Allergy Review Series VII: Intracellular signaling and regulation of allergic reactions

Molecular regulation of class switch recombination to IgE

through e germline transcription

S. Monticelli1, D. Vercelli1,2

1Respiratory Sciences Center and 2Department of Cell

Biology and Anatomy, College of Medicine, University

of Arizona, Tucson, AZ, USA

Dr D. Vercelli

Respiratory Sciences Center

College of Medicine

University of Arizona

Tucson, AZ 85724

USA

Accepted for publication 21 August 2000

The role of germline (GL) transcription in class switch

recombination

The ability of B cells to express immunoglobulinswith identical antigen speci®city but different effectorfunctions results from their capacity to undergo classswitch recombination (CSR). This process consists ofan intrachromosomal DNA recombination event thatinvolves repetitive switch (S) regions located 5k ofeach CH gene, except Cd, and results in thejuxtaposition of different downstream CH genes tothe expressed VDJ gene (reviewed in Ref. 1). The Sregion involved in recombination is not targeted atrandom. Instead, CSR appears to be directed by T-helper-cell-derived cytokines in conjunction with theregulation of B-cell proliferation and differentiation.Thus, human B cells activated by CD40 cross-linkingin the presence of IL-4 or IL-13 switch from IgM toIgE (2, 3).

Molecular analysis has shown that cytokine-depen-dent induction of CSR to a particular CH gene almost

invariably correlates with the transcriptional activa-tion of the same gene in germline (i.e., unrearranged)con®guration. All GL transcripts share the sameoverall structure, and originate from TATAA-lesspromoters a few kilobases upstream of the S region,proceed through short noncoding exons (IH exons),the S regions and CH exons, and are polyadenylatednear the normal poly(A) site for mature IgH mRNAs.The IH exon is then spliced to the normal acceptorsite in the ®rst exon of the CH gene. During CSR, theregion containing the GL promoter and the IH exonis deleted as part of a switch circle. GL transcripts are``sterile''; i.e., they do not appear to be translated intoprotein, because they contain stop codons in allreading frames (reviewed in Ref. 1).

Targeted mutagenesis experiments have indicated thatintegrity of the GL transcription units (promoters and/orexons), rather than transcription through the S regions, isessential for CSR (4±8). More recently, splicing ofprimary GL transcripts has been shown to constitutean essential step in CSR activation (8±10). This ®ndingimplies that a spliced-out RNA species may be an integralpart of the CSR enzymatic machinery, and/or some ofthe factors responsible for RNA splicing may also beinvolved in DNA recombination (see below). Supportingthe hypothesis of a functional connection between RNAsplicing and CSR are the identi®cation of proteins

Abbreviations. BSAP: B-cell-speci®c activator protein; C/EBP: CAAT

enhancer binding protein; CSR: class switch recombination; GL:

germline; IL-4R: IL-4 receptor; IL-4RE: IL-4-responsive element; NF-

kB: nuclear factor-kB; S region: switch region; STAT: signal transducer

and activator of transcription.

Allergy 2001: 56: 270±278Printed in UK. All rights reserved

Copyright # Munksgaard 2001

ALLERGYISSN 0105-4538

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involved in RNA processing as components of S regionDNA-binding complexes (11, 12) and the precedent ofyeast factors involved in both splicing and meioticrecombination (13). Involvement of the splicing machin-ery is also suggested from studies on the distribution ofCSR junctions in CH12F3-2 cells that undergo ef®cientcytokine-dependent CSR in vitro (14). CSR junctionswere found not only within but also outside the S region.However, they were rarely found within the I exon,suggesting that breakpoints of CSR are restricted to theintron region of GL transcription. If processing of tran-scripts and recombination of DNA are really coupled asthey appear to be (see below), CSR would be a veryunique system with unprecedented character (15).

The full extent to which GL transcripts participate inCSR remains unclear, but their role is currently beingreinterpreted. For a number of years, the invariableassociation between GL transcription and isotypeswitching was taken to support the accessibility model,according to which chromatin opening coupled withtranscription allows the CSR recombinase to access thetarget DNA. However, this model could not account forisotype switching induced by stimuli (e.g., glucocorti-coids) that do not affect GL transcription (16). Inaddition, it was convincingly argued that, since GLtranscripts must be spliced together for CSR to occur,they cannot be simply involved in chromatin remodeling(17). Indeed, recent evidence suggests that the choice ofantibody class in CSR may be regulated by GLtranscription through the formation of RNA/DNAhybrids that select the sites of recombination. This viewis supported by biochemical experiments demonstratingthat transcripts of the S regions in immunoglobulin genesremain associated with the template DNA after tran-scription in vitro (18). Mouse and human S regionscontain G-rich sequence repeats. The G-rich transcribedRNA appears to form a stable hybrid with the C-richDNA template, leaving the other DNA strand (thenontemplate strand) on its own in a single-stranded state(19). Most importantly, after B cells from mouse spleenare induced to undergo CSR in vivo, RNA/DNA hybridsform within the S regions of the DNA. These RNA/DNAhybrids appear to be mechanistically important for CSR,because mice transgenic for ribonuclease H (an enzymethat speci®cally snips the RNA in the hybrids) showed amarked reduction in hybrid formation and, in parallel,lower IgG levels and inhibition of CSR in their splenic Bcells (20).

What structural features of the S region are requiredfor formation of the stable hybrid during transcriptionremains to be established. Although mouse and human Sregions are G-rich, and runs of guanosines are known toform unusual structures, this may not be a crucial featurebecause the S region upstream of the Ig Cm gene inXenopus is A-T rich (21). It is possible that the stretches ofpurine-pyrimidine asymmetry, the repeated sequences,and the numerous palindromes (sequences that read

identically in both directions) in the S regions of mouse,man, and Xenopus are essential for CSR. How RNA/DNA hybrids regulate CSR is also unclear, but it is likelythat they serve as the recognition target for a CSR mach-inery still uncharacterized. Splicing may be required tostabilize the hybrids by removing extra nucleotidesincapable of participating in their formation (17).

The counterintuitive conclusion warranted by thesedata is that the intronic region spliced out of the primaryGL transcript, rather than the processed IH-CH tran-script itself, may represent what is required to targetrecombinase activity to the appropriate S region. Thecurrent model for the control of CSR to IgE through GLtranscription is presented in Fig. 1. In the absence ofcytokine stimulation and CD40 engagement, a naive Bcell expresses IgM and has the IgH locus in GL(unrearranged) con®guration. Even under these condi-tions, Im-Cm GL transcripts are constitutively expressed(step 1). After stimulation with IL-4 or IL-13 (andpossibly with the cooperation of CD40 cross-linking), theeGL promoter is activated (see below), and transcriptionis initiated (step 2). Primary e GL transcripts contain theIe exon, Se, and Ce (step 3), and share the overallstructure, albeit not the sequence, of primary mGLtranscripts. Upon splicing of the primary GL transcripts(step 4), mature IH-CH transcripts are released, whereasthe spliced intron corresponding to S region sequencesremains associated with template DNA. The resultingstable RNA/DNA hybrid represents the target for a stillunidenti®ed endonuclease that might initiate CRS.Joining of the two S regions involved in CSR, followedby DNA repair mediated by a number of factors,including Ku proteins (22) and DNA mismatch repairingenzymes (23), results in the formation of a hybrid Sm/Seregion, bringing Ce in close proximity to the rearrangedVDJ complex. The excised DNA forms a circle and israpidly degraded (step 5).

Regulation of the e GL promoter

The postulated mechanisms through which S regiontranscription and splicing may cooperate to target CSRreiterate the critical role of cytokine-dependent inductionof GL transcription in determining the isotype speci®cityof switching. Thus, it is necessary to understand howindividual cytokines activate speci®c GL promoters inorder to understand how the isotype speci®city of CSR isachieved.

Full activation of the human e GL promoter inresponse to IL-4 and CD40 engagement requires aconstellation of nuclear factors that are either consti-tutively expressed or recruited to the promoter byspeci®c activating signals. In the last few years, ourgroup has performed a systematic characterization ofthe proximal region of the human e germline promoterby electrophoretic mobility shift assays (EMSA) and

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reporter analysis. EMSA analysis provides informationon the number and af®nity of speci®c binding sites fornuclear proteins in a DNA fragment. The speci®city ofprotein±DNA interactions is assessed with oligonucleo-tide competitors to compare the af®nities of differentDNA sequences for the protein(s) of interest. Anotheruseful feature is the ability to identify the protein(s)

involved in the formation of the complex of interest,using antibodies to speci®c transcription factors in anattempt to modify the mobility of the protein/DNAcomplex, or to prevent its formation. Once a proteinbinding to a given DNA motif has been identi®ed by thepattern of oligonucleotide competition and antibody-mediated supershifting, the original probe may be tested

Figure 1. Induction of class switch recombination to IgE through GL transcription. The model is extensively discussed in the text. Thedifferent steps in CSR are labeled in blue. RNA is indicated in red. 1) Constitutive expression of primary m GL transcripts; 2) cytokine(CD40)-dependent activation of the e GL promoter; 3) cytokine-dependent expression of primary e GL transcripts; 4) splicing ofprimary GL transcripts, release of mature I-CH GL transcripts, and formation of stable hybrids between the S region and the intron ofGL transcripts; the hybrids target the switch recombinase to the appropriate S region; 5) Sm/Se recombination and formation of circlescontaining the excised DNA. ss: splice site.

COLOURFIGURE

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for its ability to bind the transcription factors of interestexpressed in recombinant form. Reporter assays, on theother hand, allow the characterization of promotersequences essential to drive transcription of a reporterconstruct with the same stimuli known to activate theendogenous gene. The functional role played byindividual transcription factors in regulating promoteractivity can be assessed with constructs that containmutations shown to abrogate binding of speci®c nuclearproteins.

The IL-4-responsive element (IL-4RE), a region that binds STAT6 and

Myb proteins

The human e GL promoter is exquisitely IL-4/IL-13-inducible. Promoter response to Th2 cytokinesappears to rely critically on a highly conserved sequence5k to the major initiation sites for e GL transcripts. Thisregion (position x109/x80 in Fig. 2) represents an IL-4RE that binds STAT6 and recombinant CAATenhancer-binding (C/EBP) proteins, and is suf®cientto confer IL-4 inducibility on a heterologous promoterin transient transfection studies (24, 25).

STAT6, whose essential role in IL-4-dependent e GLtranscription (26) and IgE switching (27±30) is wellestablished, belongs to the recently identi®ed family ofsignal transducers and activators of transcription(STAT) (31). Binding of IL-4 to its receptor (IL-4R)leads to activation by tyrosine phosphorylation of tworeceptor-associated cytoplasmic tyrosine kinases, Januskinase (JAK)3 and JAK1 (reviewed in Ref. 32). Thesekinases are believed to induce rapidly phosphorylationof critical tyrosine residues in the intracellular domainof the IL-4Ra chain. These phosphorylated tyrosineresidues provide docking sites for the latent cytosolictranscription factor STAT6. STAT6 binds to thesedocking sites via its SH2 domain, and is in turntyrosine-phosphorylated by the receptor-associatedJAKS. Phosphorylated STAT6 then homodimerizes,translocates to the nucleus, and binds to the promoterof a number of genes, contributing to the activation of

transcription (33). STAT6 preferentially binds dyadsymmetric half-sites separated by four base pairs (TTC-N4-GAA) (34). DNA binding speci®city is localized to aregion of 180 amino acids at the N terminal side of theSH3 domain (35). The discovery of JAKs and STATsprovided an explanation for the apparent paradox thatthe IL-4R, as well as several other cytokine receptors,lacks kinase domains, and yet couples ligand binding totyrosine phosphorylation.

While the x109/x80 region functions as an IL-4RE,IL-4 inducibility was surprisingly lost in constructscontaining a slightly shorter sequence (position x105/x83), even though this region contained intact bindingsites for both STAT6 and C/EBP (36). This discrepancyraised the possibility that deletions introduced at thetwo ends of the IL-4RE in the x105/x83 constructdestroyed unidenti®ed binding sites for nucleopro-tein(s) important in the regulation of e GL promoteractivity. Indeed, we have recently been able to showthat the IL-4RE contains at its ends two motifs thatstrongly resemble the consensus binding site for theMyb family of transcription factors (PyAACG/TG)(37), and bind endogenously expressed Myb proteins.The myb1 site overlaps the 5k half of the putative C/EBP site, whereas myb2 overlaps the 3k half of theSTAT6 element (Monticelli et al., submitted forpublication) (Fig. 2).

The presence of binding sites for Myb proteins atthe two ends of the human IL-4RE in the e promotermay provide a mechanistic explanation for the long-sought link between germline transcription, switchrecombination, and B-cell proliferation (38). Indeed,full expression of the transactivation potential of twomyb family members, A- and B-Myb, requiresunmasking of their transactivation domain throughphosphorylation of the carboxy-terminal regulatorydomain (39, 40). Phosphorylation appears to bemediated by cyclin A/cdk2 and cyclin E/cdk2 kinases,which are speci®cally expressed at the G1/S transition(40±42). Thus, the carboxy-termini of A- and B-Myb

Figure 2. The proximal human e GL promoter. Position +1 corresponds to the major e RNA start site in BL-2 cells (65). Binding sitesfor transcription factors identi®ed in the promoter are boxed and labeled. The IL-4RE and its boundaries are indicated by dashed lineand arrowheads.

