Despite myriad challenges, clinicians see room for progress.
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Edited by Matthew D. Scharff, Albert Einstein College of Medicine of Yeshiva Uni, Bronx, NY, and approved February 7, 2014 (received for review October 31, 2013)
Article Figures & SI
Figures
- Fig. 1.
AID variants with dominant-negative effect on CSR. (A) Schematics of AID (with predicted secondary structure), AID variants found in AD or AR HIGM2 patients, and artificial variant ΔE5. E4 and E5 denote regions encoded by AICDA exons 4 and 5, respectively. The table indicates CSR and SHM status of homozygous carrier patients. (B) Experimental design of CSR dominant-negative assay with illustrative flow cytometry primary data from WT (Aicda+/+) mouse splenic B cells expressing retrovirally transduced AID- or variant-ires-GFP and stimulated with LPS and IL-4. The proportion of IgG1+ cells was calculated for the GFP− and GFP+ populations (shown above) and then divided to calculate the CSR ratio. (C) Compiled CSR ratios for IgG1 and IgG2b in WT and Aicda+/− B cells. Each symbol represents the CSR ratio for B cells from an individual mouse. A dotted line indicates a ratio of one. Horizontal bars are means. Significant P values from ANOVA with Dunnet posttest are indicated. ΔE5 and ΔE5-E58A were compared by t test.
- Fig. 2.
CSR-deficient and dominant-negative AID variants are hypermutagenic. (A) Relative rpoB mutation frequency of AID variants in E. coli. Means + SDs of the relative medians obtained from three independent experiments normalized to AID are shown. Significant P values by ANOVA with the Holm–Sidak posttest are indicated. P20 and R174S are significantly less active than AID by t test (pHis vs. R174S, P = 0.0002; AID vs. R174S, P = 0.0002; pHis vs. P20, P = 0.0032; AID vs. P20, P < 0.0001). (B) Diagram and representative flow cytometry plots of the surface IgM-loss assay for estimating SHM frequency in IgM+ DT40 ΔψVL Aicda−/− B cells complemented with AID or variant-ires-GFP. (C) Relative IgM-loss capacity of AID variants. Each symbol is the median of ≥12 cultures from one to three experiments performed per variant normalized to their AID control median value. Horizontal lines are means. Statistics are the same as in A. (D) Western blot of AID variants in DT40 B cells. A nonspecific band was used as loading control (Ctrl). (E) Mutation load of the DT40 IgVλ in complemented cells. Pie chart slices represent the proportion of sequences with the indicated number of mutations, with the total number of sequences analyzed at the center. Deletion events and the proportion of transition (Ts) mutations at C:G pairs are indicated. (F) Real-time PCR ChIP analysis of UNG occupancy at the Igh or Gapdh control in mouse Aicda−/− B cells transduced with pMXs empty (Ctrl), AID, or ΔE5-ires-GFP vectors 21 h postinfection. The Igh amplicons and their first nucleotide position are shown on the scheme (according to the National Center for Biotechnology Information contig NG_005838.1). Means + SDs of three independent experiments are plotted. P value by t test. (G) Mutation load (pie charts; plotted as in E) of a 628-bp Sμ fragment (nucleotides 177959–178587 of NG_005838.1) in transduced Aicda−/− Ung−/− B cells. Bars show means + SDs mutation frequencies from four experiments. P value by t test. (H) Sγ1 region sequences from transduced Aicda−/− Ung−/− mouse B cells (1,805-bp fragment, nucleotides 90156–91960 of NG_005838.1). Each line is a sequence, with mutations at G indicated by stars and mutations at C indicated by dots.
- Fig. 3.
CSR-deficient dominant-negative AID variants are genotoxic. (A) Follow-up of the ratio of K562 BCR-ABL+ cells originally sensitive to imatinib transduced with AID-ires-GFP (GFP+) or untransduced (GFP−). Cells were grown in the presence of imatinib (Left), where the increase in the GFP+/GFP− cells ratio indicates acquisition of imatinib resistance, or without selection (Right), where decrease in the GFP+/GFP− cells ratio reflects the effect of each AID variant on cell fitness. One representative experiment out of three experiments performed is shown. (B) Effect of AID variants on the expansion of retrovirally complemented Aicda−/− splenic B cells evaluated from the relative proportion of GFP+ (complemented) cells in the culture at various times postinfection. Mean + SEM proportion of GFP+ cells in B-cell cultures from two independently infected mice. One of two similar experiments is shown. (C) DNA damage accumulation in HeLa cells transiently expressing AID- or variant-ires-GFP was measured by the presence of γH2AX nuclear foci 48 h posttransfection. Means + SDs proportions of GFP+ cells with ≥10 foci from three to five independent experiments are plotted for each variant with ANOVA and Dunnet posttest P values. Representative confocal images are shown below.
- Fig. 4.
