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Supplementary Figures 1 - 12, Tables 1 - 6 from 53BP1 Is Limiting for NHEJ Repair in ATM-deficient Model Systems That Are Subjected to Oncogenic Stress or Radiation

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posted on 2023-04-03, 16:07 authored by Ivana Rybanska-Spaeder, Taylor L. Reynolds, Jeremy Chou, Mansi Prakash, Tameca Jefferson, David L. Huso, Stephen Desiderio, Sonia Franco

PDF file - 7618K, S1. Analysis of the genetic interaction between 53BP1 and ATM in organismal growth and development. S2. Analysis of ploidy in Trp53bp1-/-/Atm-/- lymphomas. S3. Analysis of Trp53bp1-/-/Atm-/- thymic lymphomas by TCRα/δ locus FISH. S4. SKY analysis of two Trp53bp1-/-/Atm-/- lymphomas. S5. Cell cycle analysis of resting lymphocytes after IR. S6. Aberrant Vd2-Dd1/Dd2-Jd1 coding junctions are not detected in Trp53bp1-/-/Atm-/- mice. S7. Aberrant Vd5-Dd2 Recombination Signal Junctions are not detected in Trp53bp1-/-/Atm-/- mice. S8. Histograms showing the frequency distribution of deletions from each coding end (A-D) and signal end (E-F) analyzed. S9. Cell cycle analysis of activated (cycling) lymphocytes after IR. S10. Analysis of genomic stability in splenic B or T cells treated with olaparib. S11. Analysis of the G2/M checkpoint. α-CD40/IL-4-activated B cells were harvested one hour after exposure to 2 Gy of IR, stained with a FITC-labeled antibody to phospho(P)-histone H3 (Ser10) and propidium iodide (PI) and analyzed by flow cytometry. S12. A second primer set detects deletions at hybrid V(D)J recombination junctions in Trp53bp1-/-/Atm-/- thymic DNA from 7 day-old mice. Table S1. Mendelian ratios in liveborn mice from 53BP1+/-/ATM+/- intercrosses (n=19 litters) Table S2. Analysis of genomic stability in HU-treated B lymphocytes deficient for 53BP1 and/or ATM. Table S3. Analysis of genomic stability in α-CD40+IL-4activated B lymphocytes deficient for 53BP1 and/or ATM. Table S4. Analysis of genomic stability in α-CD40/IL-4 activated B cells deficient for 53BP1 and/or ATM. Metaphase spreads were obtained 24 hr after IR and stained with a telomere probe. Table S5. Analysis of genomic stability in LPS-activated B cells deficient for 53BP1 and/or ATM. Metaphases were obtained 24 hr after exposure to IR and stained with a telomere probe. Table S6. Analysis of genomic stability in T lymphocytes deficient for 53BP1 and/or ATM.

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ARTICLE ABSTRACT

The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1−/−/Atm−/−), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm−/− mice (average survival, 14 and 23 weeks for Trp53bp1−/−/Atm−/− and Atm−/− mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)—mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1−/−/Atm−/− primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm−/− thymocytes but is required to suppress large deletions in a subset of hybrid joints.Implications: The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.Visual Overview: http://mcr.aacrjournals.org/content/11/10/1223/F1.large.jpg.Mol Cancer Res; 11(10); 1223–34. ©2013 AACR.

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    Molecular Cancer Research

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    CarcinogenesisDNA damage and repairCell SignalingProtein serine-threonine kinasesDna Damage And RepairGenomic instabilityHematological CancersLeukemiasPreclinical ModelsAnimal models of cancer

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