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act as cell-cycle sensors. Furthermore, RNA for A-and B-myb is expressed in a cell cycle-dependentmanner during late G1 and early S-phase (41, 43).This dual control mechanism ensures that maximal A-and B-Myb activity peaks in the G1/S phase. Mybbinding may also contribute to the early steps of eGL gene activation. Indeed, recent data indicate thatboth Myb and STAT6 proteins interact with thecoactivators p300 and CBP, thereby recruiting histoneacetyltransferase activity to their cognate sites (44,45). Thus, binding of STAT6 and Myb to adjacentsites in the IL-4RE may play a key role in chromatinremodeling, as well as in the interaction with thebasal transcription machinery.

BCL-6

STAT6-binding sites share homology with the DNArecognition motif for BCL-6, a transcriptionalrepressor expressed at high levels in germinal centerB cells (46). Indeed BCL-6, a POZ/zinc-®nger protein,has been shown to bind the STAT6 site in the CD23bpromoter and block IL-4-dependent transcription ofthis gene in transient transfection assays (47).Furthermore, it has been proposed that BCL-6-mediated repression through the STAT6 site maycontribute to silencing the IL-4-inducible e GLpromoter under basal conditions (48). This hypothesiswould be consistent with the ®nding that mutationsof the STAT6 recognition element in the e GLpromoter may result in markedly increased levels ofbasal transcription (49, 50), suggesting a disruption inthe recruitment of a repressor to these sites. Weshowed that BCL-6 is expressed in the BL-2 cell lineused in our experiments. However, this factorappeared to play no role in the regulation oftranscription from the human proximal e GLpromoter. Indeed, EMSA analysis failed to showBCL-6 binding to the STAT6 site; furthermore,mutations of the STAT6 element in the e GL reporterconstruct abrogated STAT6 binding but had no effecton the basal activity of the reporter gene in transienttransfection assays.

It is noteworthy that the recognition sites for BCL-6and STAT6 are similar but not necessarily identical.Unlike the STAT6 elements in the proximal region ofthe e promoter, an optimal BCL-6 site de®ned in vitro(46) and the one found in the CD23b promoter (51)contain three, not four, nucleotides between the twopalidromic half-sites. It is possible that endogenousBCL-6 binds with high af®nity only to elements thatcontain a 3-bp spacer, i.e., to sites recognized by mostSTAT proteins. In contrast, STAT6 is known to bindsites with 3-bp spacers, but, in addition, has a uniqueability to interact with 4N sites (34, 35). This wouldimply that BCL-6 may interact with some but not all

STAT6-binding sites, but at the same time couldmodulate events triggered by other STAT proteins aswell.

NF-kB/Rel proteins and their association with STAT6

The region immediately downstream of the IL-4REcontains two sites that bind NF-kB/Rel proteins withcomparable af®nity (Fig. 2) and appear to be essentialfor promoter function (see below). The upstream site(NF-kB1) bound essentially p50/p65 heterodimers. c-Rel binding was also detectable in cells stimulated withIL-4 and/or anti-CD40 mAb. The downstream NF-kBsite (NF-kB2) bound the same NF-kB/Rel familymembers. In addition, we detected p50 homodimersin extracts from cells treated with anti-CD40 mAb, withor without IL-4, and p50/RelB heterodimers inuntreated and IL-4-stimulated cells.

Perhaps the most interesting outcome of the NF-kBstudies was the demonstration that NF-kB/Relproteins directly associate with IL-4-inducibleSTAT6, and synergize with it in activating IL-4-dependent transcription (see below). Similar ®ndingswere reported for the murine e and c1 GL promoters,in which a STAT6 element is located upstream of twoor three NF-kB binding sites (52±54). EMSA analysiswith a probe (position x97/x57) that spans both theSTAT6 and the NF-kB1 elements revealed threemajor speci®c complexes in cells treated with IL-4,with or without anti-CD40 mAb (Fig. 3). Complex 2was IL-4-inducible, and contained STAT6, because itwas speci®cally competed by an oligonucleotide(STAT6, x97/x72) containing the STAT6 bindingsite, but not by an NF-kB consensus sequence(kBH2), nor by an unrelated oligonucleotide(COUP). Furthermore, complex 2 was completelysupershifted by an anti-STAT6 antiserum. Complex 3contained NF-kB/Rel proteins, because it was speci-®cally competed by kBH2, but not by STAT6.Complex 3 was supershifted by an anti-NF-kB p50antiserum and inhibited by an antiserum to p65, butwas unaffected by addition of anti-STAT6 antiserum.Most importantly, an additional band (complex 1)was detected. Complex 1 contained both STAT6 andNF-kB nucleoproteins, because it was speci®callycompeted by STAT6 and kBH2 oligonucleotides, andsupershifted or inhibited by antisera to STAT6 andNF-kB family members (p65, p50, and c-Rel). Thesedata show that, consistent with ®ndings in the murinemodel (55), STAT6 and NF-kB physically associatewhen binding to their adjacent cognate sites in thehuman e GL promoter.

