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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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.).

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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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