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Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57–65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

The rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes requires the activity of an as yet undefined V(D)J recombinase. One component of the recombinase appears to be a terminal transferase which may be involved in the addition of untemplated nucleotides (N regions) to the V(D)J joints. It has been observed that rearranged Ig and TCR genes isolated from fetal liver have few if any N regions, whereas in the adult mouse, these genes have a large number of untemplated nucleotides. The presence of N regions greatly alters the composition of the third hypervariable, complementarity determining region of the respective proteins, thus playing a major role in the conformation of the binding site. It was possible that, for functional reasons, N region-containing Ig and TCR genes were not permissible at the fetal stage of development. We have produced transgenic mice with a rearrangement test gene which, after V-J recombination, does not result in the production of functional Ig or TCR proteins. We report here that the rearrangement products have no N regions in fetal liver, but that the majority of joints in adult lymphoid tissues have N additions. The study is also an interesting demonstration of the randomness of rearrangements and the enormous variability that can be created from a single pair of V and J sequences.

We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cis-acting elements are present within the 10-16 kb of a transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may be the BCL6 gene, which was mutated in human but not in mouse B cells. Transcription of a region is required for its mutability. When the transcriptional promoter located upstream of the variable region is duplicated upstream of the constant region, this region also becomes mutable. This suggests a model in which a mutator factor associates with the RNA polymerase at the promoter, travels with the polymerase during elongation, and causes mutations during polymerase pausing. The DNA repair systems, nucleotide excision repair and DNA mismatch repair, are not required. Our recent data with an artificial substrate of somatic mutation suggest that pausing may be due to secondary structure of the DNA or nascent RNA, and the specific mutations to preferences of the mutator factor.

Immunoglobulin (Ig) genes are rearranged in pre-B cells. Pre-B cells that express Ig heavy (H) and light (L) chain genes whose V(D)J recombination results in a functional reading frame mature into B cells that exit the bone marrow. The V(D)J recombination process creates a large repertoire of different variable regions from a restricted pool of germline genes. Additional variablity arises during the process of somatic hypermutation in mature B cells proliferating in germinal centers of lymphoid organs (reviewed in French et al. 1989). B cells that have mutated to express high-affinity antibodies are selected and develop into plasma cells or memory cells. B cells with mutations that decrease the affinity of the expressed Igs or that prevent Ig expression die by apoptosis. The somatic point mutations are located within the variable region and their proximate upstream and downstream flanks, but not generally within the constant region.

Somatic hypermutation of Ig genes is beginning to be understood in molecular terms. Ig transgenes have served as model test genes and been shown to mutate, just as endogenous genes, with a peak of mutation over the VJ region and a sparing of the C region. The levels of somatic mutation appear to be related to the expression of the transgenes. DNA hypermethylation of modified Ig transgenes interferes with both expression and somatic hypermutation. Test substrates consisting of bacterial lacZα or supF tRNA inserted within κ transgenes were shown to be rescuable as expressible bacterial plasmids, but did not seem to be targeted. A synthetic sequence consisting of alternating restriction enzyme sites, that cannot be subject to methylation, was found to be a reliable transgenic substrate for easy assay of somatic mutation. The generation of a transgenic mouse with a κ transgene in which the transcriptional promoter has been duplicated upstream of the C region has given new clues to the mutation mechanism. In this transgene, transcripts initiate from both the C region and the V region promoters, and both regions, but not the sequences between them, are hypermutated. These results suggest that somatic hypermutation is linked to the initiation of transcription. A model is proposed in which somatic mutation is dependent on transcription coupled DNA repair.

Transgenic mice are discussed which carry a rearrangment test transgene. The methylation status of the transgene varies, depending on the background mouse strain. When the transgene is bred into the C57BL/6 strain, it is completely methylated and not rearranged in lymphoid organs. After several generations of crossing into DBA/2 or SJL the transgene becomes unmethylated and rearranges at high frequency. A strain specific modifier of DNA methylation (Ssm-1) was mapped close to the Friend virus susceptibility locus (Fv-1) on mouse chromosome 4.