Switch region identity plays an important role in Ig class switch recombination

Abstract

Ig class switch recombination (CSR) is regulated through long-range intrachromosomal interactions between germline transcript promoters and enhancers to initiate transcription and create chromatin accessible to activation-induced deaminase attack. CSR occurs between switch (S) regions that flank Cmu and downstream C(H) regions and functions via an intrachromosomal deletional event between the donor Smicro region and a downstream S region. It is unclear to what extent S region primary sequence influences differential targeting of CSR to specific isotypes. We address this issue in this study by generating mutant mice in which the endogenous Sgamma3 region was replaced with size-matched Sgamma1 sequence. B cell activation conditions are established that support robust gamma3 and gamma1 germline transcript expression and stimulate IgG1 switching but suppress IgG3 CSR. We found that the Sgamma1 replacement allele engages in micro-->gamma3 CSR, whereas the intact allele is repressed. We conclude that S region identity makes a significant contribution to CSR. We propose that the Sgamma1 region is selectively targeted for CSR following the induction of an isotype-specific factor that targets the S region and recruits CSR machinery.

FIGURE 1

Targeted replacement of the endogenous Sγ3^a region. A, Insertion of Sγ1 into the γ3 locus. Genomic organization of the mouse Sγ3 loci on the targeted a allele (γ3^a) or the WT b allele (γ3^b). Vertical arrow heads R1, EcoRI; open triangles, loxP sites; tk, gene encoding the thymidine kinase; neo, neomycine resistance gene; rectangular blocks, Iγ3 and Cγ3; horizontal arrows, Iγ3 promoter; ovals, S regions (WT or targeted); and rectangular bars, probes for Southern analysis. The arrow at Iγ3 indicates the physiological orientation of transcription. B, Southern blot analysis of genomic DNA digested with EcoRI and hybridized with the 5′ probe (lanes 1–4) or 3′ probe (lanes 5 and 6). The 5′ probe on F1 ES cells detects 18-kb bands that represent the endogenous γ3 locus from both the a and b alleles. Following Cre-mediated deletion of the neo gene, the 5′ but not the 3′ homology arm is further reduced in size from 6.3-4.5 kb.

FIGURE 2

Expression of γ3 GLT from intact and Sγ1 replacement alleles. GLT γ3, γ1, and AID expression were analyzed by semiquantitative RT-PCR using cDNAs derived from purified B cells or splenocytes that were either unstimulated (U) or activated with LPS (L) alone or LPS plus IL-4^lo (1 ng/ml) or LPS plus IL-4^hi (10 ng/ml) for 48 h. RT-PCR analyses of the Gapd or Hprt transcripts were used as loading controls. A, Purified B cells were unstimulated or stimulated and analyzed as indicated. B, WT F1 and chimeric (Chi 28) splenocyte cultures, activated as indicated, and then analyzed for γ3 or γ1 GLT expression. C, LPS-activated WT F1 and chimeric (Chi D, E, F) splenocyte cultures are analyzed for γ3 GLTs. Allele-specific products were distinguished by PstI digestion, which generates 180- and 125-bp restriction fragments derived from the C57/B6 (b) and 129 (a) alleles, respectively. Samples in lanes 3–7 were digested with PstI. Samples are m.w. marker, M (lane 1), intact γ3 GLT (lane 2), and γ3 GLT from the I29 a allele (lane 3), the C57BL/6 b allele (lane 4), the 129 × C57BL/6 (F1) a and b alleles (lane 5), and Chi mice (lanes 6–8). D, WT F1 and chimeric (Chi 1) splenocyte cultures activated as indicated were assessed for allele-specific γ3 GLT by RT-PCR followed by Southern blot analysis.

FIGURE 3

Active μ→γ3 CSR in Sγ1 replacement splenocytes following LPS plus IL-4 induction. Purified B cells (A) or splenocytes from C57BL/6 × 129 (WT-F1) or Sγ1 replacement chimeric mice (B) were cultured for 5 d with LPS (L) in the presence or absence of IL-4^lo or IL-4^hi, as indicated. PST Iμ-Cγ3 and Iμ-Cγ1 were analyzed by semiquantitative RT-PCR. RT-PCR analyses of the Gapd or Hprt transcripts were used as loading controls. A, Purified B cells were unstimulated (U) or stimulated and analyzed as indicated. B, WT-F1 and chimeric (Chi D, E, F) splenocyte cultures, activated as indicated, and then analyzed for γ3 or γ1 PST expression.

FIGURE 4

DC-PCR analysis demonstrates that μ→γ3 CSR occurs on the Sγ1 replacement allele in response to LPS plus IL-4 activation. A, The DC-PCR strategy for detection of CSR at the endogenous γ3 locus is schematically illustrated. EcoRI sites (RI) flank the 5′ and 3′ ends of the S regions. Genomic DNA is digested with EcoRI and ligated under low concentration conditions that favor fragment circularization. The positions and orientations of the nested primer sets are shown postligation. The nAChR gene is used as a control. B, Allele-specific μ→γ3 DC-PCR is demonstrated using allele-specific primers and genomic DNA from the 129 a allele (μ→γ3^a) or C57BL/6 b allele (μ→γ3^b) mice. C and D, DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4^lo or IL-4^hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles. C, Allele-specific μ→γ3^a (WT) or μ→γ3^a(KI) (Sγ1 replacement allele) and μ→γ3^b (intact b allele) and μ→γ1 (WT a and b alleles) DC-PCR products. Templates were concentrated 5-fold and reanalyzed for the intact b allele as indicated (μ→γ3^bp). D, Allele-specific DC-PCR products μ→γ3^a(KI), μ→γ3^b, or μ→γ1 for Chi 1.

FIGURE 5

S/S junction frequency analysis indicates IL-4–induced targeting of the γ3^a(KI) allele for μ→γ3 CSR. Splenocytes were stimulated with LPS in the presence or absence of IL-4^lo for 5 d. S-S junctions from WT F1 (A) and chimeric (B) mice were amplified from genomic DNA using primers located 5′ of Sμ and in the Sγ3-Cγ3 intron as indicated by the arrows under each locus schematic. The locations of the recombination breakpoints were determined by DNA sequence analysis of the cloned PCR products and are indicated by the symbols beneath the locus diagrams. S-S junction sequences are shown in Supplemental Figs. 2 and 3 and are analyzed in Table I. In the Sγ1 replacement allele, indicated as γ3^a(KI), the remaining loxP site is shown by the black triangle upstream of the Sγ1 replacement region.