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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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Transgenic IgG expression in vitro. Supernatants of cultured B cells (with cytokines as indicated in each panel) from various transgenic and nontransgenic mice were tested. (A) Transgenic Flag+ IgG2a expression; (B) transgenic IgG1a expression; and (C) transgenic AD8+ (idiotype+) IgA expression. Each data point represents an independent culture, derived from independent transgenic mice. In this and subsequent figures, the 774 line and 820 line were tested in separate experiments from the 336 line, the 234 line, the 231 line, and the 761 mice, as the latter four mice were established 1 yr later.
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fig2: Transgenic IgG expression in vitro. Supernatants of cultured B cells (with cytokines as indicated in each panel) from various transgenic and nontransgenic mice were tested. (A) Transgenic Flag+ IgG2a expression; (B) transgenic IgG1a expression; and (C) transgenic AD8+ (idiotype+) IgA expression. Each data point represents an independent culture, derived from independent transgenic mice. In this and subsequent figures, the 774 line and 820 line were tested in separate experiments from the 336 line, the 234 line, the 231 line, and the 761 mice, as the latter four mice were established 1 yr later.

Mentions: B cells from each transgenic line with an intact heavy chain transgene-secreted transgenic IgG1, IgG2a, and IgA after culture with the appropriate inducing agents (Fig. 2). The amount of antibody secreted varied, in part because of the exact inducing agent used, but mostly because of unavoidable variation in the B cell cultures. However, B cells from mice with deletion of the 3′ enhancers from the transgene (820Δ, 774Δ, etc.) rarely secreted amounts of transgenic IgG2a and IgG1 above that of control nontransgenic B cells (Fig. 2, A and B). The one exception to this was IgA; specifically, for two of the transgenic lines, 336Δ and 234Δ (each with two copies of the transgene), transgenic B cells sometimes expressed transgenic IgA amounts that were slightly in excess of the amounts secreted by control, nontransgenic B cells (Fig. 2 C). Supernatant fluids from B cells from both Δ mice and nontransgenic mice were shown to express significant amounts of endogenous Ig (Fig. S3) and supernatants from Δ mice expressed transgenic IgM (not depicted), demonstrating that these B cells underwent activation and differentiation.


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)

Transgenic IgG expression in vitro. Supernatants of cultured B cells (with cytokines as indicated in each panel) from various transgenic and nontransgenic mice were tested. (A) Transgenic Flag+ IgG2a expression; (B) transgenic IgG1a expression; and (C) transgenic AD8+ (idiotype+) IgA expression. Each data point represents an independent culture, derived from independent transgenic mice. In this and subsequent figures, the 774 line and 820 line were tested in separate experiments from the 336 line, the 234 line, the 231 line, and the 761 mice, as the latter four mice were established 1 yr later.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806627&req=5

fig2: Transgenic IgG expression in vitro. Supernatants of cultured B cells (with cytokines as indicated in each panel) from various transgenic and nontransgenic mice were tested. (A) Transgenic Flag+ IgG2a expression; (B) transgenic IgG1a expression; and (C) transgenic AD8+ (idiotype+) IgA expression. Each data point represents an independent culture, derived from independent transgenic mice. In this and subsequent figures, the 774 line and 820 line were tested in separate experiments from the 336 line, the 234 line, the 231 line, and the 761 mice, as the latter four mice were established 1 yr later.
Mentions: B cells from each transgenic line with an intact heavy chain transgene-secreted transgenic IgG1, IgG2a, and IgA after culture with the appropriate inducing agents (Fig. 2). The amount of antibody secreted varied, in part because of the exact inducing agent used, but mostly because of unavoidable variation in the B cell cultures. However, B cells from mice with deletion of the 3′ enhancers from the transgene (820Δ, 774Δ, etc.) rarely secreted amounts of transgenic IgG2a and IgG1 above that of control nontransgenic B cells (Fig. 2, A and B). The one exception to this was IgA; specifically, for two of the transgenic lines, 336Δ and 234Δ (each with two copies of the transgene), transgenic B cells sometimes expressed transgenic IgA amounts that were slightly in excess of the amounts secreted by control, nontransgenic B cells (Fig. 2 C). Supernatant fluids from B cells from both Δ mice and nontransgenic mice were shown to express significant amounts of endogenous Ig (Fig. S3) and supernatants from Δ mice expressed transgenic IgM (not depicted), demonstrating that these B cells underwent activation and differentiation.

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