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Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR

Abstract

Antibody specificity and diversity is generated in B cells during germinal center maturation through clonal expansion while they undergo class-switch recombination and somatic hypermutation. Here we demonstrate that the transcriptional repressor Bcl-6 mediates this phenotype by directly repressing ATR in centroblasts and lymphoma cells. ATR is critical in replication and DNA damage–sensing checkpoints. Bcl-6 allowed B cells to evade ATR-mediated checkpoints and attenuated the response of the B cells to exogenous DNA damage. Repression of ATR was necessary and sufficient for those Bcl-6 activities. CD40 signaling 'rescued' B cells from those effects by disrupting the Bcl-6 transcription-repression complex on the promoter of the gene encoding ATR. Our data demonstrate a transcriptional regulatory loop whereby Bcl-6 mediates the centroblast phenotype through transient silencing of ATR.

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Figure 1: ATR is a direct target gene of Bcl-6.
Figure 2: Bcl-6 represses ATR and attenuates an ATR-dependent DNA damage–sensing pathway in primary human centroblasts.
Figure 3: Bcl-6 mediates survival and DNA-damage resistance in DLBCL cells through ATR.
Figure 4: Regulation of ATR is necessary and sufficient for mediation of the Bcl-6-mediated phenotype.
Figure 5: Bcl-6 represses ATR and DNA-damage sensing and repair in human tonsillar pre-GC naive B cells.
Figure 6: CD40 signaling terminates Bcl-6 silencing of ATR by disrupting the Bcl-6–N-CoR repression complex.

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References

  1. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).

    Article  CAS  Google Scholar 

  2. Arakawa, H., Hauschild, J. & Buerstedde, J.M. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295, 1301–1306 (2002).

    Article  CAS  Google Scholar 

  3. Nagaoka, H., Muramatsu, M., Yamamura, N., Kinoshita, K. & Honjo, T. Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Smu region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation. J. Exp. Med. 195, 529–534 (2002).

    Article  CAS  Google Scholar 

  4. Berek, C., Berger, A. & Apel, M. Maturation of the immune response in germinal centers. Cell 67, 1121–1129 (1991).

    Article  CAS  Google Scholar 

  5. Jacob, J., Kelsoe, G., Rajewsky, K. & Weiss, U. Intraclonal generation of antibody mutants in germinal centres. Nature 354, 389–392 (1991).

    Article  CAS  Google Scholar 

  6. Pasqualucci, L. et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412, 341–346 (2001).

    Article  CAS  Google Scholar 

  7. MacLennan, I.C. Germinal centers. Annu. Rev. Immunol. 12, 117–139 (1994).

    Article  CAS  Google Scholar 

  8. Kelsoe, G. Life and death in germinal centers (redux). Immunity 4, 107–111 (1996).

    Article  CAS  Google Scholar 

  9. Klein, U. et al. Transcriptional analysis of the B cell germinal center reaction. Proc. Natl. Acad. Sci. USA 100, 2639–2644 (2003).

    Article  CAS  Google Scholar 

  10. Ye, B.H. et al. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat. Genet. 16, 161–170 (1997).

    Article  CAS  Google Scholar 

  11. Dent, A.L., Shaffer, A.L., Yu, X., Allman, D. & Staudt, L.M. Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 276, 589–592 (1997).

    Article  CAS  Google Scholar 

  12. Reljic, R., Wagner, S.D., Peakman, L.J. & Fearon, D.T. Suppression of signal transducer and activator of transcription 3-dependent B lymphocyte terminal differentiation by BCL-6. J. Exp. Med. 192, 1841–1848 (2000).

    Article  CAS  Google Scholar 

  13. Huynh, K.D., Fischle, W., Verdin, E. & Bardwell, V.J. BCoR, a novel corepressor involved in BCL-6 repression. Genes Dev. 14, 1810–1823 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Polo, J.M. et al. Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells. Nat. Med. 10, 1329–1335 (2004).

    Article  CAS  Google Scholar 

  15. Pasqualucci, L. et al. Molecular pathogenesis of non-Hodgkin's lymphoma: the role of Bcl-6. Leuk. Lymphoma 44 Suppl 3, S5–S12 (2003).

    Article  CAS  Google Scholar 

  16. Cattoretti, G. et al. Deregulated BCL6 expression recapitulates the pathogenesis of human diffuse large B cell lymphomas in mice. Cancer Cell 7, 445–455 (2005).