BSAP

The B-cell-speci®c activator protein (BSAP, Pax-5) is ahomeodomain-class transcription factor speci®callyexpressed in the B-cell lineage from pro-B to mature

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B cells, but not in plasma cells (56). Binding sites forBSAP have been found in various promoters of B-cell-related genes (e.g., CD19, VpreB1, l5, and blk),including the Ig gene locus (reviewed in Ref. 57).BSAP binds to a highly conserved region immediatelyupstream of the major Ie transcription initiation site inboth murine (58) and human (59) B cells (Fig. 2).Notably, among the factors that regulate e GLtranscription, BSAP is unique in that it is speci®callyexpressed in B cells. GL transcription through theimmunoglobulin locus is a B-cell-speci®c event as well.Thus, it is tempting to speculate that BSAP may beinvolved in the tissue-speci®c activation of immunoglo-bulin GL promoters, several of which contain BSAP-binding sites.

Functional analysis of the human e GL promoter

Having identi®ed a set of transcription factors that bindthe human e GL promoter, we then assessed thefunctional role played by these nucleoproteins inregulating promoter ativity. For this purpose, luciferasereporter constructs containing the human e GLpromoter (either wild-type or mutated at speci®cbinding sites) were transiently transfected into BL-2cells. Reporter activity was measured in cells leftuntreated or stimulated with IL-4 and/or anti-CD40 mAb. As shown in Fig. 4, the wild-type promo-ter was activated 3.6-fold by IL-4, 2.3-fold by CD40engagement, and 31.6-fold by a combination of the twosignals. Thus, CD40 cross-linking enhanced IL-4-dependent transcription by 8.7-fold, pointing to astrong synergism between the two stimuli.

Mutations introduced in the reporter constructs hadprofound but different effects on the activation of thepromoter in response to IL-4 and/or CD40 engagement.Mutations of the STAT6 and NF-kB1 binding sites haddramatic effects on promoter activity; i.e., theyabolished both the response to IL-4 and theCD40-dependent enhancement of e transcription(80% and 70% inhibition, respectively). Mutation ofthe NF-kB2-binding site impaired IL-4 responsivenessonly minimally (19% inhibition), and decreased thesynergism between IL-4 and CD40 signaling by only40%, indicating that NF-kB proteins bound moredownstream may not be primarily involved in the

Figure 3. Association between NF-kB/Rel and STAT6 proteins.Nuclear extracts (5 mg) from serum-starved BL-2 cells incubatedin the presence or absence of IL-4 (100 U/ml) and/or anti-CD40 mAb (5 mg/ml) for 30 min were analyzed by EMSA, usingas probe an oligonucleotide spanning both the STAT6 and NF-kB1 sites in the e GL promoter (position x97/x57). Extractsfrom BL-2 cells stimulated with IL-4+anti-CD40 mAb werepreincubated with rabbit antibodies (2 mg/reaction) speci®c forindividual NF-kB/Rel proteins or unrelated factors for 30 min onice before adding the probe. Competitors were added at 100-foldmolar excess. Speci®c complexes containing STAT6 and/or NF-kB proteins are indicated by arrows.

Figure 4. Reporter analysis of the proximal e GL promoter. BL-2 cells were transiently cotransfected with the reporter constructsand an RSV-b-gal control plasmid using DEAE-dextran, andsplit into aliquots that were incubated in the presence of IL-4 (100U/ml: gray bars), anti-CD40 mAb (5 mg/ml: white bars), or acombination of the two stimuli (black bars). Luciferase activitywas assessed 48 h later and normalized for transfection ef®ciencyand total protein content. Values obtained in mock transfectedcells were subtracted as background. Fold induction representsthe ratio of relative luciferase activity (cpm/mg of protein)between treated and untreated cells. The results show themeantSE of at least three independent experiments, eachperformed in duplicate.

