MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1714-1718 0270-7306/90/041714-05$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 10, No. 4

Detection of an Immunoglobulin Switch Region-Specific DNABinding Protein in Mitogen-Stimulated Mouse Splenic B Cells ROBERT A. WUERFFEL, ASHER T. NATHAN,+

AND

AMY L. KENTER*

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60680 Received 20 September 1989/Accepted 7 December 1989

We have detected a nuclear protein from lipopolysaccharide- and dextran sulfate-stimulated mouse splenic B cells which binds specifically to the immunoglobulin switch ,u (S,u) sequence. We have termed the binding protein NF-S,u. DNA containing the S,u repeated sequence, GAGCTGGGGTGAGCTGAGCTGAGCT, was used as a probe in electrophoretic mobility shift assays. Methylation interference analysis indicated that binding centers on the run of four guanine residues. Competitions with mutated S,u sequences confirmed the importance of the run of G residues and revealed that optimal binding occurs when they are flanked by GAGCT. The kinetics of the expression of NF-S,u in splenic B cells treated with lipopolysaccharide and dextran sulfate parallels the induction of recombinational activity at S,u in these cells. On the basis of these data, we suggest that NF-S,u may be an effector of switch recombination.

When B cells are stimulated by antigen and the appropriate lymphokines, they produce antibodies of new isotypes while maintaining the original antigen-binding specificity (2,

ted for publication). This intra-S,u recombination is exquisitely regulated, occurring during the S phase of the cell cycle (15; Kenter et al., submitted). We have considered the possibility that DNA-binding proteins are effector molecules of switch recombination. We report here the detection of a protein, NF-S,i, which is induced in mitogen-stimulated mouse splenic B cells and specifically binds to S,u DNA.

3, 8). This phenomenon, the immunoglobulin heavy-chain (IgH) class switch, permits expression of a mature variable (V) region with constant (C) regions other than C,u (for a review, see reference 10). The isotype switch occurs by a DNA rearrangement, which brings one of six downstream CH genes (26) proximal to the V gene. This results in the deletion of the C,u gene and the intervening genomic material (11, 18, 25). The recombination event focuses on switch (S) DNA, regions of tandemly repetitive sequence located upstream of each CH gene, with the exception of the CB gene (23), and produces a new hybrid DNA combination (Spu-S') (5, 12, 20). Sequence analyses of the switch recombination joints of switched IgH genes have revealed that the SF. (donor) breakpoints fall within or upstream of the tandem repeats (13, 19). In other S regions (acceptors) the breakpoints fall within the tandemly repeated sequence (13, 20-22, 28). There are no obvious consensus recombination signal sequences, although the pentamers GAGCT, GGGGT, and GGTGG, components of all the switch regions, are often found at or close to recombination joints (21). Sequence comparison of S DNAs has shown that while all are highly repetitive, there has been significant sequence divergence between them (12, 20). There are two views on the mechanism of switch recombination. The first emphasizes the sequence divergence of S regions and suggests that there are switch region-specific recombinases which are responsible for the recombination (5). The second highlights the short nucleotide repeats which are common to all the switch regions and suggests that switch recombination is due to homologous recombination (12, 20). Little, however, is known regarding the molecular details of this mitotic recombination event. Mitogen stimulation of resting splenic B cells induces proliferation (4) and a high frequency of intra-S,u recombination (A. L. Kenter, J. V. Watson, and K. Bauer, submit-

MATERIALS AND METHODS Cells and culture conditions. Single-cell suspensions prepared from the spleens of BALB/c mice were stimulated in culture with lipopolysaccharide (LPS) and dextran sulfate (DxS) as previously described (16). It has been established by immunofluorescence that B cells constitute 93% of these cultures at 48 h (Kenter et al., submitted). Nuclear extracts. Nuclear extracts were prepared by a modification of the procedure of Dignam et al. (6). Cells (2 x 108 to 5 x 108) were harvested and washed in cold phosphate-buffered saline, suspended in 1.3 ml of swelling buffer (10 mM Tris hydrochloride [pH 7.5], 10 mM NaCl, 5 mM MgCl2), and kept on ice for 30 min. The cells were then transferred to a Dounce homogenizer containing 0.7 ml of lysis buffer (10 mM Tris hydrochloride [pH 7.5], 10 mM NaCI, 5 mM MgCl2, 5% Nonidet P-40) and homogenized with 25 strokes of a type B pestle. The cell lysate was centrifuged through a sucrose cushion (25 mM Tris hydrochloride [pH 7.5], 5 mM MgCl2, 250 mM sucrose). The pelleted nuclei were suspended and lysed in 0.8 ml of buffer C (20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.9], 25% glycerol, 0.42 M KCI, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 p,g of leupeptin per ml, 0.5 mM dithiothreitol) and stirred gently on a magnetic stirrer for 30 min at 4°C. The nuclear lysate was then treated as described by Dignam et al. (6). Protein concentrations were determined by the microassay procedure with the protein assay dye from Bio-Rad Laboratories, Richmond, Calif. Preparation of oligonucleotides, plasmids, and probes. The S,u region is approximately 3 kilobases long and is composed almost entirely of tandem repititions of (GAGCT), (GGGGT), where the most common value of n is 3 (19).

