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The Journal of Immunology, 2002, 168: 1259-1266.
Copyright © 2002 by The American Association of Immunologists

Regulation of Human Ig{lambda} Light Chain Gene Expression by NF-{kappa}B1

Gabriele Combriato and H.-Gustav Klobeck2

Adolf Butenandt Institut Molekularbiologie, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human Ig{lambda} enhancer consists of three separated sequence elements that we identified previously by mapping DNase I-hypersensitive regions (HSS) downstream of the C region of the Ig{lambda} L chain genes (HSS-1, HSS-2, and HSS-3). It has been shown by several laboratories that expression of the H chain genes as well as the {kappa} genes, but not the {lambda} genes, is dependent on constitutive NF-{kappa}B proteins present in the nucleus. In this study we show by band-shift experiments, in vivo footprinting, and transient transfection assays that all three hypersensitive sites of the human Ig{lambda} enhancer contain functional NF-{kappa}B sites that act synergistically on expression. We further show that the chicken {lambda} enhancer also contains a functional NF-{kappa}B site but the mouse {lambda} enhancer contains a mutated, nonfunctional NF-{kappa}B site that is responsible for its low enhancer activity. It is possible that the inactivating mutation in the mouse Ig{lambda} enhancer was compensated for by an expansion of the Ig{kappa} L chain locus, followed by a contraction of the Ig{lambda} locus in this species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During B cell development the Ig loci undergo a series of DNA rearrangements starting with the H chain locus, followed by recombinations in the {kappa} and {lambda} L chain loci. Sequence-specific recognition of the signal sequences of Ig gene segments is conducted by the Rag proteins (reviewed in Ref. 1), which are also involved in V(D)J recombination of the TCR loci. Despite the fact that the recombination signal sequences in the Ig and TCR system are identical, in a B cell only the Ig loci are recombined in an ordered fashion. This led to the idea that it is the cell type-specific regulation of accessibility of a given locus that determines which gene segments are recombined in a B cell or a T cell (2). However, the factors involved in the targeting of chromatin-remodeling machines to open the chromatin structure of entire Ig loci are still unknown.

It has been shown by a number of laboratories that the Ig enhancer elements, initially identified by their ability to enhance transcription of reporter genes, have multiple functions during B cell development. Besides their role in transcriptional control of fully assembled Ig genes, they are also involved in accessibility control, because deletion of enhancer elements leads to a failure in recombination (3, 4). The somatic hypermutation mechanism leading to affinity maturation of Abs also seems to depend on the presence of enhancer elements (reviewed in Ref. 5).

One approach to study the diverse functions of the enhancer elements is first to identify the factors binding to sequence elements within the enhancers and then determine their role during B cell development. A number of transcription factors that are known to interact with the Ig enhancer and promoter elements and/or to regulate other B cell-specific genes have been investigated for their influence on proper B cell development (reviewed in Refs. 6 and 7). One of these factors critically involved in B cell differentiation and Ig gene transcription is NF-{kappa}B, which was originally described as a pre-B cell-inducible factor binding to the Ig{kappa} L chain (Ig{kappa})3 intron enhancer and is constitutively activated in mature B cells (8, 9), but is now known to be a ubiquitous factor that can be induced by various stimuli (reviewed in Refs. 10 and 11). The NF-{kappa}B family of transcription factors forms heterodimers that are retained in the cytoplasm due to an association with I{kappa}B proteins that mask the nuclear localization signal, thereby preventing nuclear uptake. A variety of extracellular stimuli lead to phosphorylation, ubiquitination, and proteolytic degradation of the I{kappa}B proteins (reviewed in Ref. 12), followed by nuclear translocation of the NF-{kappa}B proteins. It has been shown that overexpression of a nondegradable form of I{kappa}B leads to inhibition of {kappa} gene transcription and rearrangement in pre-B cells (13). NF-{kappa}B is known to be constitutively activated in mature B cells, and it binds to the IgH intron and the Ig{kappa} intron enhancers in mouse and human. However, the Ig{lambda} L chain (Ig{lambda}) genes do not seem to be regulated by NF-{kappa}B (14, 15).

To identify putative regulatory sequence elements (with and without a direct influence on transcriptional activity) that are involved in various aspects of regulation during B cell development, we mapped chromatin accessibility in the vicinity of the human C region of the Ig{lambda} L chain (C{lambda}) gene segments and could demonstrate the presence of three B cell-specific DNase I-hypersensitive sites (HSS) downstream of the Ig{lambda} constant region genes (HSS-1, -2, and -3) (16). HSS-3 contains a powerful enhancer element that synergizes in transcriptional enhancement with HSS-1 and HSS-2, which have no enhancer activity of their own (17). We have now extended our investigation to include in vivo footprinting experiments to identify factors binding to the enhancer modules. Surprisingly, we found NF-{kappa}B sites within all three enhancer modules and show in this study for the first time that these sites are functional and that the human Ig{lambda} enhancer is regulated by NF-{kappa}B such as the H chain and {kappa} L chain Ig gene intron enhancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell culture

All cells were maintained in RPMI 1640 medium (c.c.pro, Neustadt, Germany) supplemented with 4 mM glutamine and 15% FCS (c.c.pro) in 5% CO2 in humidified air. The B cell lines MN-60 (18) and Daudi (19); the pre-B cell lines Nalm-6 (20), BV-173 (21), and Reh (22); and the T cell line CCRF-CEM (23) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Stimulation of cells with PMA (Sigma, Deisenhofen, Germany) was conducted in full medium containing 100 ng/ml PMA.