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interaction with STAT6. Mutation of the BSAP-binding site strongly impaired both the IL-4-inducedactivation and the CD40-dependent enhancement of eGL promoter activity (59). Simultaneous mutation ofboth NF-kB sites, with or without the BSAP element,abrogated not only CD40 responsiveness but also IL-4-dependent activation, even though the STAT6element was left intact (data not shown). The reporterconstruct containing mutations of the STAT6 and NF-kB1 and/or NF-kB2 sites was functionally indistin-guishable from the empty pGL3 vector (data notshown).

Our results clearly indicate that regulation of humane GL transcription involves complex interactionsbetween factors recruited through different signalingpathways. NF-kB/Rel proteins cooperate with STAT6for the synergistic activation of IL-4-dependent humane GL transcription. NF-kB activity was critical not onlyfor the CD40-dependent enhancement of IL-4-inducede GL transcription, but also for IL-4 responsiveness inthe absence of antibody-mediated CD40 cross-linking.Both effects are likely to re¯ect the physical associationbetween NF-kB/Rel proteins and STAT6. Uponinteraction with NF-kB, STAT6 appears to undergocritical quantitative and functional changes: DNA-binding af®nity is substantially enhanced, and, mostimportantly, transactivating ability is induced (35).Thus, direct STAT6/NF-kB interactions are necessaryfor the synergistic activation of transcription frompromoters containing both cognate sites. Notably,activation of neither STAT6 nor NF-kB requires denovo protein synthesis, allowing the rapid transmissionof the IL-4/CD40 signal to the nucleus. More recently, ithas been shown that the upstream NF-kB site overlapswith a PU.1-binding motif. Abrogation of DNAbinding of both PU.1 and NF-kB resulted in loss ofIL-4 inducibility of IgE GL promoter constructs (60).This result suggested that PU.1, as well as NF-kBfactors, may cooperate functionally with STAT6 tomediate IL-4 induction of the e GL promoter.

BSAP may contribute to IgE switching not onlythrough direct activation of the e GL promoter, but alsothrough differential regulation of GL transcription.BSAP inhibited a GL transcription and switching toIgA in sIgM-positive murine I.29m cells, but enhanced eGL transcription and switching to IgE (61). The

opposite effects of BSAP on e and a GL transcriptionexemplify the ability of this factor to activate or inhibittranscription, depending on the protein(s) with which itinteracts at a particular binding site (62, 63), and theaf®nity for its different binding motifs. During a B-cellimmune response, BSAP maintains its activator func-tions but is relieved of its repressor functions. Thisselective targeting of BSAP activities was shown to beregulated by a concentration-dependent mechanismwhereby activator motifs for BSAP had a 20-fold higherbinding af®nity than repressor motifs. An exchange ofactivator and repressor motifs, however, showed thatthe context of the motif, rather than the af®nity,determined whether BSAP operated as an activator orrepressor (63).

The role of C/EBP proteins in regulating human eGL transcription through the putative site in the IL-4RE remains unclear. Because sites for C/EBP andSTAT6 proteins are often adjacent or overlapping inthe promoters of IL-4-regulated genes, this arrange-ment was considered to serve a functional purpose(64). Although both factors can bind DNA indepen-dently, C/EBPb would stabilize STAT6 binding,thereby synergistically activating IL-4-dependent tran-scription (25). Indeed, the 5k portion of the IL-4REbinds recombinant C/EBPb. However, we found noevidence for interactions with endogenous members ofthis family in BL-2 cells. Of note, production of IgEupon stimulation with IL-4 and a trimeric CD40ligand was normal in C/EBPb-de®cient mice (27). Ig eGL transcription in this model was not speci®callyassessed; however, these ®ndings imply that C/EBPbmay not be necessary for IL-4-induced IgE switchingin vivo.

In conclusion, multiple factors contribute to theregulation of e GL transcription upon binding theproximal region of the promoter. It will be interestingto characterize additional regulatory elements in thegenomic region 5k to the 110 bp analyzed in thepresent study, as well as long-range interactionsinvolving the 3ka2 enhancer. Furthermore, it will beimportant to compare nucleoprotein binding andfunctional properties of the e and c4 GL promoters,since they both respond to IL-4 and CD40 cross-linking, but show distinct patterns of activationin vivo.

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