NF-kappa B p50-dependent in vivo footprints at Ig S gamma 3 DNA are correlated with mu-->gamma 3 switch recombination

Abstract

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 mu-->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 gamma3 germline transcripts in p50-deficient B cells, but mu-->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.

FIGURE 1

Analysis of γ3 germline transcript expression in p50^+/– and p50^–/– mitogen-activated B cells. A, γ3 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 aαδdex plus IL-5 or LPS plus αδdex plus IL-5 plus IL-4 plus TGF-β were detected by RT-PCR. The intensity of the signal derived from GAPDH RT-PCR was used for normalization. B, The RT-PCR of γ3 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.

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 × 10⁵ cells/ml, activated with LPS plus αδdex plus IL-5 plus IL-4 plus TGF-β or with addex 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.

FIGURE 3

DC-PCR analysis of μ→γ3 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/Sγ3 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/γ3 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 Sγ/Sγ3 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 αδdex plus IL-5 or LPS plus αδdex plus IL-5 plus IL-4 plus TGF-β as indicated. Detection of the Sμ/ Sγ1 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.

FIGURE 4

In vivo footprint analysis of Sγ3 DNA in nu/nu, p50^+/–, and p50^–/– B cells. A, A partial restriction map of the Sγ3 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 Sγ3 and above the line for the coding strand at the 3′ end of Sγ3. 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 Image-Quant software (Molecular Dynamics). B and C, In vivo footprinting was performed on the coding and noncoding strands of Sγ3 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-γB. 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 Sγ3 sequence MUSIGHANA. Boxes indicate the positions of the SNIP and SNAP binding motifs. Residues that are strongly (>30%; ●) or moderately (20–30%; ○) 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 un-stimulated nu/nu (D) and unstimulated p50^–/– (E) splenic B cells.

FIGURE 5

Quantitative comparison of the aberrant Sγ3 footprints observed in p50^+/– and p50^–/– resting splenic B cells. A, Densitometry traces of the LMPCR results (shown in Fig. 4B) 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. 4C) 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 hyper-methylated (*) 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 un-stimulated 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.