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The Journal of Immunology, 2001, 166: 4552-4559.
Copyright © 2001 by The American Association of Immunologists

NF-{kappa}B p50-Dependent In Vivo Footprints at Ig S{gamma}3 DNA Are Correlated with µ->{gamma}3 Switch Recombination1

Robert A. Wuerffel, Limei Ma and Amy L. Kenter2

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60680


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NF-{kappa}B has been demonstrated to play critical roles in multiple aspects of immune responses including Ig H chain isotype switching. To better define the specific roles the p50 subunit of NF-{kappa}B plays in µ->{gamma}3 switch recombination (SR), we systematically evaluated p50-deficient B cells for activities that are strongly correlated with SR. B cell activation with LPS plus anti-IgD-dextran plus IL-5 plus IL-4 plus TGF-{beta} produced normal levels of proliferation and {gamma}3 germline transcripts in p50-deficient B cells, but µ->{gamma}3 SR was impaired. In vitro binding studies previously showed that NF-{kappa}B p50 homodimer binds the switch nuclear B-site protein (SNIP) of the S{gamma}3 tandem repeat. Ligation-mediated PCR in vivo footprint analysis demonstrates that the region spanning the SNIP and switch nuclear A-site protein (SNAP) binding sites of the S{gamma}3 region are contacted by protein in normal resting splenic B cells. B cells that are homozygous for the targeted disruption of the gene encoding p50 (-/-) show strong aberrant footprints, whereas heterozygous cells (+/-) reveal a partial effect in S{gamma}3 DNA. These studies provide evidence of nucleoprotein interactions at switch DNA in vivo and suggest a direct interaction of p50 with S{gamma}3 DNA that is strongly correlated with SR competence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
When naive B cells are activated by antigenic stimuli, they proliferate and differentiate into memory cells and Ab-secreting plasma cells. During this response, B cells switch the class of heavy chain they produce while maintaining the original Ag-binding specificity arising from V(D)J joining. This phenomenon of isotype switching is mediated by an intrachromosomal DNA rearrangement called switch recombination (SR)3 (reviewed in Refs. 1 and 2). SR focuses on tandemly repeated DNA sequences termed switch (S) regions, which are found upstream of all of the CH genes except C{delta} and produce a new composite S DNA configuration, Sµ/Sx. The hybrid Sµ/Sx DNA is formed on the chromosome while the intervening genomic material is looped out and excised as a circle. Sequence analyses of recombination joints from normal B cells have demonstrated that SR breakpoints fall within the tandem repeats of both the donor and acceptor S regions (3, 4, 5, 6, 7), demonstrating the functional importance of the tandem repeat sequences. The presence of double strand breaks (DSBs) in S DNA (8) and the requirement for the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (9), Ku80 (10), and Ku70 (11), components of DNA-PK involved in double strand break repair, strongly suggest that SR is resolved through a nonhomologous DNA end-joining process (reviewed in Ref. 12). However, the factors that mediate SR have not been defined.

The specificity of the SR process is achieved in part through production of germline transcripts (13, 14, 15, 16, 17). Germline transcripts are RNA transcripts from specific unrearranged CH genes that are induced before SR, initiate upstream of the SR, terminate downstream of the CH gene, and are apparently not translated. Although the production of germline transcripts is required for SR, accumulating evidence indicates that there are additional factors that contribute to the isotype specificity of the reaction. For example, B cells lacking the transactivation domain of c-Rel are capable of expressing both {alpha} and {epsilon} germline transcripts but are able to carry out only µ->{alpha} and not µ->{epsilon} SR (18). Similarly, B cells that are deficient in the p50 subunit of NF-{kappa}B can produce {gamma}1 and {alpha} germline transcripts but switch only µ->{gamma}1 and not µ->{alpha} (19). Moreover, recent analyses that assay for SR on extrachromosomal S plasmids demonstrate the existence of distinct switching activities that independently mediate µ->{gamma}3 and µ->{alpha} SR (20). These studies demonstrate the existence of transacting factors that influence the specificity of SR independently of germline transcript expression. The identities of these factors remain unknown.

It is interesting to note that while I.29 µ, a B cell lymphoma cell line, constitutively expresses both the {alpha} germline transcript as well as the µ->{alpha} switching machinery detected by the S plasmid assay, it must be stimulated with LPS to induce SR at the endogenous S{alpha} loci. This suggests that additional factors or epigenetic changes are required for endogenous SR. The requirement for epigenetic changes has been demonstrated to play a role in the developmental regulation of V(D)J recombination (21, 22). We have considered the possibility that epigenetic changes permitting SR might be mediated by S region-specific nucleoprotein complexes because formation of complex nucleoprotein structures is a common feature of site-specific recombination systems (23, 24, 25). The detection of the S{gamma}3 region-specific DNA binding proteins, S nuclear B-site protein (SNIP) and S nuclear A-site protein (SNAP), and the S nuclear µ protein (SNUP) by in vitro binding assays is consistent with the idea that the assembly of recombination-proficient nucleoprotein structures at S DNA is a prerequisite for endogenous SR (26, 27). In this report, we present direct evidence of nucleoprotein interactions at S{gamma}3 DNA in normal splenic B cells in vivo.

