Abstract
Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this “collateral damage” model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.
Highlights
In mice and humans, antibodies are produced by B cells and are composed of two light chains, coded for by either Igk or Igl, and two heavy chains, coded for by Ig heavy chain gene (Igh)
Since this study found many genes whose mutation frequency was significantly higher in Msh22/2Ung2/2 germinal center (GC) B cells than in WT GC B cells, the authors proposed that there are three groups of genes: those not detectably deaminated by Activation Induced Cytidine Deaminase (AID), those deaminated by AID but repaired predominantly by high-fidelity processes, and those deaminated by AID and further mutated by error-prone repair pathways [9,14]
Ig genes in GC B cells by 3D-fluorescence in situ hybridization (3D-FISH) using GC B cells isolated from spleens of B1-8 heterozygous mice immunized 10 days prior with the hapten NP conjugated to chicken gamma globulin (NP-CGG)
Summary
Antibodies are produced by B cells and are composed of two light chains, coded for by either Igk or Igl, and two heavy chains, coded for by Igh. There are several different Igh constant regions, conferring different effector functions, and the B cell can undergo class switch recombination (CSR), a rearrangement of germline DNA, to switch from one constant region to another. The regions of DNA that break during CSR are called switch regions and, with the exception of IgD, every constant region has its own upstream switch region [1]. Both CSR and somatic hypermutation (SHM) are initiated by and dependent on the enzyme Activation Induced Cytidine Deaminase (AID). GC B cells express AID, which deaminates deoxycytidine in the variable region and switch region DNA, thereby generating deoxyuracil:deoxyguanine (dU:dG) mismatches. The dU:dG lesions can be recognized by the base excision repair protein Uracil DNA Glycosylase (UNG) and the mismatch repair proteins Msh2/6 [2,3]
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