhance or decrease the anti-inflammatory response.

An exciting issue is the recent discovery that Brd4 itself is an atypicalprotein kinase which, like P-TEFb, specifically phosphorylates the Pol IICTD on serine 2 residues [108]. Taking the interaction of Brd4 and p65into account, it would be interesting to elucidate how this kinase activ-ity is integrated into the regulation of NF-κB target genes at the level ofelongation.

An additional aspect concerns the multi-faceted nature of NF-κB,which has the ability to modulate chromatin structure and to pro-mote initiation, re-initiation, elongation andmRNAprocessing throughinteraction with a large number of co-regulators. As functional κB sitescan be found over a broad range relative to the TSS — from less than100 bp proximity up to tens of kilo-bases away, one is tempted to spec-ulate that distal and proximal κB sites may utilize distinct mechanismsand interaction with co-regulators to activate target genes.

Finally, as abnormal NF-κB activity is associated with chronicinflammatory diseases and cancer and in light of the central role thattranscription elongation plays in its activity, it is possible that de-regulation of elongation factors contributes to a subset of pathologiesinvolving NF-κB. For example, in normal cells P-TEFb activity is tightlyregulated by the HEXIM1-7SK complex so downregulation of these in-hibitory proteins may facilitate strong pro-inflammatory activity. Like-wise, alterations in the activity of Brd4 and DSIF subunits may alsocontribute to the severity of NF-κB associated diseases. We anticipatethat future studies into the transcriptional function of NF-κB will pro-vide additional insights into normal and abnormal behavior of thispathway, and that novel findings will be taken into considerationwhen designing drugs to influence specific subsets of NF-κB targetgenes.

Acknowledgements

We would like to thank Shaked Ashkenazi for her suggestions andthe anonymous referees for their comments and advise. This work wassupported by grants from the Israel Science Foundation (#816/09), IsraelCancer Research Fund and Israel Cancer Association. R.D. is the incum-bent of the Ruth and Leonard Simon Chair of Cancer Research.

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