Our previous studies demonstrated that the SNIP binding protein is indistinguishable from NF-{kappa}B p50 homodimer (27, 28). We hypothesize that µ->{gamma}3 SR will be defective in NF-{kappa}B p50-deficient B cells because p50 will be unavailable for interaction with the SNIP binding sites in S{gamma}3 DNA. The expression of a specific germline transcript is a strict requirement for targeted SR to that S region, and previous analyses of SR in NF-{kappa}B p50-/- B cells indicated that {gamma}3 germline transcript expression was impaired in those cells (19). Under conditions where the {gamma}3 germline transcript is not expressed, it is not possible to determine the direct impact of p50 deficiency on µ->{gamma}3 SR. We have now defined conditions in which {gamma}3 germline transcript expression is comparable in p50+/- and p50-/- B cells. We find that while this stimulus also results in robust proliferation in p50-/- B cells, µ->{gamma}3 SR of the endogenous IgH locus remains drastically reduced. By in vivo footprinting, we show that the SNIP and SNAP binding sites of S{gamma}3 are occupied in normal resting B cells, but the footprint is significantly altered in p50-deficient B cells. Taken together, these data provide evidence that p50 may play a role in configuring endogenous S{gamma}3 DNA into a structure that facilitates µ->{gamma}3 SR.


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

Single-cell suspensions of splenocytes from BALB/c nude (nu/nu) mice and nfkb1 mice, in which the p105 gene encoding the p50 subunit of NF-{kappa}B is disrupted (29), were prepared as previously described (30). Splenic B cells were stimulated in culture with various combinations of LPS (50 µg/ml) (Salmonella typhimurium, phenol extract; Sigma, St. Louis, MO), anti-IgD Ab conjugated to dextran ({alpha}{delta}dex; 3 ng/ml), IL-5 (150 U/ml), IL-4 (3000 U/ml), and TGF-{beta} (3 ng/ml; gifts from Dr. C. Snapper). Enrichment of B cells from the spleens of nfkb1 mice was accomplished by depletion of T cells using rat mAbs specific for mouse Thy1.2 and Dynabead M-450 magnetic beads coated with sheep anti-rat IgG (Dynal, Great Neck, NY). The purity of the cell population was confirmed by FACS analysis.

RT-PCR analysis

Semiquantitative RT-PCR was performed as described (19) with modifications. RNA was extracted from cells using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). cDNA was prepared by reverse transcription (RT) of 5 µg RNA in a 25-µl reaction mix containing 9 U of AMV-RT (Promega, Madison, WI), 1x buffer, 0.5 µg random hexameric primer (Promega), 40 U RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), and 0.1 mM of each dNTP. Three microliters of the RT reaction was taken for PCR amplification with 2.5 U of Taq polymerase (Boehringer Mannheim), 0.2 mM of each dNTP, 3.0 µCi [{alpha}-32P]dCTP (3000 Ci/mmol; NEN, Boston, MA), and 0.4 µM of each primer. Primers for the {gamma}3 germline transcripts were designed from the {gamma}3 genomic sequence (GenBank accession number D78343) and overlap the original primers described by Snapper and coworkers (19), with the sense primer sequence (5'-GTGGATCTGAACACACACAAC-3'; nucleotides 1107–1127) located in the I exon and the antisense primer sequence (5'-CCATTTTACAGTTACCGGC-3'; nucleotides 6356–6338) located in CH1. PCR was conducted for 30 cycles (0.5 min at 94°C, 0.5 min at 53°C, and 2 min at 68°C) and resulted in amplification of a 332-bp product as predicted for the {gamma}3 germline transcript. The PCR product was sequenced to confirm the specificity of the reaction. Amplification of the GAPDH control was performed as described (19).

Digestion circularization-PCR

Digestion circularization (DC)-PCR was performed as described (31) with modifications. Two rounds of PCR using nested primer sets were performed to increase sensitivity for detection of DC-PCR SR products. The Sµ and S{gamma}1 primer sets overlap primers previously described (10). The Sµ primers are: dc-µ.1, 5'-GAAGCCCTTCACGCCACTGACTGACTG-3', and dc-µ.2, 5'-GAATGGAGACCAATAATCAGAGGGAAG-3'. The S{gamma}1 primers are: dc-{gamma}1.1, 5'-AGACCAGGCTGAGCAGCTACCAAGGATCAG-3', and dc-{gamma}1.2, 5'-CACAGAGAGCAGGGTCTCCTGGGTAGGTTA-3'. The amplified product for Sµ->S{gamma}1 DC-PCR is 206 bp. The S{gamma}3 primers were designed from unpublished genomic DNA sequence generously provided by Dr. E. Max and are: dc-{gamma}3.1, 5'-TTGATCTTACAGCACAAAGGCCACG-3', and dc-{gamma}3.2, 5'-CTCCCTGGGTCGAGAGATATACAAGCC-3'. The amplified product for Sµ->S{gamma}3 DC-PCR is 195 bp. Following restriction endonuclease digestion and ligation, 4 ng DNA was taken for PCR amplification. The first round was accomplished by denaturing at 95°C for 3 min and then five cycles of 1 min at 94°C, 1 min at 61°C, and 2 min at 72°C and 15 additional cycles of 1 min at 94°C, 1 min at 65°C, and 2 min at 72°C with a final 7-min elongation at 72°C. One-tenth of the first round reaction mix was used to program the second round of PCR; initial denaturation for 3 min at 95°C was followed by five cycles of 1 min at 94°C, 1 min at 61°C, and 2 min at 72°C and 31 cycles of 1 min at 94°C, 1 min at 63°C, and 2 min at 72°C with a final 7-min elongation at 72°C. Control DC-PCR of the nicotinic acetylcholine receptor (nAChR) gene was conducted as described (31).