Plasmid constructs

All plasmids used for transient transfection experiments are based on the pGL3 series of vectors from Promega (Madison, WI). The clones pGL3{lambda}Pro and pGL3{lambda}/HSS3-2 have been described previously (17). pPL-1 is derived from pGL3{lambda}Pro after insertion of an 8.8-kb partial BamHI-BglII fragment containing HSS-1, -2, and -3 in its genomic context. Mutations of the NF-{kappa}B sites within the 8.8-kb insert were made using a PCR-based megaprimer method (24). For easy monitoring each single mutation introduced a BglII site instead of the original NF-{kappa}B site. The proofreading thermostable DNA polymerase Pfu (Promega) was used in all PCR experiments, and the resulting mutants were controlled by restriction enzyme digestion and sequencing. Multiple mutations of NF-{kappa}B sites were achieved by shuffling fragments from the single mutants and/or another round of megaprimer PCR.

The mouse Ig{lambda} enhancer E{lambda}2-4 was amplified from genomic C57/BL DNA by PCR (using Pfu polymerase) with the 5' primer LME-5 (5'-CCCCGGATCCTATATGATAGAGTTGGCC-3') and the 3' primer LME-3 (5'-CACATGACCACCACTGTCTG-3'). The resulting fragment was gel-purified, digested with BamHI (site underlined in LME-5) and BglII, and inserted into the BamHI site of the luciferase vector pGL3{lambda}Pro.

The chicken Ig{lambda} enhancer (CLE) was amplified from genomic White Leghorn DNA by PCR using Pfu polymerase and the 5' primer CLE-1 (5'-ACAGGAATTCCAGGAAGGCACAGCgctg-3') and the 3' primer CLE-2 (5'-GGGTGTCGACGTGGTGGGAGCGGGC-3'). The amplification product was gel-purified, digested with EcoRI and SalI (site underlined in CLE-2), and introduced into the vector pGL3{lambda}Pro. Mutations in the NF-{kappa}B site of the chicken enhancer and the germline-mutated site of the mouse enhancer were introduced using the megaprimer approach (24).The identities of the germline mouse and chicken enhancer fragments and the NF-{kappa}B mutants were controlled by sequencing.

The trans-dominant-negative I{kappa}B{alpha} clone pCI-I{kappa}B{alpha}({Delta}N1–36) was made after reverse transcription of poly(A) mRNA isolated from the B cell line Daudi (primer I{kappa}B-4, 5'-CTAGGCAGTGTGCAGTGTGG-3'), first PCR amplification with the primers I{kappa}B-2 (5'-AGCTCGTCCGCGCCATGTTC-3') and I{kappa}B-5 (5'-CTTTCAGCCCCTTTGCACTC-3'), and second amplification using the 5' primer I{kappa}B-3, which contains an EcoRI site and a ribosomal attachment site and starts at the internal methionine 37 of the I{kappa}B{alpha} sequence (5'-GGGGAATTCCTCGTCCGCGCCATGAAAGACGAGGAGTACGAG-3') and the 3' primer I{kappa}B-6, which contains the sequences from the 3' coding region of I{kappa}B{alpha} and an XbaI site (5'-GGTCTAGATCATAACGTCAGACGCTGGCCT-3'). Pfu DNA polymerase was used in the PCR. The EcoRI-XbaI fragment from the second PCR was then introduced into the eukaryotic expression vector pCI (Promega).

PCR was conducted in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Weiterstadt, Germany) as described previously (16).

Transient transfection and dual luciferase assays

Transfections were conducted by electroporation using a Bio-Rad Gene Pulser with capacitance extender (Bio-Rad, Munich, Germany). All plasmids used were purified with Genomed columns (Genomed, Research Triangle Park, NC). As an internal control for transfection efficiency the clone pRL-Tk (Promega), carrying the Renilla reniformis luciferase, was cotransfected in each experiment, and luciferase reporter activity was corrected accordingly. Details of the electroporation procedure and the dual luciferase assays (Dual Luciferase Reporter Assay System; Promega) have been described previously (17).

EMSA, Abs, and nuclear extracts

EMSAs were performed essentially as described previously (25). The nuclear extracts were prepared from untreated cells or cells pretreated with PMA (100 ng/ml) or L-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK; 50 µM) according to a rapid extraction method (26). The NF-{kappa}B p50 Ab (goat polyclonal IgG) used for supershifting was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

In vivo footprint analyses

In vivo footprint analyses were performed as described previously (27), with some modifications. Cells (106/ml) were incubated in medium supplemented with 0.1% dimethylsulfate (Merck, Darmstadt, Germany) for 2–10 min at room temperature. DNA was isolated and treated with 1 M piperidine (Merck) at 90°C for 30 min to cleave at methylated G residues. Three micrograms of the DNA was subjected to a primer extension reaction using 1 µM of a specific primer and the proofreading thermostable DNA polymerase Pfu (Promega) in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems) in a total volume of 50 µl (95°C for 2 min, optimal annealing temperature for 2 min, 72°C for 10 min). The mixture was cleaned using a QIAquick PCR Spin column (Qiagen, Hilden, Germany) and eluted with 30 µl of water. Fifty picomoles of a partially double-stranded TAG-oligonucleotide (made from 5'-GCGGTGACCCGGGAGATCTGAATTC-3' and 5'-GAATTCAGATC-3') was then ligated to the DNA in a volume of 40 µl using T4 DNA ligase (Roche, Mannheim, Germany). Two microliters of the ligation reaction was subjected to PCR using 1 µM of a nested primer of the region of interest and 1 µM of a TAG-specific oligonucleotide (5'-GCGGTGACCCGGGAGATC-3') for 30 cycles in a final volume of 50 µl using AmpliTaq Gold (Applied Biosystems). Finally, 2 µl of this PCR was subjected to a primer extension reaction using a radiolabeled nested primer from the region of interest and AmpliTaq Gold (Applied Biosystems) in a total volume of 20 µl (94°C for 12 min, followed by 15 cycles of 95°C for 20 s, optimal annealing temperature for 20 s, 72°C for 3 min). The resulting products were separated on a 6% sequencing gel and autoradiographed (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The human Ig{lambda} enhancer HSS-3 contains a functional NF-{kappa}B site