* Corresponding author. t Present address: Department of Genetics, The Hebrew University, Jerusalem, Israel.

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A. Probes AGCTTGCATGCCTGCAGGTCGACTCTAGAGGATC

AGCTTGCATGCCTGCAGGTCGAGCTGGGGTGAGCTGAGCTGAGCTGACTCTAGAGGATC

B. Competitors Sp oligomer

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FIG. 1. (A) S,u- and S,u+ probes used in the mobility shift and competition assays. pl.S,u- and pl.S,u+ were prepared as described in Materials and Methods. The bracket shows the position of the S,u sequence. (B) Seven chemically synthesized oligomers used in competition experiments. Dashes indicate positions of identity with the S,u oligomer sequence.

Oligonucleotides corresponding to the major S,u repeated sequence and variations of this sequence (Fig. 1B) were chemically synthesized with a model 8700 DNA synthesizer (MilliGen/Biosearch, Burlington, Mass.). Concentrations of the double-stranded oligomers were determined spectrophotometrically, under conditions where a concentration of 20 ,ug/ml yields an optical density at 260 nm of 1.0. The double-stranded SF oligomer was cloned into pUC19 at the HincIl site. Orientation of the sequence was verified by DNA sequencing. This recombinant plasmid served as the source of all S,u+ probes used in these studies. Probes were labeled with ct-32P-deoxyribonucleotides to a specific activity of 5 x 107 cpm/,ug by the Klenow fill-in reaction and then gel purified. Probes pl.S,u+ and pl.S,u- were generated by cutting the recombinant plasmid and pUC19, respectively, with BamHI and HindIll (Fig. 1A). The probe for methylation interference analysis was derived from the EcoRI-SphI fragment of the recombinant plasmid. The radioactivity of the probes was measured by Cerenkov counting. Gel mobility shift assay. The specific binding activity was detected as described by Singh et al. (27). All bindingreaction mixtures contained 2 jig of poly(dI-dC) to counteract nonspecific interactions between probe and extract proteins. In competition experiments, specific competitor DNAs were added 5 min before the probe. In all cases the DNA probe (10,000 cpm) was added last. The complete reaction mixtures were incubated for 15 min at room temperature. DNA-protein complexes were resolved by electrophoresis through a low-ionic-strength polyacrylamide gels as described previously (27). Methylation interference assay. Methylation interference assays were performed as described previously (1). Probe containing the Sji oligomer was labeled on one strand, partially methylated, and then used in scaled-up mobility shift assays. The bound and free DNAs were eluted from the gel and treated with piperidine. Equal numbers of counts of

each DNA sample were run on an 8% sequencing gel and then autoradiographed. Densitometric analyses were performed with a model TR-9OT1G densitometer (LKB Instruments, Inc., Rockville, Md.). RESULTS

The presence of a nuclear protein specific for the S,u tandem repeat was detected by the gel retardation assay (27). When nuclear extracts, prepared from normal splenic B cells stimulated with LPS-DxS for 44 h, were incubated with probe, pl.S,u+ (Fig. IA), containing a cloned 25-base-pair bp oligomer which represents the Sji tandem repeat, a prominant bound band was observed (Fig. 2, lane 1). No retarded complex was seen with an equivalent probe, pl.S,- (Fig. 1A), which lacks the S,u sequence (data not shown). Treatment of the extracts with proteinase K abolished retardation, confirming that the retarded moiety represents a complex of protein and DNA (A. T. Nathan, unpublished results). To examine the specificity of the interaction, we performed competition-binding assays. The Sji oligomer (Fig. 1B) successfully competes with pl.S,u+ for binding (Fig. 2, lanes 2 to 4). To begin delineation of the minimum sequence required for binding, we synthesized oligo.1, a 25-mer with the first 15 bp of the Sji repeat followed by randomly chosen sequences derived from the pUC19 polylinker (Fig. 1B). This oligomer was capable of efficient competition with pl.S,u+ (Fig. 2, lanes 5 to 7). This demonstrates that the recognition motif is located within the first 15 bp of the Sji 25-bp oligomer. The methylation interference procedure was used to map the guanine residues involved in essential protein contacts. The probe (see Materials and Methods) was partially methylated and incubated with nuclear extract, and the bound and free probes were separated by gel retardation electro-