In vivo footprinting

Viable cells were isolated by centrifugation on Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) step gradients according to manufacturer’s instructions. Small resting B cells form a layer at the 70% boundary, and larger stimulated B cells layer at the 65% boundary. The recovered cells were then resuspended in full medium at 37°C for treatment with dimethyl sulfate (DMS) (0.1%). After 1 min of methylation, the cells were immediately washed two times with cold RPMI 1640 containing 1% FBS, and genomic DNA was prepared using the Cell Culture DNA kit (Qiagen, Chatsworth, CA). Methylated DNA samples were cleaved with piperidine, and ligation-mediated PCR (LMPCR) was performed in accordance with the protocol of Mueller and coworkers (32) in buffers containing 7 mM MgSO4. The primers were derived from the S{gamma}3 germline sequence (MUSIGHANA) upstream and downstream of the tandem repeats. The upstream primers were previously described (8). The downstream primers are: first strand synthesis primer (FSDN), 5'-TACCCTGACCCAGGAGCTGCATAACCT-3' (nucleotides 2627–2603), amplification primer (APDN), 5'-CCTGGGACCCTGTGATCTGATAGCC-3' (nucleotides 2604–2579), and labeling primer (LPDN), 5'-CCTGGGACCCTGTGATCTGATAGCCCCAG-3' (nucleotides 2604–2575). The amplified products were separated on 4% sequencing gels. Quantitation of band intensities was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The signals of the coding strand residues were normalized to the intensity of residue 229 in the long spacer sequence. Because the G residues of the noncoding strand fall exclusively in the SNIP and SNAP binding motifs, we chose to use residue 166, at the 3' end of the SNAP motif, for normalization. To classify residues as strongly protected or enhanced, we required that a >30% difference in signal intensity be reproducibly observed for a given band in the in vivo-methylated sample relative to the same band in the in vitro-methylated sample. Residues reproducibly diminished in intensity by 20–30% were designated as moderately protected.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B p50-deficient B cells can be activated to express {gamma}3 germline transcript and to proliferate

To investigate the roles p50 might play in SR, we studied various aspects of µ->{gamma}3 isotype switching in B cells from mice carrying a targeted disruption of the p105 gene, which encodes the p50 subunit of NF-{kappa}B (29). It is well established that germline transcript expression is a prerequisite for targeted SR (2). Analysis of {gamma}3 germline transcript expression by semiquantitative RT-PCR indicates that germline transcripts are significantly reduced in {alpha}{delta}dex plus IL-5-activated p50-/- B cells, but are highly expressed in B cells from p50+/- B cells (Fig. 1GoA, lanes 1 and 2), confirming previous findings (19).



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  		FIGURE 1. Analysis of {gamma}3 germline transcript expression in p50+/- and p50-/- mitogen-activated B cells. A, {gamma}3 germline transcripts in p50+/- (lanes 1 and 3) and p50-/- (lanes 2 and 4) splenic B cells stimulated in culture for 44 h with either {alpha}{delta}dex plus IL-5 or LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} were detected by RT-PCR. The intensity of the signal derived from GAPDH RT-PCR was used for normalization. B, The RT-PCR of {gamma}3 germline transcripts from p50+/- and p50-/- B cells is in the linear range of detection. The RT-PCR signal obtained with half (1:2) the amount of input p50-/- cDNA used in A, lane 4, was compared with a RT-PCR standard curve derived by serial 2-fold dilutions of cDNA from 1.B4.B6 cells stimulated with LPS plus CD40L. The arrow indicates the signal intensity for the p50-/- RT-PCR and shows that it is within the linear range.

 
The absence of induced {gamma}3 germline transcript production in p50-/- B cells in response to {alpha}{delta}dex plus IL-5 activation prevents a determination of the role for p50 in µ->{gamma}3 SR. To circumvent this problem, the B cell activator combination of LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} was used to induce {gamma}3 germline transcript expression in p50+/- and p50-/- B cells (Fig. 1GoA, lanes 3 and 4). This activator combination was previously shown to be an excellent inducer of {alpha} germline transcripts and µ->{alpha} SR (18).

It was important to ensure that the detection of {gamma}3 germline transcripts in p50+/- and p50-/- B cells was in the linear range. We performed RT-PCR on a set of 2-fold serial dilutions of 1.B4.B6 cDNA to prepare a standard curve (Fig. 1GoB). 1.B4.B6 is a transformed B cell line that can be induced to express the {gamma}3 germline transcript and is competent for µ->{gamma}3 SR (53) and our unpublished data). In the same experiment, we included a sample containing half the normal input amount of cDNA from p50-/- cells (Fig. 1GoA, lane 4) stimulated with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} (Fig. 1GoB, lower panel). The arrow shown on the linear regression plot (Fig. 1GoB, top panel) indicates the signal intensity for the p50-/- RT-PCR and shows that it is well within the linear range of detection. We conclude that stimulation of p50-/- B cells with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} results in a level of {gamma}3 germline transcript expression that is comparable to the level observed in p50+/- B cells (Fig. 1GoA, lanes 3 and 4). The presence of equivalent levels of {gamma}3 germline transcripts in p50+/- and p50-/- B cells permits a direct evaluation of the influence of p50 on the SR process.

The requirement of B cell proliferation for the SR reaction has been noted (33, 34, 35). It was therefore important to establish that p50-/- B cells proliferate when activated with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta}. In a time course experiment, we found this activator combination stimulated a higher degree of proliferation in the p50-/- B cells than did {alpha}{delta}dex plus IL-5 in p50+/- B cells, which carry out µ->{gamma}3 SR (see below) (Fig. 2Go). This demonstrates that proliferation is not a limiting factor for SR in p50-/- B cells stimulated with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta}.



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  		FIGURE 2. Comparison of proliferation responses of p50+/- and p50-/- B cells to mitogens and cytokines. Splenic B cells from p50+/- and p50-/- mice were seeded in culture at 5 x 105 cells/ml, activated with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} or with {alpha}{delta}dex plus IL-5 only, as indicated, and analyzed for proliferation. The number of viable cells in each culture was determined at various times by counting the number of cells that excluded trypan blue.

 
p50 deficiency leads to a reduction of SR

The induction of {gamma}3 germline transcript expression and robust proliferation in response to LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} indicates that p50-/- B cells are proficient in two of the activities associated with SR competence. To evaluate the level of µ->{gamma}3 switching in p50+/- and p50-/- B cells, we used the semiquantitative DC-PCR method (31). The overall DC-PCR strategy to analyze the relative frequency of S events that produce Sµ/S{gamma}3 hybrid molecules is shown (Fig. 3GoA). SR results in the deletion of genomic DNA located between two S regions. EcoRI sites, which flank the 5' end and the 3' end of the Sµ and S{gamma}3 regions, respectively, are preserved following SR. After digestion with EcoRI, the DNA is ligated under conditions that favor circularization of the restriction fragments. The region spanning the circle joint is amplified using primers specific for sites at the 5' end of Sµ and the 3' end of S{gamma}3 and yields a 195-bp product. The nAChR gene was used as a control for digestion and ligation reactions, and all of the samples tested were positive for the nAChR DC-PCR product (Fig. 3GoB). DNA from the IgG3-expressing hybridoma, TIB114, served as a positive control for the Sµ/S{gamma}3 DC-PCR product (Fig. 3GoB, lane 4), whereas DNA from unstimulated nu/nu splenic B cells served as the negative control (Fig. 3GoB, lane 1). To ensure that our DC-PCR primers specifically detected only µ->{gamma}3 SR events, we assayed cells that were stimulated in the presence of IL-4 (Fig. 3GoB, lanes 2 and 3). Inclusion of IL-4 in cultures of B cells stimulated with either LPS or {alpha}{delta}dex plus IL-5 causes the inhibition of switching to {gamma}3 and the induction of switching to {gamma}1 (31, 36, 37, 38). The absence of the Sµ/S{gamma}3 DC-PCR product and the presence of the Sµ/S{gamma}1 DC-PCR product in B cells stimulated with LPS plus IL-4 or {alpha}{delta}dex plus IL-5 plus IL-4 demonstrates the specificity of the DC-PCR primer sets (Fig. 3GoB, lanes 2 and 3).



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  		FIGURE 3. DC-PCR analysis of µ->{gamma}3 switching at the endogenous loci in mitogen-activated nu/nu, p50+/-, and p50-/- B cells. A, Schematic diagram showing the strategy for DC-PCR. A portion of the IgH locus is depicted before and after switching. Following digestion with EcoRI, the DNA is ligated under conditions favoring intramolecular ligation. A PCR product results only if Sµ/S{gamma}3 recombination has occurred. DC-PCR of the nAChR gene serves as a positive control for digestion and ligation because it does not require rearrangement to yield a product. The positions and orientations of the µ/{gamma}3 nested primer sets and the single nAChR primer set are shown before and after ligation. B, DNA from nu/nu splenic B cells that were either unstimulated or stimulated in the presence of IL-4 serve as negative controls for the Sµ/S{gamma}3 DC-PCR (lanes 1–3). DNA from the IgG3-expressing hybridoma TIB114 serves as a positive control (lane 4). DC-PCR was performed on DNA prepared from p50+/- (lanes 5 and 7) and p50-/- (lanes 6 and 8) B cells that were stimulated in culture for 4 days with either {alpha}{delta}dex plus IL-5 or LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} as indicated. Detection of the Sµ/S{gamma}1 DC-PCR product in B cells stimulated in the presence of IL-4 (lanes 2 and 3) demonstrates the specificity of the DC-PCR primer sets.