To determine which factor(s) is responsible for targeting a chromatin-remodeling machinery to the Ig{lambda} enhancer region it is necessary first to know the factors binding to the DNase I-hypersensitive site in B cells in vivo. We therefore determined the minimal DNA fragment from HSS-3 that still contains the full enhancer activity as judged by transient transfection experiments and found that a 400-bp SfiI-SacI fragment is sufficient for this (17) (see Fig. 1GoA for the map). These experiments were then complemented by in vivo footprint analyses of B cell and non-B cell lines.



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  		FIGURE 1. The human Ig{lambda} enhancer HSS-3 contains a functional NF-{kappa}B site. A, A schematic of the human Ig{lambda} enhancer HSS-3 is shown together with the DNA sequence around the newly identified footprint designated FP. The G residues protected from in vivo methylation by DMS are marked by dots; a G that is preferentially methylated in vivo by DMS is marked by an arrow (cf, B for the in vivo footprint). The sequences of the oligonucleotides used in the EMSA shown in C and D are depicted below the HSS-3 map, nucleotides mutated in FPmut are in bold type, and the NF-{kappa}B binding site is boxed. B, The in vivo footprint of the region of the upper stand depicted in A after DMS treatment of cells from the B cell lines MN-60 and Daudi and the T cell line CCRF-CEM, followed by ligation-mediated PCR. The G-methylation pattern of free DNA from placenta AF is shown for comparison (lane DNA); the location of the binding sites for Mef2, NF{kappa}-B, and {lambda}E1 are shown as boxes; protected G residues are marked by dots; enhanced methylation is marked by an arrow. C and D, Band-shift experiments using nuclear extract from Daudi cells and the radioactively labeled oligo FP are shown. Addition of an anti-p50 Ab orthe unlabeled competitor oligos FPmut and increasing amounts of FP to the binding reaction are indicated. D, The effect of treatment of the B cell line Daudi and the T cell line CCRF-CEM with PMA or TPCK before preparation of the nuclear extract is shown in an EMSA using oligo FP for binding. The positions of the labeled oligos not bound by protein are not shown.

 
Intact cells were treated with dimethyl sulfate (DMS) to methylate accessible G residues; the DNA was then purified, cleaved by piperidine, and subjected to a ligation-mediated PCR (27, 28) with some modifications (see Materials and Methods). We identified several B cell-specific sequence elements containing G residues protected from methylation or G residues that show an enhancement in methylation. Our data are summarized in Fig. 1GoA. The protein binding boxes {lambda}E1, HELP, {lambda}E2, {lambda}D, and {lambda}E3 have previously been described by Glozak and Blomberg (29) and were confirmed in our laboratory by in vivo footprinting (data not shown, but see Fig. 1GoB for an example) and are labeled according to their nomenclature. The box designated NEW by Glozak and Blomberg (29) corresponds to two protein binding sites identified in the mouse system (PU.1 and Pip) (30, 31). The Mef2 binding site is placed according to sequence homology to the binding site identified in the E{lambda}2-4 by Satyaraj and Storb (32).

We identified a new binding site (Fig. 1GoB) between the {lambda}E1 and the Mef2 sequence elements (FP in Fig. 1GoA) that turned out to perfectly match a NF-{kappa}B consensus sequence (Transfac database (33)) and which is identical in sequence to the {kappa}B site found within the human {kappa} intron enhancer element. This was surprising, because it was shown previously that neither the mouse (14) nor the human (15) Ig{lambda} enhancer is regulated by NF-{kappa}B. EMSAs using nuclear extracts from the B cell line Daudi show that a factor present in B cells readily binds the FP oligo (Fig. 1GoC). Addition of an Ab specific for the p50 subunit of NF-{kappa}B to the binding reaction leads to a supershift, and a mutated oligonucleotide (FP mut) cannot compete for binding as the addition of a nonlabeled FP oligo does (Fig. 1GoC). Furthermore, the binding factor can be inhibited by treatment of the B cells before preparation of nuclear extract with TPCK, a known inhibitor of I{kappa}B degradation (25), and can be induced by phorbol ester (PMA) stimulation in the T cell line CCRF-CEM (Fig. 1GoD). Accordingly, the in vivo footprint can be extinguished after treatment of B cells with TPCK before DMS methylation (data not shown), while it cannot be induced after PMA stimulation of the cell line CCRF-CEM. Taken together these data unambiguously show that NF-{kappa}B binds in vivo to a site located within the Ig{lambda} enhancer element HSS-3.

At least three NF-{kappa}B sites located in the Ig{lambda} enhancer modules act synergistically and are functional in transient transfection experiments

