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The Journal of Immunology, 2000, 164: 795-804.
Copyright © 2000 by The American Association of Immunologists

Comparison of Mouse and Rabbit Ei{kappa} Enhancers Indicates That Different Elements Within the Enhancer May Mediate Activation of Transcription and Recombination1

Isabelle Coquilleau, Patricia Cavelier, François Rougeon and Michele Goodhardt2

Unité de Génétique et Biochimie du Développement, Unité de Recherche Associée 1960, Centre National de la Recherche Scientifique, Département d’Immunologie, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The intronic Ig {kappa}-light chain enhancer (Ei{kappa}) has been implicated in regulation of transcription and V{kappa}-J{kappa} recombination at the {kappa} locus. To identify sequences within the Ei{kappa} enhancer which are involved in control of recombination, we have made use of the finding that the Ei{kappa} element from the rabbit b9 {kappa} locus is capable of inducing rearrangement, but not transcription of {kappa} genes in mouse lymphoid cells. We have therefore compared the binding of murine nuclear proteins to the mouse and rabbit Ei{kappa} elements. DNase I footprinting and gel mobility shift assays indicate that only the {kappa}B, {kappa}E1, and {kappa}E2 sites of the rabbit enhancer are able to interact with murine trans-acting factors. Moreover, although the rabbit {kappa}B site binds murine NF-{kappa}B p50/p50 and p50/p65 complexes with high affinity, this site is not capable of mediating transcriptional activation of transient transfection reporter constructs in mouse B lineage cells. These results therefore suggest that, in contrast to the maintenance of {kappa} enhancer transcription which requires all of the Ei{kappa} sites, only the {kappa}B, {kappa}E1, and {kappa}E2 sites may be necessary for the recombinational activity of the enhancer. Furthermore, NF-{kappa}B-mediated effects on transcription and recombination appear to involve separate downstream activation pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Unlike other genes, expression of Ag receptor genes in B and T cells requires the assembly of a functional gene prior to transcription. Assembly occurs via a series of site-specific recombination events, which juxtaposes component V, D, and J segments to form a complete Ig or TCR variable region gene. Rearrangement of Ig heavy and light ({kappa} and {lambda}) chain genes occurs in B cell precursors and is regulated during lymphocyte development (reviewed in Ref. 1). Transgenic and gene-targeting studies have shown that control of transcription and rearrangement may be mediated by common cis-acting elements, notably transcriptional enhancers (reviewed in Refs. 2, 3).

The Ig {kappa} locus contains two known enhancer elements: one intronic (Ei{kappa}) and the other located at the 3' end of the locus (4, 5). The Ei{kappa} is B cell specific and becomes active in late B cell precursors at the time of {kappa} gene rearrangement. Although originally described as a transcriptional activator, Ei{kappa} has more recently been implicated as a critical element in promoting rearrangement of {kappa} variable region genes (6, 7, 8). Thus, transgenic studies have shown that deletion of the intronic enhancer region results in a 100-fold reduction in the level of {kappa} gene rearrangement (7). The {kappa} enhancers are also required for somatic hypermutation (9) and B lineage-specific demethylation of {kappa} genes (10), suggesting that the pleiotrophic effects of Ei{kappa} may be mediated by local changes in chromatin structure provoked by binding of trans-acting factors to the enhancer element.

The Ei{kappa} element contains multiple binding sites for nuclear proteins, some of which, such as {kappa}EO, {kappa}A, and {kappa}B, interact with B cell-specific factors, whereas others bind ubiquitous factors such as the E boxes ({kappa}E1, {kappa}E2, and {kappa}E3) and the {kappa}E* site (reviewed in Ref. 11). These sites all contribute to Ei{kappa} transcriptional activity, although the {kappa}B site appears to be a particularly crucial element for the activation of {kappa} gene transcription in B lineage cells (12, 13). Much less is known concerning the elements within Ei{kappa} that are involved in control of {kappa} gene rearrangement. However, mutation of the {kappa}B site was found to abolish the rearrangement of a transgenic recombination substrate (14), while depletion of nuclear {kappa}B-binding factors prevents the rearrangement of {kappa} genes in pre-B cells lines (15), suggesting that the {kappa}B site may also play a critical role in regulating rearrangement.

The {kappa}B motif interacts with the NF-{kappa}B/Rel family of transcription factors, including the p50, p52, p65, c-Rel, and RelB proteins (16). These proteins can form homo- and heterodimers through the shared amino-terminal Rel homology domain, which is also responsible for DNA binding as well as interaction with inhibitor {kappa}B proteins. In most cells, NF-{kappa}B is inactive due to its sequestration in the cytoplasm by the I{kappa}B inhibitor proteins; however, in B cells, NF-{kappa}B is constitutively nuclear and transcriptionally active. The most abundant NF-{kappa}B complex is the p50/p65 heterodimer. The C-terminal region of p65 contains a transcriptional activation domain, which is lacking in the p50 subunit. Individual NF-{kappa}B/rel proteins display selective target site specificities; therefore, the transactivating potential of the different NF-{kappa}B complexes may differ depending on the specific {kappa}B motif (17, 18, 19).