 
Using DC-PCR, p50+/- and p50-/- cells were analyzed for the level of µ->{gamma}3 SR in response to {alpha}{delta}dex plus IL-5 and LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta}. We found that with {alpha}{delta}dex plus IL-5 stimulation, SR was significantly reduced for p50-/- B cells as compared with p50+/- B cells (Fig. 3GoB, lanes 5 and 6). This was expected because, under these conditions, p50-/- cells are greatly impaired in their {gamma}3 germline transcript expression (Fig. 1GoA, lane 2). It was surprising to find that p50-/- B cells were unable to carry out µ->{gamma}3 SR when the {gamma}3 germline transcripts were induced by stimulation with LPS plus {alpha}{delta}dex plus IL-5 plus IL-4 plus TGF-{beta} (Fig. 3GoB, lanes 7 and 8). Therefore, even under conditions where the p50-/- B cells proliferate well and express {gamma}3 germline transcripts at levels comparable to p50+/- B cells, the SR frequency was severely reduced.

SNIP and SNAP sites are occupied by protein in vivo

Formation of DNA-multiprotein complexes is a common feature of such processes as transcription, site-specific recombination, and the initiation of DNA replication (23, 24, 25). Using in vitro methylation interference footprinting and competition binding assays, we previously identified two protein complexes, termed SNIP and SNAP, which bind specifically to two distinct motifs in the S{gamma}3 tandem repeats that we designated as the SNIP and SNAP binding sites (27). SNIP protein was defined as NF-{kappa}B p50 homodimer by in vitro binding and supershift analysis (27). SNAP protein was found to contain epitopes in common with the helix-loop-helix transcription factor, E47 (39).

To determine whether protein occupancy at the SNIP and SNAP binding sites of S{gamma}3 DNA occurs in vivo, we used the LMPCR protocol (32, 40). This technique exploits the ability of bound proteins to alter the susceptibility of local DNA residues to methylation by DMS. Because S DNA is comprised of multiple tandem repeats, the LMPCR primers were designed to anneal to positions flanking the repeats (Fig. 4GoA). Therefore, the noncoding strand at the 5' end and the coding strand at the 3' end of S{gamma}3 are the only regions amenable to in vivo footprint analysis. We have evaluated protein occupancy at two typical repeats at each end of S{gamma}3 DNA.



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  		FIGURE 4. In vivo footprint analysis of S{gamma}3 DNA in nu/nu, p50+/-, and p50-/- B cells. A, A partial restriction map of the S{gamma}3 region of the IgH locus (52 ) is shown. The relative positions of the primers used for first strand synthesis FSUP and FSDN, amplification APUP and APDN, and labeling LPUP and LPDN are indicated flanking the repetitive switch sequence. The regions amenable to in vivo footprinting are indicated with brackets below the line for the noncoding strand at the 5' end of S{gamma}3 and above the line for the coding strand at the 3' end of S{gamma}3. Restriction sites are abbreviated as: B, BglII; H, HindIII; K, KpnI; and S, SacI. B cells were treated with DMS for 1 min at 37°C. The methylated DNAs were cleaved, and the fragments were amplified and labeled according to the LMPCR protocol (32 ). The amplified fragments were resolved on a 4% denaturing gel and analyzed with a PhosphorImager using ImageQuant software (Molecular Dynamics). B and C, In vivo footprinting was performed on the coding and noncoding strands of S{gamma}3 DNA prepared from unstimulated BALB/c nu/nu splenocytes and from unstimulated splenic B cells of mice that were heterozygous (+/-) or homozygous (-/-) for targeted disruption of the p105 gene encoding the p50 subunit of NF-{kappa}B. DNA that was methylated in vitro serves as the reference for methylation in the absence of bound protein. Numbers indicate the positions relative to the 3' end of the labeling primers, where position 1 for the coding strand is equivalent to nucleotide 2574, position 260 for the coding strand is equivalent to nucleotide 2314, and positions 1 and 207 for the noncoding strand are equivalent to nucleotides 505 and 711, respectively, of the germline S{gamma}3 sequence MUSIGHANA. Boxes indicate the positions of the SNIP and SNAP binding motifs. Residues that are strongly (>30%; •) or moderately (20–30%; {circ}) protected from methylation or are hypermethylated (*) in vivo as determined by densitometry are shown. The positions of sequences related to the Ikaros recognition motif, TGGGAA, are indicated next to the gels. D and E, Densitometry traces comparing the LMPCR results for the coding strand of in vitro-methylated DNA to in vivo methylated DNA from unstimulated nu/nu (D) and unstimulated p50-/- (E) splenic B cells.