In previous experiments we could show that the complete human Ig{lambda} enhancer consists of the enhancer element HSS-3 and two additional modules (HSS-1 and -2), which have no enhancer activity of their own but act synergistically with HSS-3 (17). A search for NF-{kappa}B sites in the genomic region encompassing the three DNase I-hypersensitive sites detected seven canonical NF-{kappa}B binding sites (numbered 1–7 in Fig. 2GoA). Four of these NF-{kappa}B binding sequences reside within the identified DNase I-hypersensitive sites (site 1 in HSS-1, sites 4 and 5 in HSS-2, and the above described site 7 in HSS-3; cf, Fig. 2GoA) and therefore are candidates for an in vivo function. To test for an in vivo function we first prepared a series of plasmid constructs containing the luciferase gene as a reporter and the genomic region containing HSS-1 to HSS-3, where each single NF-{kappa}B site was mutated by a megaprimer technique. These plasmids were then transiently cotransfected with a vector expressing the luciferase from R. reniformis (pRL-Tk), which allows correction for transfection efficiencies. It is important for the experiments described below to note that the Renilla luciferase gene in pRL-Tk is under control of the thymidine kinase promoter and expressed independently of NF-{kappa}B. As shown in Fig. 2GoB a single mutation in sites 1, 2, 3, and 6 does not lead to a decrease in enhancing activity compared with the nonmutated construct. Mutation of site 4, 5, or 7, however, lowers the enhancing activity by one-third (sites 4 and 5) or even one-half (site 7). It should be mentioned in this context that the addition of HSS-1, -2, and -3 leads to a several-hundredfold enhancement of expression over the promoter only construct in B cells (17).



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  		FIGURE 2. Three functional NF-{kappa}B sites within the Ig{lambda} modules HSS-2 and HSS-3 regulate Ig{lambda} expression. The luciferase reporter construct containing a human V{lambda} promoter and the HSS-1 to -3 region is shown schematically in A. {square}, Location of the hypersensitive sites. Arrows mark the positions of the seven putative NF-{kappa}B sites found in this region. B, MN-60 cells were transiently transfected with the firefly luciferase reporter gene constructs and the Renilla luciferase control vector pRL-Tk, either cotransfected with an empty expression vector ({blacksquare}) or the trans-dominant-negative I{kappa}B{alpha} clone pCI-I{kappa}B{alpha}{Delta}(N1–36) (). The normalized activity of the firefly luciferase construct containing the unmutated NF-{kappa}B sites was set at 1.00 relative light units (R.L.U.). The NF-{kappa}B site mutated in each construct is shown underneath the diagram schematically represented by {blacksquare}. C, Effect of cotransfection of the trans-dominant-negative I{kappa}B{alpha} clone together with the luciferase reporter vectors containing two to four mutations in the NF-{kappa}B sites depicted below the diagram, as in B. To identify all functional NF-{kappa}B sites in the region containing the hypersensitive sites the activity of each reporter clone cotransfected with an empty expression vector is set at 1.00 relative light units. B and C, A clone carrying a deletion of the entire genomic region up to NF-{kappa}B site 6 is marked by a {Delta} in the scheme underneath the graphs.

 
In a second set of experiments we additionally cotransfected the clone pCI-I{kappa}B{alpha}({Delta}N1–36), which contains an I{kappa}B{alpha} gene lacking the sequence coding for the first 36 aa that contain the serine residues known to be phosphorylated by I{kappa}B kinase, which leads to degradation of the I{kappa}B{alpha} protein and translocation of NF-{kappa}B to the nucleus. The protein expressed from pCI-I{kappa}B{alpha}({Delta}N1–36) therefore acts as a trans-dominant-negative regulator of NF-{kappa}B that is irreversibly retained in the cytoplasm (13). Fig. 2GoB shows that the activity of the native (unmutated) clone is thereby lowered by a factor of 2, demonstrating that NF-{kappa}B is involved in regulating the human Ig{lambda} enhancer activity.

All clones with single mutated NF-{kappa}B sites ({blacksquare} in Fig. 2GoB), with the exception of the clone containing a mutated site 7, can be further inhibited to the expression level of the native clone plus inhibitor by cotransfection of the trans-dominant-negative I{kappa}B{alpha} ( in Fig. 2GoB). It is interesting to see that the clone containing the mutated site 7 (which is located within HSS-3; see Fig. 2Go) cannot be further inhibited in these experiments, although it still contains the functional NF-{kappa}B sites in HSS-2 (and HSS-1). We then wanted to know how important each of the identified functional NF-{kappa}B sites are for expression and constructed a series of plasmids containing combinations of multiple mutations in the NF-{kappa}B sites of interest (sites 1, 4, 5, and 7). We wanted to see which of the clones can no longer be inhibited by cotransfection of pCI-I{kappa}B{alpha}({Delta}N1–36). It is evident from Fig. 2GoC that each clone that contains a mutation in site 7 in whatever combination with mutations of other sites cannot be further inhibited by the trans-dominant-negative I{kappa}B{alpha}. One has to conclude that site 7 (located in HSS-3) is the most important site for expression and that the other sites are of importance only if the site 7 is intact. This can be interpreted in light of the known synergistic activities of the modules HSS-1 and HSS-2.

Taken together, the transient transfection experiments show that NF-{kappa}B is involved in regulation of the activity of the human Ig{lambda} enhancer, as are the Ig{kappa} genes and IgH genes. The functional sites that we now identified are those located within the DNase I-hypersensitive regions HSS-1, -2, and -3 and not the additional sites found by chance within the more tightly packed chromatin of the region.

The human and chicken, but not the mouse, Ig{lambda} enhancers are regulated by NF-{kappa}B

Our experiments described above show that the human Ig{lambda} enhancer activity is dependent on NF-{kappa}B. It was shown previously that the mouse Ig{lambda} enhancer is independent of NF-{kappa}B, because it does not contain an NF-{kappa}B consensus site, and its activity is not altered in a cell line lacking NF-{kappa}B (14). It has also been demonstrated that the mouse Ig{lambda} enhancer (E{lambda}2-4) is a relatively weak enhancer (14) and does not function in human B cells (15). We now wanted to test these observations and first conducted a sequence comparison of the human and mouse Ig{lambda} enhancers. We also included the CLE sequence (34, 35) in our comparison, which serves (in evolutionary terms) as a more distantly related Ig{lambda} enhancer sequence. The maps in Fig. 3Go show that all three enhancers have a general conserved core structure consisting of {lambda}E1 (14, 15, 36), a putative Mef2 binding site (32), and the PU.1 (31) and Pip (30) binding sites. The NF-{kappa}B site between {lambda}E1 and the Mef2 binding site is present in chicken and human, but not in the mouse enhancer. The human enhancer element seems to be more complex than the chicken and mouse enhancers, containing the additional binding sites HELP, {lambda}D, and {lambda}E3 (Ref. 29 and our own experiments), while mouse and human share the {lambda}E2 site.