The intronic enhancer is located in a region of striking homology among mouse, human, and rabbit genes (20). Nevertheless, transgenic and transfection studies have shown that the rabbit b9 intronic enhancer is capable of activating rearrangement (7, 21), but not transcription (22), of {kappa} genes in mouse lymphoid cells. We have exploited this finding to identify sequences within the Ei{kappa} element involved in control of rearrangement. Although the mouse and rabbit sequences are highly homologous over the Ei{kappa} region, sequence identity within each trans-acting factor binding site differs (15–100%). We have therefore analyzed the binding of murine nuclear proteins to the different sites of the rabbit enhancer. Our results indicate that only the {kappa}B, {kappa}E1, and {kappa}E2 sites are able to interact with mouse trans-acting factors. Furthermore, we show that although the rabbit {kappa}B site is able to bind murine NF-{kappa}B p50/p50 and p50/p65 complexes with high affinity, it cannot mediate transcriptional activation in mouse lymphoid cells. These results explain why the rabbit enhancer is not capable of activating transcription in mouse B lineage cells and suggests that a different set of elements within the Ei{kappa} enhancer may mediate transcription and recombination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell extracts

The following cell lines were used: BASP-1 (23), PD31 (24), 70Z/3 (ATCC TIB-158) mouse pre-B cells, S194 (ATCC TIB-19), X63Ag8 (ATCC TIB-9), HDR37 (6) mouse B cells, and COS (ATCC CRL-1650) monkey kidney cells. COS cells and S194 cells were grown in DMEM (Life Technologies, Rockville, MD) supplemented with 10% FCS and 1 mM pyruvate. All other cell lines were grown in RPMI 1640 (Life Technologies) with 10% FCS containing 50 µM 2-ME, except for X63Ag8, where the 2-ME was omitted.

COS cells (5 x 106) were transfected by electroporation (260 V, 960 µF, Gene Pulser; Bio-Rad, Richmond, CA) with either 10 ng of p50 expression vector alone or a combination of 5 ng of p50 and 15 ng of p65 expression vectors in 400 µl of medium containing 10% FCS, 10 mM HEPES (pH 7.2), and 30 mM NaCl. After 36 h, the cells were collected and whole-cell extracts were prepared by lysing cells in 3 vol of extraction buffer [350 mM NaCl, 10 mM HEPES (pH 8), 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.1 mM DTT, 1.25% Triton X-100, and the following protease inhibitors: 50 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml PMSF]. Extracts were centrifuged (10 min at 14,000 rpm, Sigma microcentrifuge; Sigma, St. Louis, MO), and the supernatants were stored at -80°C in aliquots.

Nuclear extracts of lymphoid cell lines were prepared essentially as described (25). Briefly, cell pellets (107 cells) were resuspended in 200 µl of lysis buffer [50 mM NaCl, 10 mM HEPES (pH 8), 500 mM sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, and 0.2% Triton X-100] and kept on ice for 1 min for lysis to occur. Nuclei were recovered by centrifugation (3 min at 6, 500 rpm, Sigma microcentrifuge), the nuclear pellets were washed once in 200 µl of buffer B [50 mM NaCl, 10 mM HEPES (pH 8), 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, and the above protease inhibitors], then resuspended in 15 µl of buffer B containing either 350 mM NaCl (for gel-shift assays) or 650 mM NaCl (for DNase I footprinting assays), and incubated for 20 min on ice before centrifugation (10 min at 14,000 rpm, Sigma microcentrifuge). Extracts for DNase I footprint analysis were subjected to a further centrifugation step (30 min at 30,000 x g) to remove residual DNA, and the supernatants were stored at -80°C in aliquots.

Plasmid constructs

A 514-bp HinfI fragment comprising the mouse Ei{kappa} enhancer (12) and a 697-bp Sau3AI fragment containing the homologous region from the b9 rabbit J{kappa}-C{kappa} intron (26) were cloned into the BglII site of pGL2-promoter vector (Promega, Madison, WI). Luciferase vectors containing trimers of the mouse or rabbit {kappa}B sites were constructed by cloning into the NheI and BglII sites of pGL2-promoter the following double-stranded oligonucleotides ({kappa}B motif is underlined): {kappa}B mouse, 5'-CTAGCACAGAGGGGACTTTCCGAGAGACAGAGGGGACTTTCCGAGAGACAGAGGGGACTTTCCGAGAGA-3'; {kappa}B rabbit, 5'-CTAGCACAGAGCGGGGTTTCCCAGGGACAGAGCGGGGTTTCCCAGGGACAGAGCGGGGTTTCCCAGGGA-3'.

The sequences of {kappa}B mutations are indicated in Table IGo and Fig. 6Go. The same phasing was observed for all constructs.


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  		Table I. Mutations in mouse and rabbit {kappa}B motifs: summary of binding capacity and transcriptional activation1

 


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  		FIGURE 6. Analysis of murine nuclear proteins binding to the rabbit {kappa}B site. A, The same NF-{kappa}B complexes interact with the mouse and rabbit {kappa}B sites. End-labeled mouse (lanes 1–7) and rabbit (lanes 8–14) {kappa}B oligonucleotides were incubated with nuclear extracts from X63Ag8 B cells, and binding was measured by gel retardation analysis. Ab-supershift assays were performed in the presence of control rabbit antiserum (lanes 2 and 9) or polyclonal Abs directed against the indicated NF-{kappa}B subunits (lanes 3–7 and 10–14). Arrows indicate retarded complexes specific for the mouse and rabbit {kappa}B sites. In some samples, a slower migrating band, which does not contain NF-{kappa}B proteins, was observed with the mouse probe. B, Murine KBF2 interacts with the rabbit {kappa}B site. Gel retardation analysis was performed as in A with the rabbit {kappa}B oligonucleotide in the absence (lane 1) or presence (lanes 2–4) of the indicated molar excess of KBF2 oligonucleotide containing the A and B sites from the HES-1 promoter (33 ). C, Mutations in the KBF2 motif does not inhibit binding of NF-{kappa}B to the rabbit {kappa}B site. Gel retardation analysis was performed as in A with the unmutated rabbit {kappa}B oligonucleotide (lane 1), or the rabbit oligonucleotide containing mutations in the KBF2 (lane 2) or {kappa}B (lane 3) site. Sequences of the oligonucleotides used are shown below the gel. Mutated nucleotides are indicated by an asterisk. D, Abolition of KBF2 binding to the rabbit {kappa}B site does not restore transcriptional activation. Luciferase vectors containing trimers of the mouse {kappa}B oligonucleotide ({kappa}B mouse), the wild-type rabbit {kappa}B oligonucleotide ({kappa}B rabbit), or the rabbit oligonucleotide containing the KBF2 mutation described above (KBF2-) were transfected into X63Ag8 cells and luciferase activity was assayed as described in Fig. 5Go.