 
Splenocytes from BALB/c nu/nu mice were either unstimulated or stimulated with LPS or {alpha}{delta}dex plus IL-5 for various times. The cells were treated with DMS, and the DNA was isolated. To identify protein-DNA contacts, the intensities of bands from in vivo-methylated DNA are compared with the corresponding bands from in vitro-methylated DNA. In unstimulated B cells, a region of protected residues of the coding strand is observed across the SNIP and SNAP recognition motifs of repeat 39, including residues 186, 188, 200, 203, 209, 210, 212, and 214 (Fig. 4GoB, compare lanes 1 and 2). In contrast, the long spacer sequences flanking the SNIP and SNAP motifs are relatively devoid of protected residues but do contain one guanine, at position 179, which is moderately hypersensitive to methylation in these cells (Fig. 4GoB, compare lanes 1 and 2). Quantitation of band intensities was conducted by densitometry, and a comparison of results obtained from DNA methylated in vitro and in vivo from normal splenic B cells is shown in Fig. 4GoD. Although the resolution of nucleotides in tandem repeat 38 is not sufficient to allow accurate densitometry, the general pattern of protein-DNA contacts across the region spanning the SNIP and SNAP sites is the same as in repeat 39 (Fig. 4GoB, compare lanes 1 and 2). B cells stimulated with either LPS or {alpha}{delta}dex plus IL-5 exhibit an overall decrease in the footprint as compared with unstimulated B cells (data not shown). The LMPCR findings for both resting and mitogen-activated B cells were observed in at least three independent experiments. It is possible that the diminution of footprints in the population of mitogen-activated B cells reflects a limitation of the in vivo footprinting method. Cultures of mitogenically stimulated B cells constitute a much more heterogeneous population than resting splenic B cells. As the heterogeneity of a population increases, specific protein-DNA interactions become progressively obscured. Thus, diminution of the in vivo footprints in stimulated B cells is difficult to interpret. Footprint studies were also conducted for the noncoding strand at the 5' end of the S{gamma}3 region, and no reproducible protein-DNA interactions were observed (Fig. 4GoC, compare lanes 1 and 2). These studies demonstrate that the pattern of protein-DNA contacts on the coding strand of S{gamma}3 DNA in resting splenic B cells focuses to the region spanning the SNIP and SNAP binding sites.

Targeted disruption of the NF-{kappa}B p105 gene is correlated with altered footprints at S{gamma}3 in vivo

In vitro binding studies indicated that NF-{kappa}B p50 homodimer binds S{gamma}3 DNA at the SNIP recognition motif (27). To determine whether the protein-DNA interactions that are found at the SNIP binding site in vivo are dependent upon expression of NF-{kappa}B p50, we performed LMPCR footprint analysis of S{gamma}3 DNA in B cells from p50-deficient mice. The absence of p50 may cause the in vivo footprints at SNIP sites to be lost or changed. These alternatives are equally possible because, in the absence of p50, other DNA-binding proteins may gain access to the region and produce new protein-DNA contacts. Comparison of the in vitro "G ladders" for nu/nu mice and p50-/- mice clearly shows that the positions of the G residues are identical in the two strains (compare lanes 1 and 3 of Fig. 4Go, B and C). Thus, all the protections and enhancements of specific residues we describe are the result of bona fide protein-DNA interaction differences and not merely due to differences in genomic DNA sequence.

In unstimulated B cells from p50-/- mice, no strongly protected residues are found on the coding strand in the region spanning the SNIP and SNAP motifs of repeat 39 (Fig. 4Go, B, lane 5, and E). However, in contrast to normal resting B cells, multiple protein-DNA contacts are observed in the long spacer sequence of p50-/- DNA (Fig. 4Go, B, lane 5, and E). The G residue at position 181 in the long spacer is highly protected from methylation in the p50-/- B cells (Fig. 4Go, B, lane 5, and E), whereas, in normal B cells, this residue is not protected (Fig. 4Go, B, lane 2, and D). Moreover, hypermethylation of the A residue at position 168, seen in the p50-/- B cells (Fig. 4GoB, lane 5), is absent in normal B cells (Fig. 4GoB, lane 2). In contrast to normal resting B cells, which exhibit no detectable footprints on the noncoding strand of S{gamma}3 DNA (Fig. 4GoC, lane 2), unstimulated B cells from p50-/- mice show protected residues at positions 153 and 157 and an enhanced band at position 199 (Fig. 4GoC, lane 5). We conclude that the pattern of protein-DNA contacts at S{gamma}3 DNA in resting splenic B cells lacking p50 is different from that found in normal B cells.

To determine whether the aberrant protein-DNA interactions observed in p50-deficient B cells are related to the concentration of p50, the in vivo footprints from p50-/- and p50+/- siblings were compared over several repeats of the coding and noncoding strands of S{gamma}3 (compare lanes 3, 4, and 5 of Fig. 4Go, B and C). Both p50-/- and p50+/- B cells reveal protections at positions 181, 134, 133, and 124 of the coding strand and positions 157 and 153 of the noncoding strand. Enhanced bands are seen at positions 168 and 131 of the coding strand and position 199 of the noncoding strand. However, the degree of both protection and enhancement appear reduced in the p50+/- DNA relative to the p50-/- DNA (compare lanes 3, 4, and 5 of Fig. 4Go, B and C).

