1. Volume 21, Issue 2, Pages 201-214 (January 2006)
H2AX Prevents DNA Breaks from Progressing to Chromosome Breaks and Translocations 
Sonia Franco, Monica Gostissa, Shan Zha, David B. Lombard, Michael M. Murphy, Ali A. Zarrin, Catherine Yan, Suprawee Tepsuporn, Julio C. Morales, Melissa M. Adams, Zhenkun Lou, Craig H. Bassing, John P. Manis, Junjie Chen, Phillip B. Carpenter, Frederick W. Alt 
Molecular Cell 
Volume 21, Issue 2, Pages (January 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Sequential Telomere/Igh FISH in H2AX-Deficient B Cells Activated for CSR
(A) Schematic of the mouse Igh and FISH probes used for sequential telomere FISH (TTAGGG probe) and FISH with a BAC spanning the 3′Igh region (left diagram). The cytogenetic readout for an intact Igh or a chromosome break at Igh is illustrated on the right.
(B and C) Partial metaphase spread of an H2AX−/− B cell after 3 days of stimulation with α-CD40/IL-4. Loss of telomere signal (left panel, yellow arrow) with intact 3′Igh BAC signal (middle panel, yellow arrow) indicates a chromosome break between the 3′ end of Igh and the 12q telomere. A normal chromosome 12 with both telomere signals (left panel, white arrow) and 3′Igh BAC signal (middle panel, white arrow) is shown for comparison. An overlay with diagrams is shown on the right panel. An example of a metaphase with Igh breaks at both chromosome 12s is shown in (C).
(D) Quantification of the relative contribution of Igh breaks to total chromosome aberrations in LPS- or α-CD40/IL-4-stimulated H2AX−/− B cells or concanavalin A-stimulated H2AX−/− T cells. Bars represent total chromosome or chromatid breaks as determined by telomere-FISH. After FISH with a 3′Igh BAC, they were further classified as labeled with the 3′Igh BAC near the break (suggesting a break within Igh, in black) or not labeled with the 3′Igh BAC (suggesting a break elsewhere, in white).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Chromosomal IgH Locus Breaks in H2AX-Deficient B Cells Activated for CSR
(A) Schematic of mouse Igh and BAC probes used for two-color Igh FISH (left diagram). The cytogenetic readout for an intact Igh or a chromosome break at Igh is illustrated on the right.
(B and C) Partial metaphase spread of an α-CD40/IL-4-stimulated B cell after two-color Igh FISH, showing split BAC signals (3′Igh, red, yellow arrow; 5′Igh, green, white arrow). A normal chromosome 12 with adjacent 3′Igh (red) and 5′Igh (green) signals is shown for comparison (yellow arrowheads). An example of a partial metaphase with Igh breaks at the two chromosomes 12 is shown in (C).
(D) Quantification of split signals for 3′Igh and 5′Igh BACs on wt, H2AX+/−, and H2AX−/− B cells stimulated with α-CD40/IL-4 for 3 days. Bars represent the mean and standard deviation of three mice per genotype.
(E) Time course of the frequency of split signals for 3′Igh and 5′Igh BACs on H2AX−/− metaphases at day 3 and day 4 of stimulation with α-CD40/IL-4. Bars represent average and standard deviation of two H2AX−/− mice at each time point.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
AID Is Required for Generation of High-Frequency Chromosome 12 Breaks within the IgH Locus in H2AX−/− B Cells
(A) H2AX−/− and control H2AX+/+ and H2AX+/− B cells were stimulated with α-CD40 alone or in combination with IL-4 for 3 days and the frequency of total chromosomal breaks measured by telomere FISH (average and standard deviation of three mice).
(B) Frequency of Igh-specific DSBs on H2AX−/− B cells stimulated with α-CD40 alone or in combination with IL-4 quantified by two-color Igh FISH at days 3 and 4 of in vitro culture (average and standard deviation of two mice).
(C–E) H2AX−/−/AID−/−, H2AX−/−, AID−/−, and wt B cells were stimulated with α-CD40/IL-4 for 3 days and the expression of surface IgG1 (sIgG1) assessed by flow cytometry (FACs). A representative experiment is shown. Similar data was obtained at 96 hr and for LPS-stimulated cells (data not shown). Metaphase spreads from these cultures were used for quantification of general and Igh-specific chromosome breaks by telomere FISH (D) and two-color Igh FISH (E), respectively. Bars represent average and standard deviation of two independent stimulations.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Chromosomal IgH Locus Breaks in H2AX-Deficient B Cells Participate in Frequent Translocations in a p53-Independent Manner
(A–F) Examples of chromosomal translocations involving a chromosome break at Igh in H2AX−/− B cells stimulated with α-CD40/IL-4 for 3 days. The involvement of a chromosome 12 with a break at Igh in the translocation is indicated by the presence of 3′Igh BAC signal (red) and the absence of adjacent 5′Igh BAC signal (green). The translocation partner can be another centric fragment (A–C), acentric fragments (D), or two other chromosomes simultaneously (E and F). A normal chromosome 12 with an intact Igh (adjacent red and green signals) is shown in (A) for comparison (white arrow). The normal chromosome and one dicentric also are illustrated in (A).
(G) Combined two-color Igh FISH and chromosome 12 paint on activated H2AX−/− B cells. Translocations of chromosome 12 to a non-12 chromosome (T1) or between the two homologous 12 chromosomes (T2) were observed.
(H) Quantification of total chromosome breaks by T-FISH in p53−/−, H2AX−/−, and H2AX−/−/p53−/− B cells stimulated with LPS, α-CD40, or α-CD40/IL-4 for 3 days. Parallel analyses of concanavalin A-stimulated T cells are shown for comparison. Bars represent average and standard deviation of two mice per genotype.
(I) After two-color Igh FISH, each Igh break was further classified as either a free untranslocated end (examples in Figure 2) or a translocated end (examples in Figures 4A–4G). Comparative analyses for 102 Igh breaks in H2AX−/− B cells and 76 Igh breaks in H2AX−/−/p53−/− B cells are shown.
(J) Relative frequency of dicentrics to all observed chromosomal translocations involving Igh breaks in H2AX−/− and H2AX−/−/p53−/− B cells (n = 24 and n = 10 translocations, respectively).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5
Role of ATM and 53BP1 in Suppressing Igh Chromosomal Breaks and Translocations
(A) Fate of broken Igh ends (free untranslocated versus translocated to another chromosome) in H2AX−/−, ATM−/−, and 53BP1−/− activated B cells. The total number of Igh breaks studied for each genotype is indicated.
(B) Comparative frequency of dicentrics with a breakpoint at Igh in H2AX−/−, ATM−/−, and 53BP1−/− activated B cells. The total number of metaphases analyzed for each genotype is indicated.
(C) Two representative examples of sequential SKY and two-color Igh FISH on α-CD40/IL-4-stimulated ATM−/− B cells. Chromosome 12 dicentrics were observed frequently (top row, left and middle panel, green arrows; see Table S1 for quantification). Reprobing of metaphases with 3′Igh and 5′Igh BACs confirmed their CSR-related origin (split 3′Igh and 5′Igh BAC signals; top right panel, green arrow). A telomeric fragment containing the 5′Igh BAC signal was present in the metaphase and is indicated by a red arrow in the DAPI (top right), spectral (middle), and Igh FISH (right) images. Translocations to chromosomes other than the homologous 12 appeared random; a t(3, 12) is shown as an example in the bottom images (green arrows).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions