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53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility

Abstract

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2fl/-) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ)1,2. Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.

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Figure 1: Requirement for 53BP1 in NHEJ of dysfunctional telomeres, but not in DNA damage signalling.
Figure 2: Optimal NHEJ of dysfunctional telomeres requires interaction of 53BP1 with H4K20me2.
Figure 3: 53BP1 promotes mobility of dysfunctional telomeres.

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Acknowledgements

We are grateful to J. Chen for his gift of 53BP1- and MDC1-deficient mice. We thank D. White for expert mouse husbandry. We thank the Bio-Imaging Facilities at Cold Spring Harbor Laboratory and at The Rockefeller University. J. Petrini is thanked for his gift of NBS1 antibody, A. Sfeir for providing the TRF1 antibody and A. Konishi for the BP1-2 construct. M. Dimitrov is thanked for mathematical advice on data analysis, and S. Jaramillo and Y. Fu for Matlab programming. Members of the de Lange laboratory are thanked for comments on the manuscript. This work was supported by a grant from the NIH (GM049046) and by the NIH Director’s Pioneer Award (OD000379) to T.d.L., and grants from the NIH to D.L.S. (EY18244 and GM42694). N.D. was supported by an HHMI pre-doctoral fellowship.

Author Contributions N.D. and T.d.L. planned and designed the experiments. N.D. performed all experiments. Y.-C.M.C. and D.L.S. provided assistance with the imaging experiments. N.D. and T.d.L. wrote the paper and made the figures.

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Correspondence to Titia de Lange.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-11 with Legends and Supplementary Movie Legends 1-4 (PDF 5335 kb)

Supplementary Movie 1

Dynamic movement of dysfunctional telomeres is diminished in TRF2F/-53BP1-/- cells. Representative TRF2F/-53BP1+/- cells imaged 72 h after Cre infection. Z-stacks of EGFP-TRF1 [green] and mCherry-BP1-2 [red] signals were acquired every 30 sec over 20 min. Video constructed from deconvolved and projected frames. (MOV 105 kb)

Supplementary Movie 2

Dynamic movement of dysfunctional telomeres is diminished in TRF2F/-53BP1-/- cells: representative TRF2F/-53BP1-/- cells imaged 72 h after Cre infection. Z-stacks of EGFP-TRF1 [green] and mCherry-BP1-2 [red] signals were acquired every 30 sec over 20 min. Video constructed from deconvolved and projected frames. (MOV 78 kb)

Supplementary Movie 3

Potential fusion events in TRF2F/-53BP1+/- but not in TRF2F/-53BP1-/- cells. Videos from deconvolved and projected frames. The movie highlights telomeres undergoing a potential fusion reaction in a TRF2F/-53BP1+/- cell. (MOV 17 kb)

Supplementary Movie 4

Potential fusion events in TRF2F/-53BP1+/- but not in TRF2F/-53BP1-/- cells. Videos from deconvolved and projected frames. The movie highlights telomeres undergoing a potential fusion reaction in a group of closely apposed, but static telomeres in a TRF2F/-53BP1-/- cell. (MOV 12 kb)

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Dimitrova, N., Chen, YC., Spector, D. et al. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524–528 (2008). https://doi.org/10.1038/nature07433

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