Quantitation of the LMPCR results for in vivo-methylated p50-/- and p50+/- S{gamma}3 DNA is presented in Fig. 5Go, A and C. When the relative intensity of each residue is expressed as the ratio of the p50-/- signal divided by the p50+/- signal, it is evident that the aberrant footprint is stronger in p50-/- B cells as compared with p50+/- B cells (Fig. 5Go, B and D). Specifically, on the coding strand at the 3' end of the S{gamma}3 region, residues 181, 134, 133, and 124 are protected to about twice the extent in p50-/- cells as compared with p50+/- cells (Fig. 5GoB). The methylation sensitivity of residues 168 and 131 of the coding strand is ~2-fold higher in p50-/- cells than in p50+/- cells (Fig. 5GoB). On the noncoding strand, at the 5' end of the S{gamma}3 region, the protection of residues 157 and 153 are ~1.5 times and 3 times stronger in the p50-/- cells than in the p50+/- cells, respectively (Fig. 5GoD). Enhancement of methylation at residue 199 is also increased ~3-fold in p50-/- B cells relative to p50+/- B cells (Fig. 5GoD). The intermediate intensity of each protected and enhanced residue found in the p50+/- as compared with the p50-/- suggests that there is a dose-response relationship between the protein-DNA contacts and p50 protein concentration. As the dose of p50 decreases, the intensity of the aberrant footprint increases. These findings are consistent with the idea that p50 is directly involved in binding to S{gamma}3 DNA in normal B cells.



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  		FIGURE 5. Quantitative comparison of the aberrant S{gamma}3 footprints observed in p50+/- and p50-/- resting splenic B cells. A, Densitometry traces of the LMPCR results (shown in Fig. 4GoB) for the coding strand of in vivo-methylated DNA from unstimulated p50+/- and p50-/- splenic B cells. The coding strand sequence is shown, and the positions of residues which are strongly protected (•) or hypermethylated (*) in the p50-/- resting B cells in vivo are indicated. The Ikaros recognition sequences are underlined. B, Histogram comparing the coding strand signal intensities of in vivo methylated DNA from unstimulated p50+/- and p50-/- splenic B cells. Quantitation was performed using ImageQuant software, and the relative intensity for each residue was expressed as the ratio of the p50-/- signal divided by the p50+/- signal. C, Densitometry traces of the LMPCR results (shown in Fig. 4GoC) for the noncoding strand of in vivo methylated DNA from unstimulated p50+/- and p50-/- splenic B cells. The noncoding strand sequence is shown, and the positions of residues that are strongly protected (•) or hypermethylated (*) in the p50-/- resting B cells in vivo are indicated. Sequences related to the Ikaros core recognition motif are underlined. D, Histogram comparing the noncoding strand signal intensities of in vivo-methylated DNA from unstimulated p50+/- and p50-/- splenic B cells. Quantitation was performed using ImageQuant software, and the relative intensity for each residue was expressed as the ratio of the p50-/- signal divided by the p50+/- signal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies indicate that p50 deficiency leads to a profound deficit in µ->{gamma}3 SR even when {gamma}3 germline transcript expression and proliferation are adequate to support switching. It was previously shown that p50-/- B cells fail to perform µ->{alpha} SR despite the fact that they express normal levels of {alpha} germline transcripts (19). Furthermore, {Delta}c-Rel B cells are incapable of µ->{epsilon} SR despite their ability to produce {epsilon} germline transcripts (18). These findings strongly suggest that the NF-{kappa}B/Rel factors play roles in controlling SR beyond the regulation of particular germline transcripts. Previously, we established that p50 homodimer interacts with the SNIP binding site in S{gamma}3 tandem repeats and suggested that these interactions may exist in vivo (27, 28). These observations together suggest that the inability of p50-deficient B cells to support µ->{gamma}3 SR may arise from perturbation of p50-dependent nucleoprotein structures normally found at S{gamma}3 DNA in vivo.

Examination of nucleoprotein interactions at S{gamma}3 DNA by in vivo footprint analyses of SR-competent normal resting nu/nu B cells revealed a protected region spanning the SNIP and SNAP recognition sites on the coding strand of S{gamma}3 DNA in vivo. The degree of protection was diminished over time following mitogenic activation of the splenic B cells (data not shown). The loss of a footprint is difficult to interpret in these circumstances because the loss may result from either diminished protein interaction or from the increasing heterogeneity of the B cell population following cell activation. Therefore, we limited the focus of our footprinting studies to resting B cells. In contrast to the in vivo footprints found spanning the SNIP and SNAP recognition motifs in normal resting B cells, no strong protections in this region were observed in resting B cells derived from p50-/- mice. However, strong protections and enhancements were observed at abnormal positions on both the coding and noncoding strands of S{gamma}3 DNA in p50-deficient B cells, resulting in the formation of aberrant footprints. Furthermore, the aberrant footprints were 2- to 3-fold stronger in p50-/- B cells than in the p50+/- B cells, demonstrating that the intensity of the aberrant footprints is inversely correlated with the concentration of p50 in the cells. These studies imply that p50 directly interacts with S{gamma}3 DNA and that the normal dose of p50 precludes the formation of aberrant footprints. Further investigation is required to directly demonstrate that p50 is located on S{gamma}3 DNA in vivo.

