- Email: [email protected]
Reshaping Chromatin Architecture around DNA Breaks
Please cite this article in press as: Caron and Polo, Reshaping Chromatin Architecture around DNA Breaks, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.12.001
Spotlight
Reshaping Chromatin Architecture around DNA Breaks
tion of discernable nuclear foci. How does the spatial organization of 53BP1 in foci at DSBs contribute to its function in protecting broken ends from degradation?
Pierre Caron1 and Sophie E. Polo1,*
Super-resolution imaging of human cells by 3D structured illumination microscopy (3D-SIM) revealed that 53BP1 and the resection-promoting factor breast cancer type 1 susceptibility protein (BRCA1) spatially organize into defined and mutually exclusive territories on damaged chromatin [3]. This spatial segregation of repair factors reflects their antagonistic functions during the repair of DSBs.
DNA double-strand breaks (DSBs) elicit major chromatin changes. Using superresolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity. DNA double-strand break (DSB) repair invokes distinct repair pathways and is orchestrated by chromatin-associated factors that couple DNA repair events with chromatin changes. The DSB response protein p53 binding protein 1 (53BP1) binds damaged chromatin through the recognition of both DNA damage-induced and cell cycle-regulated histone modifications. 53BP1 accrual is thus favored in pre-replicative chromatin and channels DSB repair to the nonhomologous end-joining (NHEJ) pathway at the expense of homologous recombination (HR) repair by inhibiting the resection of broken DNA ends. Mechanistically, 53BP1 cooperates with its effector replication timing regulatory factor 1 (RIF1), which elicits the assembly of the Shieldin complex protecting broken DNA ends from resection [1] (Figure 1A). Such a scenario implies a role for the 53BP1dependent axis directly at the vicinity of the broken ends, where the competition between NHEJ- and HR-mediated repair occurs. However, 53BP1 accrual on damaged chromatin is not restricted to the broken ends but spreads over megabases [2], leading to the forma-
However, it was still unknown whether the tridimensional organization of chromatin in the nuclear space may control 53BP1 focal accumulation and whether 53BP1 accrual may in turn impact chromatin organization in the vicinity of DSBs. In a recent issue of Nature, Lukas and coworkers explore the reciprocal relationship between chromatin topology and the formation of repair foci in response to DSBs [4]. Using 3D-SIM and 2D-stimulated emission depletion super-resolution microscopy techniques in various human cell lines exposed to ionizing radiation, they reveal that 53BP1 foci consist of several nano-domains that assemble into a circular micro-domain (Figure 1B). This peculiar 53BP1 pattern forms on chromatin as it mirrors the spatial distribution of gH2A.X [5] and of core histones and surrounds a central interchromatin space. Intriguingly, even though the size of 53BP1 foci is reduced in postreplicative conditions [3], circular micro-domains of 53BP1 form similarly in pre- and post-replicative chromatin [4], arguing that this tridimensional organization of repair foci is cell cycle-independent. This observation is consis-
tent with the fact that 53BP1 foci not only foster NHEJ in G1 but more generally prevent mutagenic repair by limiting hyper-resection throughout the cell cycle. Live-cell SIM further reveals sequential steps in 53BP1 accrual, with first 53BP1 spreading around the break to form nano-domains, and subsequent circular arrangement of the nano-domains into a micro-domain. Mechanistically, the formation of 53BP1 micro-domains relies on 53BP1 interaction with RIF1 but not on the Shieldin complex [4]. RIF1 depletion indeed disrupts circular 53BP1 micro-domains, which instead display an elongated shape, and a mutated form of 53BP1 that fails to bind RIF1 also does not organize into micro-domains. Moreover, depletion of RIF1 similarly disrupts the higher-order organization of damaged chromatin as visualized by gH2A.X staining. Together, these findings demonstrate that RIF1 drives the circularization of 53BP1 nano-domains on damaged chromatin. Besides 53BP1 foci arising in response to DSBs, 53BP1 nuclear bodies form in G1 cells on under-replicated DNA from the previous cell cycle and these bodies also contain RIF1 [6]. It is thus tempting to speculate that a similar reshaping of chromatin architecture by RIF1 might sustain the formation of 53BP1 nuclear bodies. Remarkably, although RIF1 binds 53BP1 and organizes 53BP1 foci, RIF1 does not colocalize with 53BP1 nanodomains, but instead accumulates at the boundaries between these domains [4]. Thus, RIF1 appears as the cornerstone that drives higher-order chromatin organization around a DSB by maturing the 53BP1 nano-domains into one micro-domain. Consistent with these findings, a function of RIF1 in shaping the 3D organization of the genome has been described in
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
1
Please cite this article in press as: Caron and Polo, Reshaping Chromatin Architecture around DNA Breaks, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.12.001
Figure 1. A New Perspective on DNA Repair Foci: 3D Reorganization of Chromatin Architecture around DNA Double-Strand Breaks (DSBs). Following DNA damage by ionizing radiation (IR), DSB response proteins form foci in the cell nucleus. (A) Linear view of DSB response protein distribution in the vicinity of a DNA break. p53-binding protein 1 (53BP1, green) binds to histone modifications on damaged chromatin at the vicinity of the DSB and recruits replication timing regulatory factor 1 (RIF1, purple), which elicits the assembly of the Shieldin complex (SHLD, blue). Shieldin protects broken DNA ends from nucleolytic degradation by resection factors (yellow). (B) 3D view of a DSB repair focus: RIF1 shapes chromatin topology around DSBs. The reorganization of DSB-flanking chromatin proceeds in two steps. (1) The spreading of 53BP1 (green) on chromatin occurs over megabases around the DSB and is shaped by chromatin topology with the formation of distinct 53BP1 nano-domains corresponding to chromatin topologically associated domains (TADs). (2) RIF1 accrual (purple) leads to the circularization of 53BP1 nano-domains into one micro-domain. RIF1 builds on a preexisting chromatin topology, defined by cohesins (purple rings), to shape chromatin architecture around DSBs. The resulting higher-order organization into a ring-shaped structure regulates the distribution of repair proteins and constrains pro-resection factors (yellow) into the interchromatin space. This provides a chromatin compartment that is amenable for DSB repair by nonhomologous end-joining factors (NHEJ, orange).