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FIG. 2. Competition assays confirm the specificity of NF-S,u binding. Mobility shift assays were performed in which different unlabeled competitor DNAs (Fig. 1B) were included in binding reactions with the pl.S,u+ probe (10,000 cpm) and activated B-cell nuclear extract (3.75 ,ug). Competitor DNAs were present at 25 ng (lanes 2, 5, 8, 11, 14, and 17), 100 ng (lanes 3. 6, 9. 12, 15, and 18), or 400 ng (lanes 4. 7, 10, 13, 16, and 19). Lane 1 shows the binding reaction in the absence of competitor. Lane 20 shows free probe. B and F indicate the positions of bound and free probe.

phoresis. The DNA recovered from the bound and free bands was then subjected to chemical cleavage at methylated purine residues. Methylation of G residues at positions 7, 8, and 9 markedly interfered with binding (Fig. 3). Guanines at other positions both within and outside the S,u sequence are also involved in protein contacts. However, the guanines at positions 16, 18, 23, and 26 are not critical for stable NF-S,u binding, since oligo.1, which lacks these residues, competes for binding as efficiently as the S,u oligomer. DNA that is methylated at the G-6 residue is overrepresented in the bound band. These results indicate that binding is centered on the G-7, G-8, and G-9 residues

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and suggest that the GAGCT context in which the guanines are embedded may contribute to stable binding. The specificity of the protein-Spl interaction and the importance of the run of G residues was further tested by competition-binding assays with a series of oligomers related to the SrL repeat sequence (Fig. 1B). Oligo.2 was virtually unable to compete for binding even at the highest concentration tested (Fig. 2, lanes 8 to 10). This oligomer carries two mutations, at positions 7 and 9, which convert the string of four guanines to the pentameric repeat, GAGCT. This demonstrates that the sequence (GAGCT)5 is insufficient for binding. Single point mutations which affect the run of G

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FIG. 3. Methylation interference analysis of the interaction between NF-S,u and the SpL repeat sequence. DNA probe, labeled on the plus strand (prepared as described in Materials and Methods), was used in methylation interference assays with 44-h mitogen-stimulated B-cell extract. (A) Autoradiograph showing cleavage products of the bound (B) and free (F) bands of the binding reaction. Maxam and Gilbert G and G+A reaction products (1) were coelectrophoresed to position the contact sites. Residues whose methylation either greatly (0) or moderately (0) interferes with or enhances (*) binding are indicated. (B) Densitometry tracings of the free (upper) and bound (lower) lanes.

IMMUNOGLOBULIN

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FIG. 4. Competition assays to further delineate NF-S,u specificity. Probe pl.SpL' (10,000 cpm) was used in a mobility shift assay along with activated B-cell nuclear extract (8 ,ug). Competitor DNAs were included at 25 ng (lanes 2, 5, and 8), 100 ng (lanes 3, 6, and 9), and 400 ng (lanes 4, 7, and 10). Lane 1 shows binding in the absence of competitor. Lane 11 shows free probe. Abbreviations: B, bound probe; F, free probe.

