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Switch recombination and somatic hypermutation are controlled by the heavy chain 3' enhancer region.

Dunnick WA, Collins JT, Shi J, Westfield G, Fontaine C, Hakimpour P, Papavasiliou FN - J. Exp. Med. (2009)

Bottom Line: Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon.In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced.Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48103, USA.

ABSTRACT
Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

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Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.
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fig6: Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.

Mentions: Past work has shown that elements within the 3′ enhancer region (specifically the HS3B and HS4 sites) were dispensable for SHM (Le Morvan et al., 2003). To determine whether deletion of the entire 28-kb enhancer region had an effect on mutation, we immunized our transgenic mice with ARS-KLH, and isolated GL7+/Fas+ (germinal center) B cells from their spleens 12 d after immunization. DNA from sorted B cells was used to amplify the VDJ exon and 3′ flanking sequences, which were then cloned and sequenced. In contrast to most heavy chain transgenes which do not mutate at wild-type levels (Giusti and Manser, 1993; Unniraman and Schatz, 2007), our BAC transgenics were fully competent for SHM, accumulating mutations at a rate of 0.3–0.8% (Fig. 6). Deletion of the enhancers had a major effect on the frequency of mutations. For example, the frequency of mutation in 820 VDJ and flanking region dropped from ∼0.46–0.06%, in the absence of the enhancers; this is close to the background rate of mutation caused by polymerase error during amplification (∼0.02% or 2/10,000 nt). Similarly, the frequency of mutation near the VDJ in the 336 line dropped from 0.36% to 0.08%, and in the 774 line dropped from 0.72% to 0.23%. In each set of paired transgenic lines, the number of clones with multiple mutations was also reduced in the absence of the enhancers (Fig. 6).


Switch recombination and somatic hypermutation are controlled by the heavy chain 3' enhancer region.

Dunnick WA, Collins JT, Shi J, Westfield G, Fontaine C, Hakimpour P, Papavasiliou FN - J. Exp. Med. (2009)

Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.
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fig6: Somatic hypermutation of transgenic VDJ exons and flanking sequences. Part of the transgenic VDJ exon and 3′ flanking sequences (from germinal center B cells) were amplified, cloned, and sequenced. Mutations in the 3′ 171 bp of VDJH2 exon and 329 bp of 3′ flanking sequences are presented as pie charts, in which the sectors of the pie are proportional to the number of sequences with a given number of mutations. The number of sequences is shown in the center of the pie chart, and the number of mutations in some sectors is noted. For lines 820, 820Δ, 774, and 774Δ, the data represent a compilation of sequences from two sorted cell samples derived from independent mice. For lines 336 and 336Δ, the data are derived from one sorted cell sample each; a second set of sequences from the same cell sample yielded 0.21% mutations in line 336 DNA and 0.02% mutations in line 336Δ DNA. For all three pairs of transgenic lines tested, the mutation frequency is greater in intact versus the enhancer-deleted line, P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). Mutation frequencies in the intronic 329-bp were: line 820, 0.36% (27 mutations in 7557 nt); line 820Δ, 0.03% (4 mutations in 13,818 nt); line 774, 0.40% (43 mutations in 10,857 nt); line 774Δ, 0.11% (14 mutations in 12,831 nt); line 336, 0.16% (44 mutations in 27,636 nt); and 336Δ, 0.03% (9 mutations in 30,268 nt). For each set of paired transgenic lines, the difference in intronic mutations was significant at P < 0.0001 (two-tailed χ2 test, with Yate’s correction and one degree of freedom). In separate experiments, transgenic V region mutations were also determined in cloned mRNA (VDJCγ1, VDJCγ2a, and VDJCγ2b pooled sequences) from spleens of immunized line 820 mice (see text). The 820 VDJ exon had 1.67% mutations (128 mutations in 7680 nt sequenced from 30 clones). The 820 Cγ region had 0.1% mutations (10 mutations in 10,041 nt sequenced in the same 30 clones). By a two-tailed χ2 test, with Yate’s correction and one degree of freedom, the difference in mutation frequency in V and C is significant at P < 0.0001.
Mentions: Past work has shown that elements within the 3′ enhancer region (specifically the HS3B and HS4 sites) were dispensable for SHM (Le Morvan et al., 2003). To determine whether deletion of the entire 28-kb enhancer region had an effect on mutation, we immunized our transgenic mice with ARS-KLH, and isolated GL7+/Fas+ (germinal center) B cells from their spleens 12 d after immunization. DNA from sorted B cells was used to amplify the VDJ exon and 3′ flanking sequences, which were then cloned and sequenced. In contrast to most heavy chain transgenes which do not mutate at wild-type levels (Giusti and Manser, 1993; Unniraman and Schatz, 2007), our BAC transgenics were fully competent for SHM, accumulating mutations at a rate of 0.3–0.8% (Fig. 6). Deletion of the enhancers had a major effect on the frequency of mutations. For example, the frequency of mutation in 820 VDJ and flanking region dropped from ∼0.46–0.06%, in the absence of the enhancers; this is close to the background rate of mutation caused by polymerase error during amplification (∼0.02% or 2/10,000 nt). Similarly, the frequency of mutation near the VDJ in the 336 line dropped from 0.36% to 0.08%, and in the 774 line dropped from 0.72% to 0.23%. In each set of paired transgenic lines, the number of clones with multiple mutations was also reduced in the absence of the enhancers (Fig. 6).

Bottom Line: Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon.In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced.Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48103, USA.

ABSTRACT
Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

Show MeSH