The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

Immunoglobulin heavy-chain class-switch recombination (CSR) occurs between highly repetitive switch sequences located upstream of the constant region genes. However, the role of these sequences remains unclear. Mutant mice were generated in which most of the I-C intron was deleted, including all the repeats. Late B-cell development was characterized by a severe impairment, but not a complete block, in class switching to all isotypes despite normal germ line transcription. Sequence analysis of the I-C intron in in vitro activatedmutant splenocytes did not reveal any significant increase in activation-induced cytidine deaminase (AID)-induced somatic mutations. Analysis of switch junctions showed that, in the absence of any S repeat, the I exon was readily used as a substrate for CSR. In contrast to the sequence alterations downstream of the switch junctions, very few, if any, mutations were found upstream of the junction sites. Our data suggest that the core E enhancer could be the boundary for CSRassociated somatic mutations. We propose that the core E enhancer plays a central role in the temporal dissociation of somatic hypermutation from class switching. ( IntroductionDuring B-cell development, the immunoglobulin (Ig) locus is the site of 2 types of rearrangements: V(D)J assembly that generates the variable (V) region exons at the heavy-and light-chain loci and class-switch recombination (CSR) at the heavy-chain (IgH) locus. Upon antigen challenge, mature B cells expressing IgM and/or IgD undergo diversification processes that affect both the V and the constant (C) genes. Point mutations and occasional insertions and deletions are introduced in the V regions during somatic hypermutation (SHM) and gene conversion eventually resulting in higheraffinity receptors. CSR specifically affects C genes through a deletional process whereby a downstream C-region gene is brought to proximity of a rearranged VDJ gene, allowing expression of one of the downstream isotypes (IgG, IgE, or IgA). 1,2 CSR generally occurs between highly repetitive, G-rich switch (S) sequences located upstream of all the C genes except C␦. S sequences differ both in size and in the nature of the repeats. In the mouse, S, S⑀, and S␣ are composed of pentameric tandem repeats such as GGGGT, GGGCT, and GAGCT, while S␥ sequences, which also contain these elements, consist of repeats of a 49-base pair (bp) sequence. 3 In addition, S sequences bind several protein complexes but the physiologic consequences of this binding are poorly understood. 2 CSR involves DNA breaks within partner S sequences followed by repair and ligation through a nonhomologous end-joining (NHEJ) mechanism with looped-out deletion of the intervening DNA. The final steps of CSR involve components of the general DNA repair machinery as well as mismatch-repair mechanisms. In contrast, the early steps requiring recognition and cleavage of S DNAs are still unclear. Both double-strand breaks and staggered single-strand breaks have been involved in the early steps of CSR. 4,5 Frequent mu...

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3′ regulatory region (3′RR) in a stimulus-dependent manner. Why the 3′RR-mediated upregulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3′RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3′RR in developing B cells. E xpression of complex loci is developmentally programmed or induced by specific stimuli and is often controlled by distant regulatory elements within relatively large chromatin domains. Transcriptional and architectural factors play an important role in the establishment and maintenance of these domains and facilitate long-range interactions between regulatory elements and target promoters (1, 2). The Ig heavy chain (IgH) locus is expressed in a lineage-and developmental stage-dependent manner. Various cis-acting elements including promoters, enhancers, and insulators control IgH locus expression and are engaged in multiple long-range interactions (3, 4).Factors such as YY1, PAX5, IKAROS, CTCF, and Cohesin play important roles in various aspects of long-range events at the IgH locus, including V(D)J recombination, CSR, and promoter/enhancer and enhancer/enhancer interactions (3-6). Multiple CTCF binding elements (CBEs) were reported along the IgH locus. The majority of these CBEs lie within the variable domain (7), and two CBEs were identified within the V H -D intergenic region (7-9). At the 3′ end of the locus, ∼10 CBEs were identified downstream of the 3′RR and are thought to delineate the 3′ border of the IgH locus (10). More recently, a discrete CBE was identified within the α constant gene (11), but its role in vivo is presently unknown.Upon antigen challenge, mature B cells can undergo CSR that allows B cells to change the heavy-chain constant domain of an IgM to IgG, IgE, or IgA. CSR to a particular isotype is induced by specific external stimuli, including antigens, mitogens, cytokines, and intercellular interactions. CSR is mediated by highly repetitive sequences called switch (S) sequences located upstream of the constant exons and is preceded by germline (GL) transcription of the S sequences that originates from GL promoters, named I promoters (12).The 3′RR is composed of four enhance...