residues in the S,u repeat, as found in oligo.3 and oligo.4, result in a 4- to 16-fold reduction in binding competition (Fig. 2, lanes 11 to 16). The Chi sequence, GCTGGTGG, enhances general recombination in bacteriophage lambda (17) and is highly related to the S,u tandem repeat (14). It was of interest to determine whether NF-S,u is also capable of binding the Chi sequence. Oligo.5, which contains the Chi permutation, exhibited a 10- to 20-fold-lower binding affinity for NF-S,u than the S,u oligomer did (Fig. 2, lanes 17 to 19). We conclude that Chi sequences in immunoglobulin genes are not recognized by NF-S,u. Furthermore, when the integrity of the string of four G residues is interrupted, specific recognition of the S,u repeat is lost. These results confirm the essential nature of the proteinguanine contacts for stable binding. These data do not rule out that four G residues flanked by thymidines are sufficient for protein binding. To test this, we synthesized oligo.6 to contain TGGGGT flanked by plasmid-derived sequences (Fig. 1B). Three competition assays were performed in which binding specificity was tested with the S,u oligomer, oligo.2, and oligo.6 (Fig. 4). It is clear that oligo.6 competes poorly for the binding of NF-S,u, requiring between 4 and 16 times as much DNA as did the S,u oligomer to achieve a comparable reduction in signal intensity of the bound band (compare lanes 9 and 10 with lane 2). These data demonstrate that optimal NF-S,u binding requires the run of G residues to be within the context of GAGCT pentamers or related sequences. B lymphocytes are induced to proliferate and differentiate upon mitogenic stimulation. Intra-S,u rearrangements, a form of switch recombination, occurs during the S phase of the cell cycle, beginning at 30 to 40 h of activation (Kenter et al., submitted). In this context, it was of interest to determine the kinetics of NF-S,u expression. Nuclear extracts were prepared from splenic lymphocytes which had been cultured in the presence of LPS-DxS for various periods. Equal amounts of total protein from these extracts were used in binding reactions with the pl.S,u+ probe. The mobility

F -3 hours: 0 20 44 68 93 FIG. 5. Kinetics of expression of NF-S,u in LPS-DxS-stimulated splenic lymphocytes. The pl.S,u+ probe was incubated with equal amounts of nuclear extract (5 ,ug) prepared from splenic lymphocytes stimulated with LPS-DxS for either 0, 20, 44, 68, or 93 h (lanes 1 to 5, respectively) and analyzed by mobility shift assay. Free probe is shown in lane 6. Abbreviations: B, bound probe; F, free probe.

shift assay indicates that NF-S,-binding activity, virtually absent in unstimulated splenic lymphocytes, is induced by LPS-DxS activation (Fig. 5). Furthermore, the amount of NF-S,u increases to a peak at around 44 h after stimulation and then diminishes.

DISCUSSION Recombination requires that the two recombining DNA helices be in close apposition. The topological and directional product of the recombination event will be determined by the precise spatial arrangement of the juxtaposed DNA sequences (24, 29). Formation of DNA-multiprotein complexes is a common feature of such processes as transcription, site-specific recombination, and the initiation of DNA replication (7). For example, the Tn3 resolvase brings into close proximity spatially distant DNA sequences which are involved in a site-specific recombinational event (9). Immunoglobulin switch recombination involves DNA rearrangements between sequences separated by 50 to 100 kilobases of genetic material (26). Since switch recombination is clearly focused on switch regions, we hypothesized that some DNA-binding protein factor(s) might be involved in specifically recognizing and facilitating the alignment of switch regions prior to recombination. We have detected an S,u sequence-specific DNA-binding protein, termed NF-S,u, in mitogen-activated B cells. In these studies we used a cloned 25-bp oligomer which contained the pentameric elements of the S,u tandem repeat. Competitive-binding assays suggested that a 15-bp sequence, GAGCTGGGGTGAGCT, was sufficient for recognition by NF-S,u (Fig. 2). A methylation interference assay identified three guanine residues within a string of four as critical protein contact sites. Several other G residues also make contact, and most are located within the 15-bp sequence. The contact sites which reside outside the 15-bp sequence are apparently not critical for NF-S,u binding, since the efficient competitor, oligo.1, lacks those residues.

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This confirms that the recognition motif necessary for NFS,u binding residues in the 15-bp sequence described. Moreover, competitive-binding assays have demonstrated that the integrity of the string of G residues is essential for recognition by NF-S,. Intra-Sp. rearrangements are mitogen induced in splenic B cells during the S phase of the cell cycle (15; Kenter et al., submitted). It is interesting that the kinetics of expression for NF-Sp. precisely parallel the onset of recombinational activity at S,u DNA in these cells. This observation supports the notion that NF-SR. may be an effector of switch recombination in activated B cells. ACKNOWLEDGMENTS We thank Leslie Morgan for expert technical assistance. This work was supported by Public Health Service grant R01 GM39231 to A.L.K. from the National Institutes of Health. A.L.K. is the recipient of an Arthritis Foundation Investigator Award. LITERATURE CITED 1. Baldwin, A. 1987. Methylation interference assay for analysis of DNA-protein interactions, p. 1-6. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl (ed.), Current protocols in molecular biology, unit 12.3. John Wiley & Sons, Inc., New York. 2. Baumhackel, H., B. Liesegang, A. Radbruch, K. Rajewsky, and F. Sablitzky. 1982. Switch from NP-specific IgG3 to IgGl in the mouse hybridoma cell line S24/63/63. J. Immunol. 128:12171220. 3. Burrows, P. D., G. B. Beck-Engeser, and M. R. Wabl. 1983. Immunoglobulin heavy chain class-switching in a pre-B cell is accompanied by DNA rearrangement. Nature (London) 306: 243-246. 4. Cebra, J. J., J. L. Komisar, and P. A. Schweitzer. 1984. CH isotype switching during normal B cell development. Annu. Rev. Immunol. 2:493-548. 5. Davis, M. M., S. K. Kim, and L. E. Hood. 1980. DNA sequences mediating class switching in oL-immunoglobulins. Science 209:

1360-1365. 6. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids 7.

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1988a. Looping out and deletion mechanism for the immunoglobulin heavy chain class switch. Proc. Natl. Acad. Sci. 85:1581-1585. Kataoka, T., T. Miyata, and T. Honjo. 1981. Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes. Cell 23:357-368. Katzenberg, D. R., and B. K. Birshtein. 1988. Sites of switch recombination in IgG2b- and IgG2a-producing hybridomas. J. Immunol. 140:3219-3227. Kenter, A. L., and B. K. Birshtein. 1981. Chi, a promoter of generalized recombination in lambda phage, is present in immunoglobulin genes. Nature (London) 293:402-404. Kenter, A. L., and J. V. Watson. 1987. Cell cycle kinetics model of LPS-stimulated spleen cells correlates switch region rearrangements with S phase. J. Immunol. Methods 97:111-117. Kenter, A. L., J. V. Watson, T. Azim, and T. Rabbits. 1986. Colcemid inhibits growth during early Gl in normal but not tumorigenic lymphocytes. Exp. Cell Res. 167:241-251. Kobayashi, I., M. M. Stahl, and F. W. Stahl. 1984. The mechanism of the Chi-cos interaction in the RecA-RecBC-mediated recombination in phage. Cold Spring Harbor Symp. Quant. Biol. 49:497-506. Marcu, K. B. 1982. Immunoglobulin heavy chain constant region genes. Cell 29:719-721. Nikaido, T., S. Nakai, and T. Honjo. 1981. Switch region of immunoglobulin C,u gene is composed of tandem repetitive sequences. Nature (London) 292:845-848. Nikaido, T., Y. Yamawaki-Kataoka, and T. Honjo. 1982. Nucleotide sequences of switch regions of immunoglobulin CE and CY genes and their comparison. J. Biol. Chem. 257:7322-7329. Petrini, J., and W. Dunnick. 1989. Products and implied mechanism of H chain switch recombination. J. Immunol. 142: 2932-2935. Petrini, J., B. Shell, M. Hummel, and W. Dunnick. 1987. The immunoglobulin heavy chain switch: structural features of -Y1 recombinant switch regions. J. Immunol. 138:1940-1946. Richards, J. E., A. C. Gilliam, A. Shen, P. W. Tucker, and F. R. Blattner. 1983. Unusual sequences in the murine immunoglobulin -u-8 heavy-chain region. Nature (London) 306:483-487. Sadowski, P. 1986. Site-specific recombinases: changing partners and doing the twist. J. Bacteriol. 165:341-347. Shimizu, A., and T. Honjo. 1984. Immunoglobulin class switching. Cell 36:801-803. Shimizu, A., N. Takahashi, Y. Yasita, and T. Honjo. 1982. Organization of the constant-region gene family of the mouse immunoglobulin heavy chain. Cell 28:499-506. Singh, H., R. Sen, D. Baltimore, and P. Sharp. 1986. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature (London) 319:154-158. Szurek, P., J. Petrini, and W. Dunnick. 1985. Complete nucleotide sequence of the murine y3 switch region and analysis of switch recombination sites in two y3-expressing hybridomas. J. Immunol. 135:620-626. Wasserman, S. A., and N. R. Cozzarelli. 1986. Biochemical topology: applications to DNA recombination and replication. Science 232:951-960.

Detection of an immunoglobulin switch region-specific DNA-binding protein in mitogen-stimulated mouse splenic B cells.

We have detected a nuclear protein from lipopolysaccharide- and dextran sulfate-stimulated mouse splenic B cells which binds specifically to the immun...
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