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  		FIGURE 3. The human and chicken, but not the mouse, Ig{lambda} enhancer are regulated by NF-{kappa}B. The Ig{lambda} enhancers of human (HSS-3), mouse (E{lambda}2-4), and chicken (CLE) were introduced into luciferase reporter vectors containing a human V{lambda} promoter and were transiently cotransfected into the human MN-60 B cell line with either an empty expression vector ({blacksquare}) or the trans-dominant-negative I{kappa}B{alpha} clone pCI-I{kappa}B{alpha}{Delta}(N1–36) (). The normalized luciferase activity measured from the human HSS-3 construct was set at 1 relative light unit (R.L.U.) as a reference point. The protein binding regions of the enhancers are shown as boxes in the respective maps. The protein binding sites shown in the CLE map are derived from computer-aided comparisons of the chicken sequence with the human and mouse sequences.

 
We cloned all three enhancers in luciferase reporter vectors and transiently transfected the human B cell line MN-60 with these clones together with the R. reniformis luciferase expressing control plasmid pRL-Tk. We either cotransfected the empty eukaryotic expression plasmid pCI or the clone pCI-I{kappa}B{alpha}({Delta}N1–36) expressing the trans-dominant-negative I{kappa}B{alpha} protein. Fig. 3Go shows that all three enhancers are active in the human B cell line, although the mouse enhancer reaches only 28% and the chicken enhancer only 19% of the human enhancer activity. The human and chicken enhancer activities can be inhibited by the trans-dominant-negative I{kappa}B{alpha}, but not the mouse enhancer. This confirms, as already suggested by the sequence comparisons, that the mouse enhancer is a relatively weak enhancer that is independent of NF-{kappa}B (14).

Because the NF-{kappa}B site present in the chicken enhancer (GG PuPyNNPyPyCC) does not match fully the classical NF-{kappa}B consensus binding site (GGPuPuNNPyPyCC), we wanted to know whether it is this sequence deviation that is responsible for the low enhancer activity in human B cells. Therefore, we replaced the NF-{kappa}B site in the human enhancer with the chicken sequence and further introduced mutations into the NF-{kappa}B site in the chicken enhancer, as shown in Fig. 4Go. Replacing the human NF-{kappa}B site with the chicken sequence reduced the human enhancer activity 2-fold, showing a relatively moderate further inhibition by cotransfecting the trans-dominant-negative I{kappa}B{alpha} clone (Fig. 4Go). This can be interpreted by a weak, but still significant, binding of NF-{kappa}B to the chicken sequence. In contrast, the human NF-{kappa}B sequence, when introduced by point mutating the respective chicken sequence in the CLE, leads to a pronounced increase in enhancer activity, now reaching ~50% of the human Ig{lambda} enhancer activity (Fig. 4Go). We conclude from these experiments that the human and CLEs are regulated by NF-{kappa}B and that the chicken enhancer contains an NF-{kappa}B site with weaker binding affinity in human B cells.



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  		FIGURE 4. The CLE contains a functional NF-{kappa}B site. The B cell line MN-60 was transiently cotransfected with luciferase reporter constructs containing the human HSS-3 or the CLE either with an empty expression vector (pCI) or the trans-dominant-negative I{kappa}B{alpha} clone pCI-I{kappa}B{alpha}{Delta}(N1–36). The normalized luciferase activity of the HSS-3 construct was set at 1.00 relative light units (R.L.U.) as a reference point for all the other constructs; the luciferase activity of the constructs containing the human or the chicken enhancer are shown as {blacksquare} and , respectively. The luciferase activity of the constructs cotransfected with the I{kappa}B{alpha} clone are depicted as . In the different constructs only the NF-{kappa}B sites were mutated, as shown above the activity diagram (arrows). The human NF-{kappa}B site is boxed and shaded; the chicken NF-{kappa}B sequence is boxed only. The protein binding sites identified in the human and chicken enhancer elements are shown as {blacksquare} and {square}, respectively (cf, Figs. 1Go and 3Go for details).

 
The mouse Ig{lambda} enhancer can be healed by point mutations in a mutated NF-{kappa}B site

A close inspection of the respective sequence in the mouse enhancer that contains the NF-{kappa}B site in the human (HSS-3) and chicken enhancer shows that the mouse sequence contains a cryptic NF-{kappa}B site (GAGAATCCAC), which differs only in two positions from the NF-{kappa}B consensus site (GGPuPuNNPyPyCC). This suggests that the mouse Ig{lambda} enhancer originally also contained a functional NF-{kappa}B site that was lost due to point mutations during evolution of the species. We therefore asked whether the enhancer activity of the mouse Ig{lambda} enhancer can be increased by correction of these mutations and prepared a number of reporter plasmids containing the mouse Ig{lambda} enhancer in which one, two, or three point mutations were introduced into the cryptic NF-{kappa}B site, leaving the rest of the enhancer intact. Again, each clone was cotransfected either with an empty expression vector or with the same vector containing the trans-dominant-negative I{kappa}B{alpha} construct to monitor directly for NF-{kappa}B dependence of the enhancer activities. Fig. 5Go shows that a mutation of the NF-{kappa}B site in the human enhancer (HSS-3) reduces the activity 2-fold and leads to a independence of the enhancer activity from the trans-dominant-negative I{kappa}B{alpha}.



