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

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 ∼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.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

“…It should be pointed out that the site-independent expression of IgH⌬3alx3b was not dependent upon the introduction of heterologous insulator sequences. In studies by others, the chicken ␤-globin locus-derived insulator was inserted at the 5Ј end of an Igh BAC to assist in generating integration site-independent expression (4). The authors noted, however, that another BAC modification that may have explained better expression of this BAC relative to earlier versions was removal of an upstream D H element, preventing deletion of the assembled V H by D H -J H recombination.…”

“…The authors noted, however, that another BAC modification that may have explained better expression of this BAC relative to earlier versions was removal of an upstream D H element, preventing deletion of the assembled V H by D H -J H recombination. As described above, IgH⌬3alx3b was designed to preclude B1-8V H deletion by D H -J H rearrangement as well, suggesting that this, rather than the presence of the ␤-globin locus-derived insulator, improved expression in the prior study (4). Notably, CTCF-associated insulator sequences lie just downstream of hs4 (11).…”

“…Most notably, deletion of the pair of elements hs3b and hs4 (Fig. 1A, 3b and 4), had a profound effect on CSR to all classes except IgG1, and deletion of the 28 kb spanning hs3a, hs1.2, hs3b, and hs4 dramatically reduced CSR to IgG1 as well (2)(3)(4)(5). Chromosome conformation capture techniques have revealed a cytokine-induced, tripartite interaction involving the Ig transcription unit, the relevant intronic promoter for germline transcripts, and the 3ЈRR, providing physical evidence for the role of 3ЈRR in the classswitch recombination process (19).…”

“…Several studies have shown that the 3Ј regulatory region (3ЈRR) plays an important role in class-switch recombination, implicating it in this recruitment process (2)(3)(4)(5). The 3ЈRR is a series of DNase hypersensitivity sites, initially identified as enhancer-like elements, that map downstream of the Ig heavy chain constant region genes (6 -11) (see Fig.…”

The IgH 3′ regulatory region and its implication in lymphomagenesis

“…Indeed, combined deletion of both hs3b and hs4 affected CSR as a consequence of impairment of the Ig constant gene germline transcription to most isotypes (except g1) [16]. Recently the complete deletion of the 3 0 RR in large transgenes [17] or in the endogenous locus [18] showed that it is a master control element of CSR in all isotypes. Endogenous 3 0 RRdeficient mice clarified that 3 0 IgH enhancers play their most crucial role at the late stages of B-cell development.…”