 
The NF-{kappa}B p50 expression vector was constructed by cloning a 1.7-kb ApaI cDNA fragment containing the murine p50 coding sequence (27) into the ApaI site of the pRc-CMV expression vector (Invitrogen, San Diego, CA). The murine p65 expression vector was a gift from Dr. A. Israël (Institut Pasteur, Paris, France).

Gel retardation assays

Gel retardation experiments were performed as described (28), except that binding of nuclear extracts (7–10 µg) or COS cell extracts (1–5 µg) was conducted in 20 µl of 20 mM HEPES buffer (pH 7.5) containing 4% Ficoll, 70 mM NaCl, 2 mM DTT, 100 µg/ml BSA, 1 µg of poly(dI-dC), and 1 ng of radiolabeled probe for 20 min at room temperature. For supershift assays, 2–3 µl of polyclonal anti-p50, anti-p52, anti-p65, anti-cRel Abs (Santa Cruz Biotechnology, Santa Cruz, CA), or KBF2 (a gift from Dr. A. Israël) was added following the binding reaction, and samples were incubated for another 35 min at room temperature. For competition experiments between mouse and rabbit {kappa}B sites, 1 µg of COS extracts was incubated for 30 min at room temperature with 1 ng of radiolabeled mouse {kappa}B probe and 0.1- to 50-fold molar excess of competing oligonucleotide. Preliminary experiments were performed to ensure that optimal binding of NF-{kappa}B factors to the {kappa}B probe had occurred under these conditions. The following double-stranded oligonucleotides were used as competitors or probes (top strand sequences only are given, and mutations introduced in the binding sites are underlined): {kappa}B mouse, 5'-GATCCAGAGGGGACTTTCCGAGAGGTTAT-3'; {kappa}B rabbit, 5'-GATCCAGAGCGGGGTTTCCCAGGGCTTAT-3'; for sequences of {kappa}B mutants see Table IGo and Fig. 6GoC; {kappa}A mouse, 5'-AAGAACTCTCAGTTTTCGTTTTTACTACCTCTG-3' (29); {kappa}A- mouse, 5'-AAGAACTCTCAGTTTTAACTTTTACTACCTCTG-3'; {kappa}A rabbit, 5'-GGATTCTTGATTTTTTCATTTTAACGATGCTCT-3'; Oct T1, 5'-TTCCCAATGATTTGCATGCTCTCA-3' (30); Oct- T1, 5'-TTCCCAATGATTTGTGGGCTCTCA-3'; and KBF2, 5'-GATCGTTACTGTGGGAAAGAAAGTTTGGGAAGTTTCACAC-3' (a gift from Dr. A. Israël).

The mouse Oct probe (corresponding to probe C in Ref. 30) was prepared by PCR amplification using the forward primer 5'-CCGGAATTCGAGTCATTAAGTTATTTAAC-3' and reverse primer 5'-GGCGAATTCAATTATGAGCAGCCTTTC-3'. The rabbit Oct probe spanning nt 813–942 of the rabbit b9 {kappa} intron was amplified using the forward PCR primer 5'-CCGGAATTCGTGCTGCCAAGTCCACTG-3' and the reverse primer 5'-GGCGAATTCGTCAGCAGACGCTCGGAC-3'.

DNase I footprinting assays

DNase I footprint reactions were conducted with 1–2 ng of radiolabeled probes in the presence or absence of 50–80 µg of S194 nuclear extracts essentially as described (31), except that digests were performed with 4–20 µg/ml DNase I (Worthington Biochemical, Lakewood, NJ ) for 1 min at 25°C in a final volume of 20 µl. DNA probes were labeled at a 3' EcoRI site (Klenow fragment labeling grade; Boehringer Mannheim, Mannheim, Germany) following subcloning of a 697-bp Sau3AI fragment containing the rabbit enhancer, or a 514-bp HinfI fragment containing the mouse enhancer region (see Plasmid contructs), into the BamHI site of pUC19 (Biolabs, Northbrook, IL). The appropriate end-labeled fragments were released by restriction and purified by acrylamide gel electrophoresis to give a 470-bp EcoRI-PstI rabbit probe (nt 776-1247 of the rabbit b9 {kappa} intron, accession number M14068) and a 345-bp EcoRI-SspI mouse enhancer probe (nt 3744–4087 of the mouse {kappa} intron, accession number V00777).

Transient transfection and luciferase assays

Cells (2 x 106) were transfected with 2 µg of luciferase vectors by incubating in 200 µl of 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, and 0.5 mM MgCl2 containing 500 µg/ml DEAE-dextran (Pharmacia, Piscataway, NJ) for 30 min to 2 h, depending on the cell line. Luciferase activity was assayed 24 h after transfection in 500 µl of lysis buffer [25 mM Tris phosphate (pH 7.8), 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 15% glycerol] containing 0.06 mM luciferin (Boehringer Mannheim) and 0.2 mM ATP using a Berthold luminometer (Berthold, Nashua, NH).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The rabbit {kappa} enhancer does not mediate transcriptional activation in mouse B or pre-B cells

Numerous trans-acting factor binding sites have been identified within the Ei{kappa} element, some of which are required for activation of transcription in B lineage cells such as {kappa}EO, {kappa}A, {kappa}B, and the E boxes, which bind the octamer proteins, kBF-A, NF-{kappa}B, and the E motif-binding proteins, respectively. Others, such as {kappa}E*, act as negative regulatory elements. Most of these sites have been conserved between the mouse and rabbit intronic enhancers (Fig. 1GoA). To determine whether the rabbit b9 intronic enhancer is capable of activating transcription in mouse B lineage cells, luciferase reporter constructs containing either a 514-bp HinfI fragment comprising the murine Ei{kappa} element (12) or a 697-bp Sau3AI fragment containing the homologous region from the rabbit {kappa} locus (20, 22) were transfected into various mouse B and pre-B cell lines. As expected, the mouse enhancer stimulated luciferase activity both in mature B cells and in pre-B cells (Fig. 1GoB), whereas no enhancer-stimulated activation was observed in any of the murine B lineage cells tested for constructs containing the rabbit intronic sequences. This therefore confirms our previous results (22) and shows that the rabbit Ei{kappa} is inactive as a transcriptional enhancer both in B and pre-B cells.



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  		FIGURE 1. The rabbit {kappa} enhancer is a poor transcriptional activator in murine B and pre-B cell lines. A, Schematic representation of the J{kappa}-C{kappa} intronic region. Position of the intronic enhancer (Ei{kappa}) and binding sites for transactivating proteins are shown. Percent homology between b9 rabbit and murine sequences are indicated below each site. -, <45% homology. Restriction sites utilized for the mouse Ei{kappa} are shown above and for the rabbit Ei{kappa} below the diagram (not drawn to scale). B, Comparison of transcriptional activation by mouse and rabbit intronic enhancers in B (S194 and X63Ag8) and pre-B (PD31 and BASP-1) cell lines. Activity of luciferase vectors containing the mouse or rabbit Ei{kappa} are expressed as fold increase in luciferase activity relative to the enhancerless pGL-2 promoter construct. Results represent averages of two or more experiments, each performed in triplicate.

 
The mouse and rabbit enhancers do not interact with the same murine nuclear proteins

To determine which of the sites in the rabbit Ei{kappa} are capable of interacting with murine trans-acting factors, we performed in vitro DNase I footprint analysis of 345 bp of murine {kappa} intronic sequences encompassing the known Ei{kappa} binding sites and a homologous 470-bp fragment from the rabbit {kappa} intron. Nuclear extracts from S194 B cells, which gave the highest level of transcriptional activation in luciferase assays, were used. Incubation of the mouse probe with S194 nuclear extracts before DNase I digestion resulted in protection over the {kappa}E3, {kappa}B, and {kappa}EO sites, as well as partial protection over the {kappa}E* site (Fig. 2GoA). A footprint was also observed over an A/T-rich sequence situated between the {kappa}E* and {kappa}EO sites which does not contain defined factor binding sites. As previously reported, we could not detect protein interaction with the E1 and E2 boxes using this technique (32).



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  		FIGURE 2. Comparison of binding of murine nuclear proteins to mouse and rabbit enhancer sequences. A, DNase I footprint analysis. 3' end-labeled DNA fragments extending from nt 3744–4087 of the mouse {kappa} intron (55 ) and nt 776-1247 of the rabbit b9 {kappa} intron (26 ) were subjected to partial digestion by DNase I in the absence (free) and presence of 60 µg of S194 nuclear extract (+). A sequencing reaction performed on each fragment (57 ) was run in parallel (G + A). Protected areas in the presence of nuclear extract are indicated by a solid line and sites of DNase I hypersensitivity by an arrow. The position of transcription factor binding sites for the mouse enhancer are indicated along the side. Longer electrophoretic runs were conducted to analyze the 3' regions in greater detail. B, Nucleotide sequence of mouse and b9 rabbit enhancer regions showing the location of DNase I-protected areas (solid line) and hypersensitive sites (asterisk). The E boxes, {kappa}EO, {kappa}E*, {kappa}A, and {kappa}B sites in the mouse sequence are bracketed. Nucleotides are numbered as in accession number V00777 for the mouse {kappa} intron and accession number M14068 for the rabbit sequence. Gaps have been introduced to maximize the alignment between the two sequences.

 
A different pattern of protection was obtained for the rabbit probe (Fig. 2GoA). There was no evidence of binding to the putative {kappa}E3 motif present at the 3' end of the rabbit probe. However, we observed a strong footprint over the region homologous to the murine {kappa}B site (I, Fig. 2Go), suggesting that the rabbit site can interact with murine NF-{kappa}B factors. Two regions located at the 5' end of the rabbit probe were also protected following incubation with mouse B cell nuclear extracts (II and III, Fig. 2Go). Footprints II and III do not correspond to consensus sites found in the mouse Ei{kappa}, nevertheless they cover two potential octamer binding sites in the cd and dc orientation: ATGCAAAG and GTTACCAT (IIIc and II, Fig. 2GoB). To determine whether the DNase I protection in this region is due to binding of octamer proteins, gel mobility shift assays were performed with DNA probes containing the 5' protected regions from the rabbit enhancer and a corresponding fragment from the mouse {kappa} intron containing the octamer binding {kappa}E0 site (30) (see Fig. 2GoB). Unlike the mouse {kappa}EO probe, no specific binding of octamer proteins to the rabbit probe was observed with nuclear extracts from either B lineage cells (data not shown) or C33 cells overexpressing the B cell-specific octamer factor Oct 2 (Fig. 3Go). Furthermore, a 50-fold excess of the rabbit probe did not inhibit binding of Oct 2 to the mouse {kappa}EO probe (Fig. 3Go), whereas, as previously reported (30), binding to the mouse {kappa}EO motif was competed by a consensus octamer site from the T1 V{kappa} promoter (Fig. 3Go). These results indicate that the rabbit enhancer does not bind murine octamer proteins.



