V DJ Ce
isotype switching to proceed. Germline transcripts are found at both the | locus and the downstream heavy chain locus to which an activated B cell is being induced to switch. At each participating switch region, the germline transcript facilitates the generation of DNA double-stranded breaks, as described later. The DNA break in the upstream (| ) switch region is joined to the break in the downstream selected switch region. As a result, the rearranged VDJ exon just upstream of the | switch region in the IgM-producing B cell recombines with the transcriptionally active downstream switch region. Cytokines determine which CH region will undergo germline transcription. For instance, IL-4 induces germline transcription through the Ie-Se-Ce locus (see Fig. 11-15). This leads first to the production of germline e transcripts in an IgM-expressing B cell and then to recombination of the S| switch region with the Se switch region. The intervening DNA is lost, and the VDJ exon is thus brought adjacent to Ce. The end result is the production of IgE with the same V region as that of the original IgM produced by that B cell.
The key enzyme required for isotype switching (and somatic mutation, described later) is activation-induced deaminase (AID). Humans and knockout mice lacking this enzyme have profound defects in isotype switching and affinity maturation. AID expression is activated mainly by CD40 signals. The enzyme deaminates cyto-sines in single-stranded DNA templates, converting cyto-sine (C) residues to uracil (U) residues (Fig. 11-16). Switch regions are rich in G and C bases, and switch region transcripts tend to form stable DNA-RNA hybrids involving the coding (top) strand of DNA, thus freeing up the bottom or nontemplate strand, which forms an open single-stranded DNA loop called an R-loop. The R-loop is where a large number of C residues in the switch DNA sequence are converted to U residues by AID. An enzyme called uracil N-glycosylase removes the U residues, leaving abasic sites. The ApeI endonuclease and probably other endonucleases cleave these abasic sites, generating a nick at each position. Some nicks are generated on the upper strand as well in an AID-dependent manner, but it is less clear how that happens. Nicks on both strands contribute to double-stranded breaks both at the S| region and at the downstream switch locus that is involved in a particular isotype switch event. The existence of double-stranded breaks in two switch regions results in the deletion of the intervening DNA and joining together of the two broken switch regions by use of the machinery involved in double-stranded break repair by nonhomologous end joining. This machinery is also used to repair double-stranded breaks during V(D)J recombination (see Chapter 8).
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