    Article  CAS  Google Scholar 

  17. Pasqualucci, L. et al. Expression of the AID protein in normal and neoplastic B cells. Blood 104, 3318–3325 (2004).

    Article  CAS  Google Scholar 

  18. Allman, D. et al. BCL-6 expression during B-cell activation. Blood 87, 5257–5268 (1996).

    CAS  PubMed  Google Scholar 

  19. Golay, J. et al. The A-Myb transcription factor is a marker of centroblasts in vivo. J. Immunol. 160, 2786–2793 (1998).

    CAS  PubMed  Google Scholar 

  20. Phan, R.T. & Dalla-Favera, R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature 432, 635–639 (2004).

    Article  CAS  Google Scholar 

  21. Reina-San-Martin, B., Chen, H.T., Nussenzweig, A. & Nussenzweig, M.C. ATM is required for efficient recombination between immunoglobulin switch regions. J. Exp. Med. 200, 1103–1110 (2004).

    Article  CAS  Google Scholar 

  22. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M. & Nussenzweig, A. H2AX: the histone guardian of the genome. DNA Repair (Amst.) 3, 959–967 (2004).

    Article  CAS  Google Scholar 

  23. Shechter, D. & Gautier, J. ATM and ATR check in on origins: a dynamic model for origin selection and activation. Cell Cycle 4, 235–238 (2005).

    Article  CAS  Google Scholar 

  24. Costanzo, V. et al. An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication. Mol. Cell 11, 203–213 (2003).

    Article  CAS  Google Scholar 

  25. Lupardus, P.J., Byun, T., Yee, M.C., Hekmat-Nejad, M. & Cimprich, K.A. A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev. 16, 2327–2332 (2002).

    Article  CAS  Google Scholar 

  26. Espinosa, L., Ingles-Esteve, J., Robert-Moreno, A. & Bigas, A. IκBα and p65 regulate the cytoplasmic shuttling of nuclear corepressors: cross-talk between Notch and NFκB pathways. Mol. Biol. Cell 14, 491–502 (2003).

    Article  CAS  Google Scholar 

  27. Espinosa, L., Santos, S., Ingles-Esteve, J., Munoz-Canoves, P. & Bigas, A. p65-NFκB synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR. J. Cell Sci. 115, 1295–1303 (2002).

    Article  CAS  Google Scholar 

  28. Monti, S. et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood 105, 1851–1861 (2005).

    Article  CAS  Google Scholar 

  29. Alizadeh, A.A. et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–511 (2000).

    Article  CAS  Google Scholar 

  30. Shechter, D., Costanzo, V. & Gautier, J. ATR and ATM regulate the timing of DNA replication origin firing. Nat. Cell Biol. 6, 648–655 (2004).

    Article  CAS  Google Scholar 

  31. Unsal-Kacmaz, K., Makhov, A.M., Griffith, J.D. & Sancar, A. Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl. Acad. Sci. USA 99, 6673–6678 (2002).

    Article  CAS  Google Scholar 

  32. Cuadrado, M. et al. ATM regulates ATR chromatin loading in response to DNA double-strand breaks. J. Exp. Med. 203, 297–303 (2006).

    Article  CAS  Google Scholar 

  33. Myers, J.S. & Cortez, D. Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J. Biol. Chem. 281, 9346–9350 (2006).

    Article  CAS  Google Scholar 

  34. Jazayeri, A. et al. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat. Cell Biol. 8, 37–45 (2006).

    Article  CAS  Google Scholar 

  35. Honjo, T., Nagaoka, H., Shinkura, R. & Muramatsu, M. AID to overcome the limitations of genomic information. Nat. Immunol. 6, 655–661 (2005).

    Article  CAS  Google Scholar 

  36. Lumsden, J.M. et al. Immunoglobulin class switch recombination is impaired in Atm-deficient mice. J. Exp. Med. 200, 1111–1121 (2004).

    Article  CAS  Google Scholar 

  37. Franco, S. et al. H2AX prevents DNA breaks from progressing to chromosome breaks and translocations. Mol. Cell 21, 201–214 (2006).

    Article  CAS  Google Scholar 

  38. Chen, Y. & Sanchez, Y. Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst.) 3, 1025–1032 (2004).

    Article  CAS  Google Scholar 

  39. Niida, H. & Nakanishi, M. DNA damage checkpoints in mammals. Mutagenesis 21, 3–9 (2006).

    Article  CAS  Google Scholar 

  40. Phan, R.T., Saito, M., Basso, K., Niu, H. & Dalla-Favera, R. BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells. Nat. Immunol. 6, 1054–1060 (2005).

    Article  CAS  Google Scholar 

  41. Margalit, O. et al. BCL6 is regulated by p53 through a response element frequently disrupted in B-cell non-Hodgkin lymphoma. Blood 107, 1599–1607 (2006).