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  		FIGURE 3. Octamer proteins do not bind the 5' region of the rabbit enhancer. Gel retardation assays were performed with end-labeled mouse (lanes 1 and 4–8) or rabbit (lanes 2 and 3) octamer probes incubated with nuclear extracts from C33 cells transfected with an Oct 2 expression vector (56 ). For competition experiments, the binding reaction was conducted in the presence of a 50-fold molar excess of unlabeled rabbit octamer probe (lane 5), mouse octamer probe (lane 7), a 24-bp double-stranded oligonucleotide encompassing the octamer site of the T1 V{kappa} promoter (lanes 3 and 6) or the T1 oligonucleotide containing mutations in the octamer site (lane 8).

 
The 5' region of the Ei{kappa} element also contains a binding site for the B cell-specific factor kBF-A, which is found in activated pre-B cells (29). The {kappa}A site corresponds to a well-conserved sequence in the mouse and rabbit enhancers (Fig. 2GoB); however, it was not possible to identify protein interaction with this site by DNase I footprint experiments, since it is situated in a highly DNase I-resistant region. We therefore conducted gel mobility shift assays with oligonucleotide probes corresponding to the mouse and rabbit {kappa}A sites to determine whether the rabbit site is capable of binding murine kBF-A. As previously described (29), the murine probe interacts with an LPS-inducible protein in the 70Z/3 pre-B cell line (Fig. 4Go). This binding activity first appears ~8 h after LPS stimulation (data not shown) and is inhibited by mutations in the {kappa}A site (Fig. 4Go), indicating that it is indeed kBF-A. Using the same extracts, we could not detect binding of an LPS-inducible protein to the rabbit probe, suggesting that it cannot interact with murine kBF-A (Fig. 4Go). This was confirmed by competition experiments which showed that a 50-fold excess of the rabbit probe does not inhibit binding of kBF-A to the mouse {kappa}A site.



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  		FIGURE 4. Gel retardation analysis of binding to the rabbit {kappa}A site. End-labeled oligonucleotides containing the mouse {kappa}A site (lanes 1, 2, and 7–10), the homologous sequence in the rabbit {kappa} intron (lanes 5 and 6), or the mouse {kappa}A oligonucleotide containing mutations in the {kappa}A site (lanes 3 and 4) were incubated with nuclear extracts from uninduced 70Z/3 cells (lanes 1, 3, 5, and 7) or 70Z/3 cells treated with 10 µg/ml LPS for 24 h (lanes 2, 4, 6, and 8–10). For competition experiments, a 50-fold molar excess of the indicated competitor was added to the binding reaction.

 
Taken together, these results indicate that relatively few sites in the rabbit enhancer can interact with murine trans-acting factors. No interaction could be detected between murine nuclear proteins and the rabbit {kappa}E3, {kappa}A, {kappa}E*, and {kappa}EO sites. Binding was only observed for the {kappa}B site and although no formal binding studies were conducted for the {kappa}E1 and {kappa}E2 motifs, there is 100% sequence identity between the rabbit and mouse sites, suggesting that they too can interact with the appropriate murine factors.

The rabbit {kappa}B site is a poor transcriptional activator in mouse B or pre-B cells

The DNase I footprint experiments showed that the rabbit {kappa}B site can interact with murine B cell nuclear proteins. To test whether this site is able to stimulate transcription in mouse cells, transient transfection experiments were conducted with luciferase reporter constructs containing three tandem repeats of the mouse or rabbit {kappa}B sites. Surprisingly, transfection of the rabbit construct in X63Ag8 B cells gave only a 4-fold activation of luciferase activity, whereas the corresponding murine NF-{kappa}B-driven construct gave a 100-fold stimulation (Fig. 5Go). A 60-fold difference in transcriptional activation between mouse and rabbit NF-{kappa}B reporter constructs was also observed in S194 cells, and similar results were obtained with other murine B and pre-B cell lines (Fig. 5Go), indicating that the rabbit {kappa}B site is a very weak transcriptional activator in murine B lymphoid cells.



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  		FIGURE 5. Comparison of transcriptional activation by mouse and rabbit {kappa}B sites in murine B and pre-B cell lines. Luciferase vectors containing trimers of the mouse ({kappa}B mouse) or rabbit ({kappa}B rabbit) {kappa}B site were transfected into S194, X63Ag8, and HDR37 B cells or 70Z/3 and PD31 pre-B cells. Luciferase activity was measured 24 h after transfection in either untreated cells or cells treated with 50 ng/ml PMA (70Z/3 + PMA) to induce nuclear NF-{kappa}B complexes. Activity is expressed as fold increase in luciferase activity relative to the enhancerless pGL2-promoter construct. Data shown are averages of two or more experiments, each performed in triplicate.

