AID-Targeting and Hypermutation of Non-Immunoglobulin Genes Does Not Correlate with Proximity to Immunoglobulin Genes in Germinal Center B Cells

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

Distances of genes from <i>Igλ</i> in germinal center and naïve B cells.

<p>(A and C) Scatter plots showing all distances (µm) measured from the gene marked on the x-axis and the nearest <i>Igλ</i> allele in (A) splenic GC B cells 10 days post-immunization with NP-CGG or (C) splenic naïve B cells collected from the same spleen samples as those used in (A). (B and D) Bar graphs summarizing data from (A) and (C) respectively. Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039601#pone-0039601-g001" target="_blank">Figure 1</a>.</p

Distance between <i>c-Myc</i> and <i>Igh</i> during the GC response.

<p>(A) Scatter plot showing the distance (µm) between <i>c-Myc</i> and <i>Igh</i> in GC B cells 8, 10, 14, and 16 days post-immunization with NP-CGG. (B) Bar graphs summarizing data from (A). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039601#pone-0039601-g001" target="_blank">Figure 1</a>.</p

Expression of endogenous <i>c-Myc</i> and transgenic <i>MYC</i> in B cells.

<p>(A, B, and C) Bar graphs represent the average expression level of endogenous <i>c-Myc</i> (top row of each panel) or transgenic <i>MYC</i> (bottom row of each panel) relative to <i>Hprt</i> expression in the same sample for either the Myc31 mouse line (left column in each panel) or the Myc24 line (right column in each panel). Error bars denote the standard error of the mean (SEM). Cell populations analyzed were: (A) Progenitor cells (B220⁻, CD43⁻, IgM^-), Pro B cells (B220⁺, CD43⁺, IgM^-), Pre B cells (B220⁺, CD43⁻, IgM^-), and Immature B cells (B220⁺, CD43⁻, IgM⁺) sorted from bone marrow cells of 4–5 week old mice; (B) Naïve (B220⁺, NIP^lo, FAS^-) and GC B cells (B220⁺, NIP^hi, FAS⁺) sorted from spleens of mice immunized 16 days previously with NP-CGG; and (C) Naïve B cells (B220⁺, CD19⁺, PNA^lo), and GC B cells (B220⁺, CD19⁺, PNA^hi) sorted from Peyer’s patches isolated from mice that had lived at least 1 month with non-autoclaved food and tap water. Data represent an average of 2–6 experiments: Panel A top row, 4; panel A bottom row, 2; panel B top row, 6; panel B bottom row, 3; panel C top left, 6; panel C top right, 4; panel C bottom left, 5; panel C bottom right, 3.</p

Mutation level in Myc31 and Myc24 transgenic mice.

<p>Bar graphs show mutations per 100,000 basepairs sequenced for various non-immunoglobulin genes in germinal center (GC) B cells of <i>MYC</i> transgenic mice. Data from the Myc31 line is shown on the left of each graph and the Myc24 line on the right of each graph. Sequence data from the first 1 kb downstream of the MYC P0 promoter is labeled Myc31 or Myc24. Sequence data from the first intron (including the end of the first exon) was done only in Msh2^−/−Ung^−/− dko Myc31 mice and is labeled Int31. A star above the bar indicates that the mutation frequency is significantly (p<0.05) above the background frequency of 1.6 mutations per 100,000 basepairs sequenced reported by Liu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039601#pone.0039601-Liu2" target="_blank">[14]</a> using a Chi-square test with Yate’s correction. A “0” in place of a bar indicates that no mutations were found in all sequences analyzed. An “N” in place of a bar indicates that the sample was not sequenced. (A) Splenic GC B cells sorted from Myc31 or Myc24 positive, B1-8 heterozygous mice immunized 16 days prior with NP-CGG. (B) Mutation frequency in Peyer’s patch GC B cells isolated from either wild type mice (red bars) or <i>Msh2</i>^−/−<i>Ung</i>^−/− double knockout mice (yellow bars) aged at least four months with non-autoclaved food and tap water.</p

Distances of genes from <i>Igh</i> in germinal center and naïve B cells.

<p>(A) Examples of cells used in analysis. Collapsed z-stacks are shown with nuclei outlined. <i>Igh</i> probes are shown in red and <i>Bcl6</i> probes are shown in blue and green (colors overlap in example shown). In all experiments, only cells in which both alleles for both genes under study were clearly visible were included in the analysis. Ig alleles were marked by one probe and other genes were marked by either one or two probes. Cells whose nuclei were cut off by the edge of the picture were excluded. (B) Schematic showing measurements taken from cells. Two cells and their nuclei are shown with red dots representing FISH probes to an Ig gene and blue and/or green dots representing two different FISH probes to a non-Ig gene. Distances were measured from each allele of the gene of interest to the closest allele of the Ig gene in question. (C and E) Scatter plots showing all distances (µm) measured from the gene marked on the x-axis to the nearest <i>Igh</i> allele. The number of measurements taken is shown in parentheses above each data set and red bars mark the median. Two genes that are not deaminated by AID are shown on the left, and 5 genes that are deaminated by AID are shown on the right. (C) Measurements taken from splenic GC B cells 10 days post-immunization with 4-hydroxy-3-nitrophenylacetyl conjugated to chicken gamma globulin (NP-CGG). (E) Measurements taken from splenic naïve B cells collected from the same spleen samples as those used in (C). (D and F) Bar graphs summarizing data from (C) and (E) respectively. Bars represent the percentage of measurements in a dataset within 0.5 µm (dark blue) or 1.0 µm (blue green). The number of measurements found in each distance range are shown in parentheses.</p