    Article  CAS  Google Scholar 

  42. Basso, K. Tracking CD40 signaling during germinal center development. Blood 104, 4088–4096 (2004).

    Article  CAS  Google Scholar 

  43. Allen, C.D., Okada, T., Tang, H.L. & Cyster, J.G. Imaging of germinal center selection events during affinity maturation. Science 315, 528–531 (2007).

    Article  CAS  Google Scholar 

  44. Fujita, N. et al. MTA3 and Mi-2/NuRD complex regulate cell fate during B-lymphocyte differentiation. Cell 119, 75–86 (2004).

    Article  CAS  Google Scholar 

  45. Ho, C.C. et al. The relative contribution of CHK1 and CHK2 to adriamycin-induced checkpoint. Exp. Cell Res. 304, 1–15 (2005).

    Article  CAS  Google Scholar 

  46. Lewis, K.A. et al. Heterozygous ATR mutations in mismatch repair-deficient cancer cells have functional significance. Cancer Res. 65, 7091–7095 (2005).

    Article  CAS  Google Scholar 

  47. Rossi, R., Lidonnici, M.R., Soza, S., Biamonti, G. & Montecucco, A. The dispersal of replication proteins after etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway. Cancer Res. 66, 1675–1683 (2006).

    Article  CAS  Google Scholar 

  48. Oberley, M.J., Tsao, J., Yau, P. & Farnham, P.J. High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays. Methods Enzymol. 376, 315–334 (2004).

    Article  CAS  Google Scholar 

  49. Gifford, L.K. et al. Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules. Nucleic Acids Res. 33, e28 (2005).

    Article  Google Scholar 

  50. Singh, N.P., McCoy, M.T., Tice, R.R. & Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191 (1988).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Schreiber and K. Cimprich for FLAG-ATR constructs, and A. Follenzi for assistance in establishing high-efficiency lentiviral transduction of primary B cells. Supported by the Cancer Research Institute (S.M.R.), the National Cancer Center (J.M.P.), the National Cancer Institute (R01 CA104348 to A.M. and R01 CA100885 to M.C.), the Leukemia and Lymphoma Society (A.M. and M.C.), the Chemotherapy Foundation (A.M.), the Samuel Waxman Cancer Research Foundation (A.M.) and the G&P Foundation (A.M.).

Author information

Authors and Affiliations

Authors

Contributions

S.M.R. designed and did experiments and wrote the paper; J.M.P. did chromatin immunoprecipitation (quantitative and on a chip); J.D. did 'comet' assays; M.S. did microarray experiments; T.K. developed critical reagents; J.G. designed microarray experiments; R.G. designed experiments; and M.C. and A.M. designed and conceived experiments and wrote the paper.

Corresponding authors

Correspondence to Martin Carroll or Ari Melnick.

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Competing interests

M.S. and R.G. are employees of NimbleGen Systems.

Supplementary information

Supplementary Fig. 1

ChIP-on-chip of BCL6 on the CCL3 and GAPDH loci. (PDF 162 kb)

Supplementary Fig. 2

Phenotypic characterization of primary B cell populations. (PDF 291 kb)

Supplementary Fig. 3

BCL6 shRNA knockdown in centroblasts and DLBCL cells. (PDF 484 kb)

Supplementary Fig. 4

Electroporation of a second BCL6 shRNA (shBCL6 II) mediates similar effects as shBCL6 I in centroblasts. (PDF 281 kb)

Supplementary Fig. 5

BPI mediates similar effects as BCL6 shRNA in centroblasts. (PDF 561 kb)

Supplementary Fig. 6

BCL6 shRNA II rescues ATR from BCL6-mediated repression and the BCL6 damage-sensing attenuation phenotype. (PDF 507 kb)

Supplementary Fig. 7

A BCL6 transcriptional circuit mediates the germinal center B cell phenotype. (PDF 312 kb)

Supplementary Table 1

The table lists the coordinates of the gene loci, probe sequences, starting position of each probe, and log-ratio enrichment of DNA probes used for ChIP on chip on the ATR, CCL3 and GAPDH loci - including all three replicates as indicated (see accompanying Excel file). (XLS 231 kb)

Supplementary Table 2

shRNA and siRNA sequences used to deplete BCL6 and ATR (shRNA loop is in bold letters). (PDF 80 kb)

Supplementary Table 3

Primers used for qPCR. (PDF 64 kb)

Supplementary Table 4

Complete list of antibodies. (PDF 80 kb)

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Ranuncolo, S., Polo, J., Dierov, J. et al. Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR. Nat Immunol 8, 705–714 (2007). https://doi.org/10.1038/ni1478

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