Complex regulation of somatic hypermutation by cis-acting sequences in the endogenous IgH gene in hybridoma cells

Abstract

To create high-affinity antibodies, B cells target a high rate of somatic hypermutation (SHM) to the Ig variable-region genes that encode the antigen-binding site. This mutational process requires transcription and is triggered by activation-induced cytidine deaminase (AID), which converts deoxycytidine to deoxyuridine. Mistargeting of AID to non-Ig genes is thought to result in the malignant transformation of B cells, but the mechanism responsible for targeting SHM to certain DNA regions and not to others is largely unknown. Cis-acting elements have been proposed to play a role in directing the hypermutation machinery, but the motifs required for targeting SHM have been difficult to identify because many of the candidate elements, such as promoters or enhancers, are also required for transcription of Ig genes. Here we describe a system in cultured hybridoma cells in which transcription of the endogenous heavy-chain Ig gene continues in the absence of the core intronic enhancer (E) and its flanking matrix attachment regions (MARs). When AID is expressed in these cells, SHM occurred at the WT frequency even when E and the MARs were absent together. Interestingly, SHM occurred at less than the WT frequency when E or the MARs were individually absent. Our results suggest that these intronic regulatory elements can exert a complex influence on SHM that is separable from their role in regulating transcription.cis-acting elements ͉ enhancer ͉ matrix attachment region D uring the adaptive immune response, the variable regions (V regions) of Ig genes undergo somatic hypermutation (SHM) to generate the high-affinity antibodies required to protect against pathogenic organisms. SHM depends on the targeting of activation-induced cytidine deaminase (AID) to the V regions of heavy and light chain genes, whereas the constant regions (C regions) of the Ig genes are protected from high rates of SHM. AID is expressed primarily in centroblast B cells in the germinal centers of secondary lymphoid organs; thus, its restricted expression spares other cells from its mutagenic effects (reviewed in ref. 1). In centroblast B cells, some highly expressed non-Ig genes do not undergo SHM, indicating that a high rate of transcription is not sufficient to make a gene accessible to AID (2). AID can also cause mutations in some highly expressed non-Ig genes, including many protooncogenes, especially in tumor cells (3-5), whereas ubiquitous expression of AID in mice resulted in tumorigenesis (6). It is therefore important to understand the mechanisms that are responsible for the preferential targeting of AID and its associated proteins to the V regions, not only because SHM is required for affinity maturation during the antibody response but also because the mistargeting of AID to non-Ig genes is thought to be associated with malignant transformation.SHM is dependent on and roughly proportional to the rate of transcription of the targeted genes in vivo and in cultured cells (7-9). Furthermore, SHM begins just downstream from the transcription st...

Forced expression of AID facilitates the isolation of class switch variants from hybridoma cells

“…We sequenced the heavy and light-chain variable regions and found that additional mutations had not accumulated, which was surprising. However, since AID is able to mutate itself, as previously reported (Martin et al, 2002b;Ronai et al, 2005), we sequenced the AID gene in these class switch variants and found that they all had the same premature stop codon (Y13*) that led to a loss of the AID enzymatic activity.…”

In a model of immunoglobulin heavy‐chain (IGH)/MYCtranslocation, theIgh3′ regulatory region inducesMYCexpression at the immature stage of B cell development

“…Both play a role in driving appropriate levels of IGH gene expression during different stages in B cell development, and it is probable that these two regulatory regions influence SHM, although how they do so remains unclear (Arulampalam et al, 1997;Stevens et al, 2000;Shi and Eckhardt, 2001;Terauchi et al, 2001;Morvan et al, 2003;Perlot et al, 2005;Ronai et al, 2005). What is clear is that errors take place in restricting these gene-damaging processes to the IGH locus.…”

Somatic Hypermutation and Junctional Diversification at Ig Heavy Chain Loci in the Nurse Shark

“…Only experiments involving the removal of the Ig enhancer and the nuclear matrix attachment region (MAR) 3 components affect the ability of the region to be mutated (11)(12)(13). The role of the intronic enhancer/MARs in hypermutation is not clear but it seems to be one separable from transcription regulation (14). It has been speculated that these (or other) cis-elements are involved in the recruitment of various factors, including activation-induced cytidine deaminase (AID) 3 and replication protein A, assembling a "mutasome" that most likely induces DNA lesions which become subject to error-prone repair (12,(15)(16)(17).…”