 
Mouse and rabbit {kappa}B sites interact with the same murine NF-{kappa}B complexes

The {kappa}B motif interacts with the NF-{kappa}B/Rel family of transcription factors comprised of homo- and heterodimers of the p50, p52, p65 (RelA), c-Rel, and RelB polypeptides; these complexes have selective target site specificities and may have distinct biological activities (16). To determine the nature of the factors binding to the rabbit {kappa}B site, which differs from the murine site by a deletion in the central AC dinucleotide (Fig. 2GoB), we conducted gel mobility shift assays with oligonucleotide probes corresponding to the rabbit and mouse {kappa}B sites. Three retarded bands were observed when the rabbit {kappa}B probe was incubated with murine B cell nuclear extracts; complexes I and II have similar electrophoretic mobility to the two complexes generated with the mouse probe, whereas complex III, the fastest migrating band, was only observed with the rabbit {kappa}B probe (Fig. 6GoA). Supershift assays using Abs specific for individual NF{kappa}B/Rel proteins showed that the two slower migrating bands contain NF-{kappa}B complexes (Fig. 6GoA). Pretreatment with anti-p65, anti-p50, and, to a lesser degree, anti-cRel Abs resulted in displacement of complex I for both mouse and rabbit probes. Complex II, generated with both the mouse and rabbit {kappa}B probes, was inhibited by anti-p50 Abs only. These results indicate that, like the mouse {kappa}B site, the rabbit site interacts with murine p50 homodimers as well as p50/p65 and p50/c-Rel heterodimers. Furthermore, we found that the mouse and rabbit sites interact with the same NF-{kappa}B/Rel complexes in all B lineage cells tested (data not shown).

The only difference observed between the mouse and rabbit {kappa}B probes was the formation of an additional complex with the rabbit site. Complex III is not recognized by NF-{kappa}B/Rel Abs (Fig. 6GoA) and is constitutively present in non-B cells (data not shown), so it is unlikely to contain a NF-{kappa}B complex. Inspection of the rabbit {kappa}B site revealed the presence of an overlapping KBF2 site. We therefore conducted competition experiments with an oligonucleotide containing the KBF2 site from the HES-1 promoter (33). As shown in Fig. 6GoB, complex III was specifically inhibited by a 5-fold molar excess of unlabeled KBF2 probe, indicating that in addition to NF-{kappa}B/Rel factors, the rabbit {kappa}B probe also interacts with KBF2 in murine B cells. This was confirmed by supershift assays using anti-KBF2 Abs (data not shown). Because the rabbit {kappa}B and KBF2 sites overlap, it was possible that the lack of transcriptional activation observed with the rabbit {kappa}B site in transfection experiments could be due to binding of KBF2. However, this was not found to be the case, since mutations in the KBF2 site, which abolish binding of KBF2 without affecting NF-{kappa}B binding (Fig. 6GoC), do not restore transcriptional activation when used to drive expression of luciferase reporter plasmids in transfection experiments (Fig. 6GoD).

The possibility remained that the difference in transcriptional activity observed between the mouse and rabbit {kappa}B sites was due to selective binding affinities of the sites for different NF-{kappa}B factors. We therefore performed competition experiments to determine the relative affinity of the mouse and rabbit sites for homodimeric p50 and heterodimeric p50/p65, the major NF-{kappa}B complexes expressed in B lineage cells. Extracts from COS cells transfected with either p50 or p50 and p65 expression vectors were incubated with radiolabeled mouse {kappa}B probe in the presence of increasing concentrations of competing mouse or rabbit {kappa}B oligonucleotides and binding was analyzed by gel mobility shift assays. As shown in Fig. 7Go, the rabbit {kappa}B site was as efficient as the murine site in competing for binding of p50 homodimers and showed only a 3-fold decrease in affinity (EC50, 4.0 + 1.0, n = 3) for binding of the p50/p65 heterodimer relative to the mouse {kappa}B site (EC50, 1.3 + 0.6, n = 3).



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  		FIGURE 7. Mouse and rabbit {kappa}B motifs have similar affinities for p50/p50 and p50/p65 complexes. Gel retardation experiments were performed using 1 µg of whole-cell extract from COS cells transfected with p50 (A) or p50 and p65 (B) expression vectors and end-labeled mouse {kappa}B oligonucleotide in the presence of the indicated molar excess of competitor oligonucleotide. The binding reactions were analyzed by electrophoresis on a nondenaturing acrylamide gel that was dried and exposed to an x-ray film as well as to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) to quantitate band intensities. Top panel, Autoradiogram of a gel. Bottom panel, Graphic representation of PhosphorImager quantifications (Molecular Dynamics). Data represent averages of three independent experiments.

 
Nature of the {kappa}B site

The above results show that the rabbit {kappa}B site is able to bind the same {kappa}B factors with virtually the same affinities as the murine site and yet cannot activate transcription. To determine whether this is due to the {kappa}B motif itself or the surrounding sequences, we introduced mutations in the murine and rabbit site and determined their effect on in vitro binding and transcriptional activation. (Table IGo). Addition of an AC dinucleotide in the rabbit site creates a {kappa}B motif identical to the mouse site and restores the capacity of the rabbit oligonucleotide to activate transcription ({kappa}B Rabbit.1, Fig. 8GoB), indicating that the lack of transcriptional activation of the rabbit {kappa}B site cannot be attributed to the sequence context of the site. Deletion of the central AC pair in the mouse {kappa}B motif, however, abolishes both binding and transcriptional activation ({kappa}B Mouse.1, Fig. 8Go). We therefore analyzed further mutations in the mouse {kappa}B motif (Table IGo). The "rabbit phenotype," i.e., binding but not activation of transcription, is restored by additional mutation of the 3'G to a C ({kappa}B Mouse.3, Fig. 8Go). This shows that the sequence GGGGTTTCCC, present in the rabbit enhancer, constitutes an unusual {kappa}B site, which is capable of binding but not transactivation in murine lymphoid cells.



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  		FIGURE 8. Mutational analysis of mouse and rabbit {kappa}B motifs. A, Binding of the mutated {kappa}B sites shown in Table IGo was analyzed by gel retardation experiments using either whole-cell extracts from COS cells transfected with p50 and p65 expression vectors (lanes 1–5) or nuclear extracts from X63Ag8 B cells (lanes 6–9) as in Fig. 6Go. B, Relative transcriptional activation by {kappa}B motifs. Luciferase vectors containing trimers of {kappa}B motifs shown in Table IGo were transfected into S194 or X63Ag8 B cells, and luciferase activity was measured as in Fig. 5Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcriptional enhancers have been shown to play a critical role in regulating V(D)J recombination (2, 3). However, it is not clear whether the same cis-acting elements within the enhancer region are involved in the regulation of transcription and recombination. We have addressed this question by the use of the rabbit Ei{kappa} enhancer, which has previously been shown to be capable of activating recombination but not transcription of Ig {kappa} light chain genes in mouse lymphoid cells (7, 21, 22). By analyzing the binding of murine nuclear proteins to the rabbit Ei{kappa} element, we do not find evidence of binding to regions homologous to the 3' murine {kappa}E3 site or to the {kappa}EO, {kappa}E*, and {kappa}A sites present at the 5' end of the mouse Ei{kappa} enhancer. Our results indicate that {kappa}B, {kappa}E1, and {kappa}E2 are the only sites within the rabbit enhancer that are able to interact with murine trans-acting factors. Mutational analysis has shown that deletions which remove 5' and 3' sequences of the mouse Ei{kappa} enhancer, but leave these three sites intact, abolish the transcriptional activity of the enhancer (12), hence explaining the lack of transcriptional activation observed for the rabbit Ei{kappa} enhancer in murine B lineage cells. This enhancer element can nevertheless promote V(D)J recombination in the mouse, suggesting that although the {kappa}B, {kappa}E1, and {kappa}E2 sites are not sufficient to activate transcription, they may be the only elements within the Ei{kappa} enhancer that are required to induce recombination.

There is previous evidence to suggest that the {kappa}B site and the NF{kappa}B/Rel transcription factors are involved in the regulation of Ig{kappa} gene rearrangement. Hence, repression of the nuclear import of p65 and c-Rel in a pre-B cell line prevents V{kappa}-J{kappa} rearrangement (15). Furthermore, mutation of the {kappa}B motif within the Ei{kappa} enhancer abolished V(D)J recombination of a linked recombination substrate, indicating that the {kappa}B motif plays a critical role in the ability of the Ei{kappa} enhancer to induce rearrangement (14). Nevertheless, the {kappa}B site alone is not sufficient to promote recombination (14). Our data suggest that induction of recombination may also require the presence of two E box motifs: {kappa}E1 and {kappa}E2. The {kappa}E2 site interacts with a family of basic helix-loop-helix proteins, primarily encoded by the E2A gene. These proteins also bind to E box motifs present in the Ig heavy chain and TCR{beta} enhancers (11, 34). The analysis of E2A-deficient mice has indicated that the E2A proteins act as positive and negative regulators of TCR V{delta} and V{gamma} gene rearrangement (35). A specific role for E2A in the regulation of Ig{kappa} gene rearrangement has recently been suggested from transfection experiments in which expression of the E2A gene along with the Rag genes allows V-J rearrangement of endogenous {kappa} genes in nonlymphoid cells (C. Murre, personal communication).

The question then becomes, how does the rabbit Ei{kappa} enhancer promote {kappa} gene rearrangement in mouse cells? Several studies have suggested that transcriptional enhancers regulate V(D)J recombination by modulating the accessibility of Ig and TCR gene segments to recombination trans-acting factors (reviewed in Ref. 3). An obvious explanation would be that enhancers induce rearrangement via activation of transcription of the Ig and TCR loci. In support of this idea is the fact that there is a temporal correlation between rearrangement at each Ag receptor locus and onset of germline gene transcription. However, it has been found that, in the presence of an active recombinase, transcription is not sufficient to obtain recombination (36), and, conversely, recombination can occur in the absence of detectable transcription (21, 37, 38), indicating that recombination and transcription are not mechanistically linked. Our data are consistent with this idea and suggest that the action of the Ei{kappa} enhancer on V(D)J recombination and transcription is mediated by a different set of trans-acting factors. An analysis of the Ig heavy chain enhancer has similarly led to the conclusion that distinct protein-binding sites within the enhancer region mediate recombination and transcription (39).

An intriguing finding of this study is that, although we observe binding to the {kappa}B motif present in the rabbit enhancer both by DNase I footprint and gel shift analysis, the rabbit {kappa}B site directs only minimal transcriptional activation in B and pre-B cells. Because the various NF-{kappa}B/Rel homo- and heterodimers have been found to differ in their target site specificities (17, 18, 19), it was possible that the mouse and rabbit {kappa}B sites do not interact with the same NF-{kappa}B complexes. However, we found no difference in the nature of the NF-{kappa}B factors bound to the two {kappa}B sites. In particular, both p50 homodimers and p50/p65 heterodimers, which are the most abundant forms in early B cells (40, 41), bind to the rabbit {kappa}B site. The C-terminal region of p65 contains a transactivation domain, that is lacking in the p50 subunits; therefore, the p50/p65 complexes are regarded as the transcriptionally active form (16). We could not detect any difference in the affinity of p50 homodimers for the two {kappa}B sites, but p50/p65 heterodimers bind with a 3-fold lower affinity to the rabbit site. The slight lower affinity of the rabbit site for p50/p65 as compared with the mouse {kappa}B site is unlikely to account for its inability to activate transcription, since the physiologically relevant {kappa}B site studied, the {kappa}B motif of the mouse Ei{kappa}, has the highest affinity for p50/p65 heterodimers. For example, the affinity of the IL-2 gene {kappa}B site for NF-{kappa}B p50/p65 is 10-fold lower than the Ei{kappa} site, yet it is transcriptionally active. Similarly, the transcriptionally active MHC class I and IFN-{beta} {kappa}B motifs are all lower affinity sites (17, 19).