Dominant-negative ability of CSR-deficient AID variants correlates with DNA damage accumulation. (A) E. coli rpoB mutation frequency by AID single point mutants or variant m7.3 (bearing mutations K34E, T82,I and E156G). Means + SEMs of the relative medians normalized to AID from two to three independent experiments are plotted with significant P values from one-way ANOVA with the Holm–Sidak posttest. (B) SHM assay by IgM-loss fluctuation analysis in complemented DT40 ΔψVL Aicda−/− B cells. Each symbol is the percent of IgM− cells in an individual population after 2 wk of expansion; horizontal lines are median values for each population, with significant P values by Kruskal–Wallis test with Dunnet posttest comparison indicated. One representative experiment of two experiments is shown. (C) IgG1 CSR activity of AID variants in complemented mouse Aicda−/− B cells 4 d after stimulation with LPS + IL-4. Mean + SEM of two mice for each variant. (D) Effect of AID variants expression on the expansion of Aicda−/− splenic B cells transduced with AID variants-ires-GFP vectors. Mean ± SEM proportion of GFP+ cells over time postinfection in B-cell cultures from two mice. One representative experiment of two experiments is shown. (E) CSR dominant-negative assays for the indicated mutants like in Fig. 1. Each symbol is the CSR ratio of B cells transduced with AID- or variant-ires-GFP to uninfected cells from an individual mouse. Means (lines) and significant P values from one-way ANOVA with Dunnet posttest are indicated. (F) Protein expression level of AID variants by Western blot in packaging cells. (G) DNA damage accumulation in HeLa cells transiently transfected with AID variants assessed by the presence of γH2AX foci at 48 h posttransfection. Mean + SEM proportion of transfected cells showing ≥10 γH2AX nuclear foci from two to three experiments for each variant is plotted.
- Fig. 5.
E5 deletion compromises end joining. (A) Real-time PCR ChIP assays for the presence of γH2AX, Nbs1, 53BP1, ATM, and Ku70 at the Igh or Gapdh control in mouse Aicda−/− B cells transduced with pMXs empty (Ctrl), AID, or ΔE5-ires-GFP vectors at 21 h postinfection. Means + SDs of three independent experiments are plotted. Significant differences vs. AID for each amplicon were calculated by ANOVA with Dunnet posttest (*P < 0.05; **P < 0.01; ***P < 0.001). Amplicons in Igh are the same as in Fig. 2F. (B) Analysis of Sμ-Sγ1 junctions amplified from LPS + IL-4–stimulated Aicda−/− B cells complemented with AID or ΔE5 72 h postinfection from three mice. B, blunt (includes 1- or 2-nt insertion); INS, ≥3-nt insertions. (C) Analysis of Sμ-Sα junctions from the CH12 B-cell line depleted of AID with shRNA, complemented, and analyzed as in B. (D) ChIP analysis for CtIP is the same as in A. (E) PCR strategy for detecting Igh-cMyc chromosomal translocation in retrovirally complemented Aicda−/− primary B cells. (F) Representative ethidium bromide-stained agarose gels from PCR assays. Each lane is a PCR performed on DNA extracted from 0.5 to 1 × 106 sorted GFP+ cells expressing each indicated construct. A positive control (+) of the PCR using a cloned translocation is shown. (G) Schematic representation of all independently amplified PCR products from two experiments after cloning and sequencing.
- Fig. 6.
Truncation of E5 leads to end resection at the Sμ. Real-time PCR ChIP assays for (A) RPA, (B) EXOI, and RAD51 and (C) γH2AX at the Igh in cell extracts of Aicda−/− B cells transduced with empty vector (Ctrl) or carrying the indicated AID variants at 21 h postinfection. Amplicons are the same as in Fig. 2F. Means + SDs of three biological replicates for each variant are plotted. Significant differences vs. AID in each amplicon by ANOVA with Dunnet posttest are indicated (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05). (D) CSR in the complemented CH12 B-cell line. Cells were depleted of AID alone or combined with CtIP or Ku70 by shRNA transfection and then transduced with empty pMXs-ires-GFP (GFP) or encoding human AID or ΔE5. Cells were stimulated, and CSR was measured 24 h later. A representative example of the raw data is shown. The fold change in CSR for AID or ΔE5 when depleting CtIP or Ku70 from three to four independent experiments is plotted. The proportion of IgA+ cells in the GFP+ population was first corrected for the background CSR in the corresponding GFP (vector only) infection. Then, the CSR value of cells expressing AID or ΔE5 in shCtIP (AID- and CtIP-depleted) or shKu70 (AID- and Ku70-depleted) cells was normalized to the CSR value of Ctrl cells (AID-depleted only) expressing AID or ΔE5, respectively. Significant differences by ANOVA with Dunnet posttest are shown for each variant. (E) Real-time PCR ChIP assays for CtIP, RPA, and RAD51 occupancy at the Sμ in the CH12 cells from D. Means + SDs of three biological replicates for each variant and condition are plotted. Significant differences by paired two-tailed t test. (F) Proposed model of the mechanisms operating at the S regions downstream from AID or CSR-deficient and dominant-negative variants (ΔE5).
Tables
Patient AICDA status Ig levels (g/L) CSR in vitro SHM in vivo Lymphadenopathies IgM IgG IgA 1-I-1* R190X/+ 4.05† 1.28† 0.1† − N − 1-II-2* R190X/+ 2.85† 0.5† <0.05† − N − 1-II-3* R190X/+ 3.02† 6.33† <0.05† − N − 1-III-1* R190X/+ 1.22 7.58 <0.05† − N − 2-I-1* R190X/+ 2.93† 2.82† <0.05† − N − 2-II-2* R190X/+ 2.34† 0.21† <0.05† − − ++ 3-II-1* R190X/+ 2.34† 0.38† <0.05† − N − 4-I-1 R190X/+ 2.03 1.30† <0.05† − ND − 5-I-1‡ V186X/+ 2.02 5.41† 1.19 − ND − 5-II-1‡ V186X/+ 0.84 NE <0.05† ND − ++ 5-II-2‡ V186X/+ 1.32 4.71† <0.05† − ND − AR-HIGM2§ −/− (range) 0.7–37† <0.2–1.3† <0.05–0.2† − − ++ (70%) Reference +/+ (range) 0.4–2.3 7–16 0.7–4 + N −
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AID links DNA damage and repair
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