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  		FIGURE 5. Full enhancer activity of the mouse Ig{lambda} enhancer can be restored by point mutations in a defective NF-{kappa}B site. The B cell line MN-60 was transiently cotransfected with luciferase reporter constructs containing the human HSS-3 or the mouse Ig{lambda} enhancer (E{lambda}2-4), with either an empty expression vector (pCI) or the trans-dominant-negative I{kappa}B{alpha} clone pCI-I{kappa}B{alpha}{Delta}(N1–36). The normalized luciferase activity of the HSS-3 construct was set at 1.00 relative light units (R.L.U.) as a reference point for all the other constructs; the luciferase activities of the constructs containing the human or the mouse enhancer are shown as {blacksquare} and , respectively. The luciferase activity of the constructs cotransfected with the I{kappa}B{alpha} clone are depicted as . In the different constructs only the NF-{kappa}B sites were mutated as shown above the activity diagram (arrows). The human NF-{kappa}B site is boxed and shaded; the defective mouse NF-{kappa}B sequence is boxed only. The protein binding sites identified in the human and mouse enhancer elements are shown as {blacksquare} and , respectively.

 
The experiments shown in Fig. 5Go further demonstrate that a full enhancer activity of the mouse Ig{lambda} enhancer can be restored after only two point mutations in the nonfunctional sequence. The "healed" mouse enhancer reaches only 60–80% activity of the human enhancer HSS-3, which might be attributed to the presence of additional factors binding to the human enhancer element (Fig. 5Go). These data suggest that the mouse Ig{lambda} enhancer lost most of its activity due to point mutations in an NF-{kappa}B site essential for optimal function of the enhancer in B cells.

The human Ig{lambda} enhancers are not critically dependent on NF-{kappa}B, but induction of NF-{kappa}B during B cell development leads to further transcriptional enhancement of the Ig{lambda} enhancer

It is well established that in pre-B cells NF-{kappa}B is sequestered in the cytoplasm by I{kappa}B protein(s) and becomes constitutive after transition from the pre-B to the B cell stage, which is accompanied by rearrangement of an Ig L chain gene. The B cell stage is then characterized by the expression of an assembled L chain Ig molecule.

The establishment of constitutive NF-{kappa}B activity during B cell development can be mimicked by treatment of human pre-B cells with phorbol esters such as PMA (9). The Ig{kappa} intronic enhancer was shown to be totally dependent on the presence of NF-{kappa}B and the integrity of the {kappa}B site within the enhancer (37). We now wanted to test whether the Ig{lambda} enhancer element(s) that contains four NF-{kappa}B sites (see above) is also critically dependent on NF-{kappa}B. We first checked human pre-B cell lines for nuclear NF-{kappa}B activity using EMSAs with the NF-{kappa}B oligonucleotide FP (cf, Fig. 1GoA). We chose the pre-B cell line REH, which does not show nuclear NF-{kappa}B activity unless stimulated with PMA (Fig. 6GoA).



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  		FIGURE 6. Induction of NF-{kappa}B during B cell development leads to further transcriptional enhancement activity of the Ig{lambda} enhancer. A, An EMSA using nuclear extract from the pre-B cell line REH and the NF-{kappa}B oligonucleotide FP (cf, Fig. 1GoA) either without or after treatment of the cells with PMA is shown. The REH cells were transiently transfected with V{lambda} promoter-driven luciferase reporter constructs containing the enhancers as indicated in B. Directly after electroporation the cultures were split in two halves, one-half being treated with 50 ng/ml PMA overnight. After 24 h the cells were harvested and luciferase activity was determined. The graph shows the ratio of luciferase activity with PMA stimulation to the respective unstimulated transfectants (fold induction). The bars represent the mean of at least four independent experiments together with the respective error bars. The sequence of the NF-{kappa}B binding site in the human HSS-3 element and the respective mutated site from the mouse enhancer E{lambda}2-4 (boxed) are shown above the bars. In E{lambda}2-4 mut () the human NF-{kappa}B binding site replaces the mutated mouse sequence in the otherwise unaltered E{lambda}2-4 sequence. In HSS-3 mut () the functional human NF-{kappa}B site was replaced by the respective mouse sequence; the rest of HSS-3 was unaltered.

