AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations

Abstract

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

Figure 1. The Myc^Δ allele is protected from translocating to the IgH

(A) Schematic representation of wild type (Myc⁺) and mutant (Myc^Δ)c-myc alleles. The dashed bracket indicates the region deleted and replaced by a loxP site (triangle) in the Myc^Δ allele. (B) Genotyping summary from the indicated Myc^Δ/+ cross. (C) RNA-FISH showing monoallelic vs. biallelic c-myc transcription in wild type (WT) vs. Myc^Δ/+ cells, respectively. (D) Diagram showing the location of the primers used in the PCR assay to detect derivative chromosome 12 (der12, black arrows) and derivative chromosome 15 (der15, green arrows) c-myc/IgH translocations. The table summarizes the number of allele-specific der12 and der15 c-myc/IgH translocations in activated Myc^Δ/+ B cells, as determined by sequencing. Three independent experiments.

Figure 2. Cre-mediated c-myc/IgH translocations

(A) Schematic representation of the Myc^Δ, Myc^I and IgH^I alleles with the PCR primers for detecting der12 c-myc/IgH translocations. Triangles represent loxP sites, circles point to recognition sequences for I-SceI. (B) Translocations by retroviral Cre. Ethidium bromide stained agarose gel with 0.5kb PCR products corresponding to precise loxP-to-loxP c-myc/IgH translocations (as verified by sequencing). B lymphocytes of the indicated genotypes were stimulated with LPS and IL-4 and infected with retroviruses that direct expression of Cre. The number of cells that were assayed in each reaction is indicated. Two independent experiments. (C) Translocations in AID^Cre B cells. Agarose gel with 0.5kb PCR products corresponding to precise loxP-to-loxP c-myc/IgH translocations (as verified by sequencing). AID^Cre B cells of the indicated genotypes were stimulated with LPS and IL-4. The number of cells assayed in each reaction is indicated. Two independent experiments.

Figure 3. I-SceI induced breaks

(A) Immuno-FISH analysis of 53BP1 DNA damage foci at IgH or c-myc loci in activated B cells. Representative images and table showing co-localization frequencies as percentage of cells analyzed in LPS and IL-4 stimulated wild type (WT) or AID^−/− B lymphocytes. The number of cells analyzed is shown in parentheses. Three independent experiments. (B) Immuno-FISH analysis of AID induced 53BP1 DNA damage foci in AID^−/− B cells infected with AID or empty virus control. Representative images and table showing co-localization frequencies at c-myc (p = 0.007 with Student’s T-Test). (C) Immuno-FISH analysis of I-SceI induced 53BP1 DNA damage foci in IgH^I/+AID^−/− (top) or Myc^I/+AID^−/− B cells (bottom). Representative images and table showing co-localization frequencies at IgH or c-myc (as indicated) in I-SceI or I-SceI* expressing B lymphocytes.

Figure 4. I-SceI induced translocations in AID deficient B lymphocytes

(A) Schematic representation of the Myc^I, Myc⁺ and IgH^I alleles with the PCR primers for detecting der12 and der15 c-myc/IgH translocations. Circles point to the position of the I-SceI sites. (B) I-SceI rescues translocations in the absence of AID. Representative agarose gels with PCR products corresponding to c-myc/IgH translocations (as verified by sequencing and/or Southern Blot, see Supplemental Figure S7). Myc^I/+IgH^I/+AID^−/− B cells were stimulated with LPS and IL-4 and infected with I-SceI or I-SceI* control retroviruses. 100,000 cells were assayed each in lane. Three independent experiments. (C) Map of translocation breakpoints from I-SceI expressing Myc^I/+IgH^I/+AID^−/− B cells (see also Supplemental Table 2). Arrows point to c-myc/IgH translocation breakpoints for der12 (black) or der15 (green).

Figure 5. I-SceI induced translocations in AID sufficient B lymphocytes

(A) Schematic representation of the Myc^I, Myc⁺ and IgH⁺ alleles with the PCR primers for detecting der12 and der15 c-myc/IgH translocations. Circle points to recognition sequence for I-SceI. (B) I-SceI directed translocations in the presence of AID. Representative agarose gels with PCR products corresponding to c-myc/IgH translocations (as verified by sequencing and/or Southern Blot, see Supplemental Figure S8). Myc^I/+ B cells were stimulated with LPS and IL-4 and infected with I-SceI (three independent experiments) or I-SceI* control. 100,000 cells were assayed in each lane. (C) Map of translocation breakpoints from stimulated Myc^I/+ B cells infected with I-SceI (see also Supplemental Table 3). Filled arrows point to c-myc/IgH translocation breakpoints from der12 (black) or der15 (green). Empty arrows indicate the distribution of breakpoints obtained in the absence of I-SceI. (D) Table shows summary of the extent of junctional microhomology from c-myc/IgH translocations (see Supplemental Tables 2, 3, and 4).

Figure 6. Translocations induced by AID, I-SceI or Cre in AID^−/− cells

(A) Schematic representation of the Myc^I, Myc⁺ and IgH^I alleles with the PCR primers for detecting der15 c-myc/IgH translocations. Triangles represent loxP sites, circles point to recognition sequences for I-SceI. (B) Representative agarose gels with PCR products corresponding to c-myc/IgH translocations (as verified by sequencing and/or Southern Blot, see Supplemental Figure S10). Myc^I/+ IgH^I/+ AID^−/− B cells were stimulated with LPS and IL-4 and infected with AID, I-SceI, or Cre, as indicated. DNA equivalents for the indicated number of cells were assayed in each reaction. Two independent experiments.

Figure 7. AID is essential for the lesion in c-myc that leads to c-myc/IgH translocation

(A) Top: schematic representation of the Myc^I, Myc⁺ and IgH⁺ alleles with the PCR primers for detecting der12 and der15 c-myc/IgH translocations. Circle indicates I-SceI site. Bottom: a representative ethidium bromide (EtBr) stained agarose gel was also Southern blotted and probed for c-myc and IgH, as indicated, to verify translocations. Myc^I/+AID^−/− B cells were stimulated with LPS and IL-4 and infected with an I-SceI encoding retrovirus. 100,000 cells were assayed in each lane. No translocations were identified out of 3.4×10⁷ cells analyzed. Three independent experiments. (B) Top: schematic representation of the Myc⁺ and IgH^I alleles with the PCR primers for detecting der12 and der15 c-myc/IgH translocations. Circle indicates I-SceI site. Bottom: a representative ethidium bromide (EtBr) stained agarose gel was also Southern blotted and probed for c-myc and IgH, as indicated to verify translocations. IgH^I/+ AID^−/− B cells were stimulated with LPS and IL-4 and infected with I-SceI. 100,000 cells were assayed in each lane. No translocations were identified in 6.9×10⁷ cells. Seven independent experiments.