Recent evidence suggests that the transcriptional activity of NF-{kappa}B complexes are regulated by their interaction with cellular coactivators. Notably, the coactivator CBP/p300 has been found to associate with p65 and to potentiate NF-{kappa}B-activated transcription (42, 43). Interaction of CBP/p300 with p65 appears to be dependent on the phosphorylation of p65 (44). In nonlymphoid cells, a variety of stimuli which lead to activation of NF-{kappa}B induce p65 phosphorylation (45, 46, 47), whereas in B cells, a fraction of p65 constitutively present in the nucleus is phosphorylated (45). At present it is unclear whether phosphorylation of p65 increases its transcriptional potential solely by promoting its association with the CBP/p300 coactivator or whether phosphorylation also affects the binding potential of p65 (45, 47). One attractive explanation for the inability of the rabbit {kappa}B site to mediate transcriptional activation would be that this site preferentially binds the nonphosphorylated and hence transcriptionally inactive form of p65.

The crystal structure of the p50/p65 heterodimer bound to the mouse Ei{kappa} {kappa}B site has shown that the p50 subunit occupies the 5' half of the site, whereas the p65 subunit interacts with the 3' end (48). By mutating the mouse {kappa}B site, we found that the {kappa}B motif in the rabbit enhancer, which confers binding of NF-{kappa}B factors but not transactivation, corresponds to the sequence 5'-GGGGTTTCCC-3'. The p50 subsite therefore changes from 5'-GGGAC-3' in the mouse site to 5'-GGGGT-3' in the rabbit site. The first three guanines, which form multiple hydrogen bonds with N-terminal residues in the p50 molecule, are conserved in all physiologically relevant {kappa}B motifs. They are also conserved in the rabbit sequence, which probably accounts for the fact that this site can bind p50 complexes. However, the last two bases, which differ in the rabbit and mouse {kappa}B motifs, also interact by hydrogen bond and Van der Waal contacts with p50. These contacts are most probably disrupted or at least altered upon binding to the rabbit sequence. Furthermore, there is a one nucleotide change between the mouse (5'-TTCC-3') and rabbit (5'-TCCC-3') p65 subsite. The nucleotide at this position hydrogen bonds with an arginine, which is itself tightly held by interactions with other p65 residues. It is therefore likely that there may be subtle differences in the conformation of the p50/p65 complexes bound to the rabbit and mouse sites. Such a change in conformation may result in the inability of the NF-{kappa}B complex bound to the rabbit {kappa}B motif to interact with transcriptional coactivators or the basal transcriptional machinery. This proposal is consistent with evidence from structural (48) and in vitro binding (19, 49, 50) studies indicating a high level of flexibility in the interaction of the NF-{kappa}B dimers with various target sites.

Because the {kappa}B site has been shown to be essential for the Ei{kappa}-mediated activation of V(D)J recombination (14, 15), the above results suggest that a nontransactivating form of NF-{kappa}B may be capable of inducing recombination. There are several ways that trans-activating factors may positively regulate V(D)J recombination other than via transcription, including local changes in chromatin structure or recruitment of the recombinase complex. Although there is no convincing evidence to date for an interaction between enhancer-binding factors and V(D)J recombination proteins, several lines of evidence suggest that one component of enhancer function could be to locally alter chromatin structure and this independently of transcriptional stimulation (10, 51, 52). For the Ei{kappa} enhancer, both the {kappa}B motif and nuclear NF-{kappa}B are essential for directing B-specific {kappa} gene demethylation (53). Furthermore, NF-{kappa}B has been found to bind to its cognate site within a chromatin template and induce changes in chromatin structure at the HIV-1 enhancer (54), suggesting that this factor may serve to recruit architectural proteins or chromatin remodeling factors to the Ig locus.

In conclusion, the results of this study indicate that the lack of transcriptional activation by the rabbit Ei{kappa} in murine cells can be explained by a combination of two defects. First, only the {kappa}B, {kappa}E1, and {kappa}E2 sites are able to interact with murine transactivating proteins. Second, even though the rabbit {kappa}B site is able to bind NF-{kappa}B factors, this site cannot mediate transcriptional activation in murine B lineage cells. Since this element can nevertheless act as a recombinational enhancer in murine lymphoid cells (7, 21), this suggests that distinct cis-acting elements within the intronic enhancer and/or different signaling pathways may mediate the effects of the Ei{kappa} enhancer on transcription and recombination.


   Acknowledgments
 
We are grateful to Silvia Cereghini for advice on DNase I footprinting techniques and C33 cell extracts, Robert Weil for help in setting up gel retardation experiments and critical comments on this work, and Jocelyne Demongeot for helpful discussions and HDR37 cells. We also thank Noëlle Doyen, Marc Delarue, and Jérome Maës for their comments on this manuscript.


   Footnotes
 
1 This work was supported by grants from the Institut Pasteur and the Centre National pour la Recherche Scientifique (Unité de Recherche Associée 1960). I.C. was a recipient of a fellowship from the Ministry of Research. Back

2 Address correspondence and reprint requests to Dr. Michele Goodhardt, Departement d’Immunologie, Unité Recherche Associée Centre de la Recherche Scientifique 1960, Institut Pasteur, 25 rue du Docteur Roux 75724, Paris Cedex 15, France. E-mail address: Back

Received for publication August 9, 1999. Accepted for publication October 26, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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