 
Transfection of the unstimulated pre-B cell line REH with a luciferase reporter construct containing a V{lambda} promoter and either HSS-3 or the entire Ig{lambda} enhancer (HSS-1, -2, and -3) leads to 22- and 34-fold enhancements of luciferase activity compared with the promoter-only construct (data not shown). This shows that the Ig{lambda} enhancer has a basal activity in pre-B cells without NF-{kappa}B being present in the nucleus. We then transiently transfected the cells with luciferase constructs containing the entire Ig{lambda} enhancer (HSS-1, -2, and -3), HSS-3 alone, the native mouse Ig{lambda} enhancer (E{lambda}2-4), and two mutant enhancer constructs where the cryptic NF-{kappa}B site of the mouse enhancer was replaced by the respective human sequence (E{lambda}2-4 mut) or the cryptic site from the mouse enhancer replaces the human NF-{kappa}B site (HSS-3 mut). Immediately after electroporation the cultures were split in half; one-half was stimulated by 100 ng/ml PMA overnight. Fig. 6GoB shows that the enhancer activity of HSS-1, -2, and -3 or HSS-3 alone can be further induced 4- to 7-fold by NF-{kappa}B, while the mouse enhancer E{lambda}2-4 as well as the human HSS-3 mut (containing the mutated mouse NF-{kappa}B site) cannot be further induced by NF-{kappa}B activation. Replacing the mutated site in the mouse enhancer by the functional human NF-{kappa}B site, however, leads to a 10-fold induction of the mouse enhancer activity. These experiments show that although the human Ig{lambda} enhancer is not critically dependent on NF-{kappa}B like the {kappa} intron enhancer the induction of NF-{kappa}B activity during B cell development leads to a further rise in enhancer activity. This is not the case with the mouse enhancer, which contains a mutated NF-{kappa}B site and therefore cannot respond to NF-{kappa}B when it becomes constitutive in B cell development. Furthermore, the experiments indicate that the induction of enhancer activity is not a secondary effect through the induction of NF-{kappa}B-dependent transcription factors, because the mouse enhancer and the human HSS-3 mut cannot be stimulated by NF-{kappa}B.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The transcription factor NF-{kappa}B was initially identified in mature B cells by its ability to bind the {kappa}B motif located in the Ig{kappa} intronic enhancer and was thought to be B cell specific (8). It soon turned out that in a variety of cell types NF-{kappa}B is sequestered in an inactive cytoplasmatic complex with inhibitor proteins of the I{kappa}B family (38). With few exceptions the members of the NF-{kappa}B family of transcription factors (p50/p105, p52/p100, RelA, RelB, and c-Rel) can form heterodimers with other family members and regulate a variety of genes, mostly concerned with cellular defense mechanisms, among which the Ig genes of the H chain and {kappa} L chain are the most prominent. In most cells, including pre-B cells, active DNA binding NF-{kappa}B can be released from the inhibitor protein by stimulation of the cells with various agents such as LPS, phorbol esters, and cytokines or cross-linking of surface molecules. The signal transducing pathways lead to phosphorylation, subsequent ubiquitination, and degradation of I{kappa}B, thus allowing translocation of NF-{kappa}B into the nucleus (reviewed in Ref. 12).

During B cell differentiation Ig genes appear to rearrange in an ordered fashion starting with the H chain genes and proceeding to the {kappa} L chain genes at the late pre-B cell stage. Only if {kappa} L chain gene rearrangement does not lead to a functional protein do the {lambda} L chain genes start to rearrange. During maturation of the pre-B cells to B cells I{kappa}B becomes unstable, leading to constitutive NF-{kappa}B activity in mature B cells (39, 40). Because L chain gene rearrangement is crucial for B cell development, and because it has been shown that Ig{kappa} gene expression correlates with the presence of NF-{kappa}B in the nucleus of mature B cells, it is thought that NF-{kappa}B is a critical factor for Ig gene expression and B cell development.

Surprisingly, the Ig{lambda} enhancers of mouse (14) and human (15) were reported to be independent of NF-{kappa}B. We here confirm that this idea is correct for the mouse enhancer, but it does not hold for the human Ig{lambda} enhancer. The dependency of the human Ig{lambda} enhancer on NF-{kappa}B proteins might have been missed in the experiments described by Blomberg et al. (15), because they were obviously designed only to test for an absolute requirement of NF-{kappa}B for the function of the human Ig{lambda} enhancer. According to our experiments the human Ig{lambda} enhancer, which consists of an enhancer element (HSS-3) and two additional elements (HSS-1 and HSS-2) that synergize with HSS-3 (17), contains three functional NF-{kappa}B sites (one site in HSS-3 and two sites in HSS-2) as shown by in vivo footprinting, transient transfection assays with reporter plasmids containing mutated sites and cotransfection experiments with a trans-dominant-negative I{kappa}B construct (Figs. 1Go and 2Go). The four sites, however, do not have an identical effect on the enhancer activity.

Mutation of each of the two NF-{kappa}B sites within HSS-2 leads to a reduction of enhancer activity to ~70%, which can still be lowered by cotransfection of a trans-dominant-negative I{kappa}B plasmid, suggesting that additional functional NF-{kappa}B sites are present in the enhancer modules. The NF-{kappa}B site located within HSS-3, however, seems to be the critical site, because a mutation of this site leads to a 2-fold reduction of enhancer activity that cannot be lowered by cotransfection of the trans-dominant-negative I{kappa}B construct (Fig. 2Go). This reflects the synergy between the hypersensitive sites, which is lost once the HSS-3 enhancer activity is critically lowered. The NF-{kappa}B site located within HSS-1 has no direct effect on overall enhancer activity.

It has been reported that the mouse Ig{kappa} intronic enhancer critically depends on the presence of the NF-{kappa}B binding site and active nuclear NF-{kappa}B (37). This is definitely not the case for the human Ig{lambda} enhancer, as we could show by transfecting relevant constructs into the human pre-B cell line REH, which lacks active nuclear NF-{kappa}B. There is basal activity of the Ig{lambda} enhancer despite the lack of NF-{kappa}B in REH cells, and a stimulation of these cells with PMA leads to nuclear translocation of NF-{kappa}B and a further severalfold increase in Ig{lambda} enhancer activity (Fig. 6Go). Our comparison of human, mouse, and CLEs show several protein binding sites that are conserved during evolution, namely the {lambda}E1, Mef2, PU.1, and Pip binding sites (cf, Fig. 3Go). These sites all lie within the PstI-SacI fragment of the human enhancer (Fig. 3Go), which represents the minimal fragment exhibiting enhancer activity (15). The NF-{kappa}B binding site, located between the {lambda}E1 and Mef2 binding sites in the human and chicken enhancer, is mutated in two positions in the mouse enhancer, rendering the mouse enhancer incapable of binding NF-{kappa}B. The mouse enhancer is a relatively weak enhancer compared with its human counterpart, which can be attributed to its inability to bind NF-{kappa}B, because two corrective point mutations in this region are sufficient to make it a potent enhancer with ~70% of the human enhancer activity. This and the fact that the distantly related chicken enhancer also contains a NF-{kappa}B site lead us to speculate that in mice the NF-{kappa}B site was mutated during evolution or inbreeding of laboratory mouse strains. In humans the total number of potentially functional L chain variable gene segments sums up to 82 gene segments (46 V{kappa} genes (41) and 36 V{lambda} genes (42)), which is similar to the 78 potentially functional V gene segments of the mouse repertoire (75 potentially functional V{kappa} genes (43, 44) and three V{lambda} genes (45)). It is striking that these numbers reflect the proportions of {kappa} to {lambda} L chains in the sera of the species (60/40 and 95/5 for human and mouse, respectively). It therefore seems as if the mouse {kappa} locus had been expanded during evolution at the expense of the {lambda} locus, which currently contains only two functional V{lambda} gene segments. It is tempting to speculate that this is linked to the occurrence of the mutation in the mouse {lambda} enhancer. The mouse {lambda} locus consists of a duplicated cluster of two J-C units, each linked to a copy of the {lambda} enhancer (45). Interestingly, both copies of the mouse Ig{lambda} enhancer shows the same point mutations of the NF-{kappa}B site, which makes it likely that the mutation of the NF-{kappa}B site occurred before the duplication event.