Expression of germline transcripts is necessary for SR to occur and was originally proposed to confer a degree of accessibility of the targeted S region to the recombination machinery (14, 17). Mounting evidence strongly suggests that other events in addition to germline transcription play roles in controlling SR (41). Recent analyses have demonstrated that switching factors that mediate plasmid-based µ->{alpha} SR are constitutively expressed in the I.29 µ, CH12LX, and 1.B4.B6 B cell lines (20). In the case of I.29 µ cells, there is constitutive expression of both the µ->{alpha} switching factor and the {alpha} germline transcript (20, 42). However, in these cells, endogenous µ->{alpha} SR only occurs following LPS induction (42). The finding that I.29 µ cells require mitogen activation to carry out SR of its endogenous loci implies that mitogen activation induces factors or epigenetic changes that are distinct from germline transcripts and the constitutively expressed activities detected by the plasmid S substrate. The access of switching factors to the endogenous loci may be regulated by modulation of chromatin configuration at S DNA. Precedence for the involvement of chromatin in the regulation of recombination has been established in V(D)J joining (21). The recombination-activating gene proteins 1 and 2 (RAG-1 and RAG-2), which constitute the V(D)J recombinase, are expressed in B and T cells during early development, but specific chromatin changes are required to allow recombinase accessibility to specific endogenous loci (43).

Chromatin remodeling through hyperacetylation of histones has been correlated with the induction of transcription (44). More recently, it has become clear that transcriptional activation is associated with hyperacetylation of histones H3 and H4 in either a localized region surrounding the promoter or over a broad region encompassing much of the gene being transcribed (45, 46). These observations may be relevant to our understanding of the presence of S region nucleoprotein complexes in the process of SR. We speculate that germline transcript expression is associated with a localized region of histone acetylation that is confined to the promoter. We hypothesize that additional independent acetylation events are required at S regions to allow SR and that nucleoprotein complexes located at S regions regulate these secondary chromatin remodeling events.

The presence of p50 at S{gamma}3 DNA in resting B cells might be necessary for recruitment of histone acetylases that open nucleosome structure and make the DNA more accessible to switching factors (47). In this context, it is of interest to note that both of the regions of the coding strand in which aberrant footprints are seen in p50-/- B cells contain the sequence TGGGAA (Figs. 4GoB and 5A). This is the core recognition motif for the Ikaros family of lymphoid-specific proteins (48). The regions of aberrant contacts observed on the noncoding strand in p50-/- cells occur at the sequences TACCCA and TGCCCA (read TGGGTA and TGGGCA on the coding strand; Figs. 4Go and 5Go, C), closely related to the Ikaros core motif. The Ikaros family are lymphoid lineage-determining transcription factors that are often found associated with chromatin remodeling proteins (49, 50). It has been shown that Ikaros family members can function as repressors of gene expression by recruiting distinct histone deacetylase complexes (51). It is possible that in the absence of p50, Ikaros and other DNA binding factors can gain access to S{gamma}3 DNA and lead to a remodeling of S{gamma}3 chromatin that is incompatible with µ->{gamma}3 SR. Based on these observations, we speculate that p50 is crucial in facilitating formation of a nucleoprotein structure spanning the SNIP and SNAP recognition motifs, which prevents the binding of factors that alter chromatin and lead to a dysfunctional S DNA configuration.


   Acknowledgments
 
We thank H. Singh and J. Stavnezer for helpful discussions, C. Snapper for cytokines, E. Max for sharing unpublished genomic DNA sequence, and W. Sha for providing p50-/- and p50+/- mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant GM 57078 and a grant from the Concern Foundation (to A.L.K.). Back

2 Address correspondence and reprint requests to Dr. Amy L. Kenter, Department of Microbiology and Immunology, University of Illinois College of Medicine, 835 South Wolcott Avenue, Chicago, IL 60680. Back

3 Abbreviations used in this paper: SR, switch recombination; S, switch; SNAP, switch nuclear A-site protein; SNIP, switch nuclear B-site protein; {alpha}{delta}dex, anti-IgD Ab conjugated to dextran; nu/nu, nude; RT, reverse transcription/transcriptase; DC, digestion-circularization; nAChR, nicotinic acetylcholine receptor gene; DMS, dimethyl sulfate; LMPCR, ligation-mediated PCR. Back

Received for publication August 15, 2000. Accepted for publication January 23, 2001.


   References
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 Materials and Methods
 Results
 Discussion
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