Experiments described by Popov et al. (46) show that introduction of a human Ig{lambda} minilocus containing 15 V{lambda} genes, the seven J-C{lambda} units, and the Ig{lambda} enhancer in a transgenic {kappa}+/{kappa}- mouse strain led to a high production of human Ig{lambda} (43%) L chains, while the endogenous mouse Ig{lambda} locus expression was unaltered. This demonstrates that a functional human Ig{lambda} enhancer leads to a competition of {kappa} and {lambda} rearrangements in these transgenic mice and supports a model that {kappa} and {lambda} rearrangements are independent, and the low Ig{lambda} expression in mice may be the result of inefficient Ig{lambda} enhancer activity. Further support for the idea that it is indeed the relative {kappa} and {lambda} enhancer efficiencies that regulate the observed {kappa} to {lambda} ratio comes from inactivation experiments of the {kappa} 3' enhancer (47), which leads to a dramatic decrease in the {kappa}/{lambda} ratio (from 20:1 in normal mice down to 1:1 in the {kappa} 3' enhancerless mice).

During the last decade the function of Rel/NF-{kappa}B/I{kappa}B proteins has been studied in detail by generating knockout and transgenic mice. Although there seems to be a considerable redundancy among the functions of individual members of the NF-{kappa}B family of proteins, the disruption of each gene has its distinct phenotype and shows the general importance of the NF-{kappa}B proteins for proper function of the immune system. These data have been reviewed in detail by Attar et al. (48). With the exception of rela-/- mice (which showed embryonic lethality due to massive apoptosis of liver cells), neither of the gene disruptions leads to a total lack of functional B lymphocytes, but, rather, to an impairment at different stages of an immune response. A more direct indication for the involvement of NF-{kappa}B in the onset and regulation of V-J rearrangement comes from in vitro experiments described by Scherer et al. (13). These authors showed that repression of NF-{kappa}B in mouse pre-B cells stably transfected with a trans-dominant I{kappa}B gene totally abolished germline transcription and V-J rearrangement of the endogenous Ig{kappa} genes. Because our experiments indicate that the mutation in the NF-{kappa}B site is responsible for the weak performance of the mouse Ig{lambda} enhancer, it is likely that the lack of a functional NF-{kappa}B site is also responsible for the inability of the {lambda} genes to compete efficiently with the {kappa} genes during the process of initiation of L chain rearrangement. According to the accessibility model of Ig gene activation, any Ig gene rearrangement takes place only after establishment of an active chromatin structure, presumably as a consequence of interaction of proteins with enhancer elements (2). It has been shown that in the mouse pre-B cell line 70Z/3, which contains a rearranged, but transcriptionally silent, Ig{kappa} gene, the {kappa} intronic enhancer is inaccessible to DNase I. LPS stimulation and nuclear translocation of NF-{kappa}B lead to the establishment of DNase I hypersensitivity at the intronic enhancer and transcription of the rearranged {kappa} gene (49). Recently, it was shown that NF-{kappa}B recruits the transcriptional coactivator CBP/p300 (50, 51), which can provide a bridge to the basal transcription machinery and has histone acetylation properties (52, 53). This suggests that histone acetylation is involved in the action of NF-{kappa}B. It is well known that histone acetylation correlates with an active chromatin structure, which might explain the above-described observations made after stimulation of 70Z/3 with LPS. It is therefore possible that NF-{kappa}B plays a similar role in the initial establishment of an active chromatin structure of the Ig{lambda} locus before the onset of DNA rearrangements.


   Acknowledgments
 
We thank Wolfram Hörz and Peter Becker for invaluable comments on the manuscript.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 190, Mechanismen und Faktoren der Genaktivierung). Back

2 Address correspondence and reprint requests to H.-Gustav Klobeck, Adolf Butenandt Institut Molekularbiologie, Schillerstrasse 44, D-80336 Munich, Germany. E-mail address: gustav.klobeck{at}bio.med.uni-muenchen.de Back

3 Abbreviations used in this paper: Ig{kappa}, Ig{kappa} L chain; Ig{lambda}, Ig{lambda} L chain; C{lambda}, C region of the Ig{lambda} L chain; DMS, dimethyl sulfate; CLE, chicken Ig{lambda} enhancer; HSS, DNase I-hypersensitive site; TPCK, L-p-tosylamino-2-phenylethyl chloromethyl ketone. Back

Received for publication October 2, 2001. Accepted for publication December 5, 2001.


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 Discussion
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