mammalian cells, where RIF1 restricts long-range chromatin interactions between domains having the same replication timing [7]. Functionally, 53BP1 micro-domains constrain the distribution of repair factors, with resection-associated proteins like BRCA1 and replication protein A (RPA) being confined to the central interchromatin space while NHEJ factors colocalize with 53BP1 nano-domains. Loss of the micro-domain structure upon RIF1 depletion leads to an invasion of BRCA1 and RPA within 53BP1 nano-domains [4]. Such spatial segre2
gation of repair factors by 53BP1 micro-domain organization may exert a repressive effect on resection and channel DSB repair to NHEJ. It will be interesting to determine whether 53BP1 liquid phase separation properties [8] may contribute to this compartmentalization of repair factors by reinforcing the higher-order topological arrangement of chromatin at DSBs. The authors further explore the connection between 53BP1 spatial arrangement and chromatin topology by using Cas9 nuclease to introduce a DSB within a topologically associated
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
domain (TAD). Immuno-fluorescence in situ hybridization analyses reveal that each 53BP1 nano-domain actually corresponds to a unique TAD. This strongly suggests that 53BP1 accrual on damaged chromatin follows chromatin topology and that 53BP1 effector RIF1 subsequently elicits the circular arrangement of several TADs flanking a given DSB. The positions of TAD boundaries are cohesin-dependent [9]. Strikingly, perturbation of chromatin topology by cohesin depletion phenocopies the loss of RIF1 in disrupting 53BP1 micro-domains [4]. Thus, RIF1 is not sufficient for shaping chromatin
Please cite this article in press as: Caron and Polo, Reshaping Chromatin Architecture around DNA Breaks, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.12.001
architecture around DSBs. RIF1 builds on a pre-existing chromatin topology, defined by cohesins. While the impact of cohesin depletion on RIF1 loading and on the distribution of resection factors still need to be examined, these findings support the idea that RIF1 stabilizes chromatin topology around DSBs. High-resolution microscopy and chromosome conformation capture techniques unveiled the 3D-organization of the genome and its regulatory function in crucial processes such as transcription [10] or replication [7]. The study by Ochs et al. [4] highlights a novel interplay between chromatin architecture and DNA damage response factors, which shape chromatin topology around DSBs, thus providing a new perspective on repair foci. Future studies, using imaging and orthogonal approaches, may provide further mechanistic insights into the formation and the resolution of these structures,
their functional relevance for the regulation of DSB responses, and their possible long-term and long-range impact on chromatin architecture.
2.
3.
Acknowledgments We thank members of our laboratory for stimulating discussions and the European Research Council (ERC-2018CoG-818625), the French National Research Agency (ANR-18-CE12-001701), and the Fondation ARC (PDF20190509195) for funding.
4. 5.
6.
1Epigenetics
and Cell Fate Centre, UMR7216 CNRS, Universite´ de Paris, F-75013, Paris, France
7.
*Correspondence: [email protected] https://doi.org/10.1016/j.tibs.2019.12.001
8.
ª 2019 Elsevier Ltd. All rights reserved. 9.
References
10.
1. Scully, R. et al. (2019) DNA double-strand break repair-pathway choice in somatic
mammalian cells. Nat. Rev. Mol. Cell Biol. 20, 698–714 Clouaire, T. et al. (2018) Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol. Cell 72, 250–262 Chapman, J.R. et al. (2012) BRCA1associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J. Cell. Sci. 125, 3529– 3534 Ochs, F. et al. (2019) Stabilization of chromatin topology safeguards genome integrity. Nature 574, 571–574 Natale, F. et al. (2017) Identification of the elementary structural units of the DNA damage response. Nat. Commun. 8, 15760 Spies, J. et al. (2019) 53BP1 nuclear bodies enforce replication timing at underreplicated DNA to limit heritable DNA damage. Nat. Cell Biol. 21, 487–497 Foti, R. et al. (2016) Nuclear architecture organized by Rif1 underpins the replication-timing program. Mol. Cell 61, 260–273 Kilic, S. et al. (2019) Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments. EMBO J. 38, e101379 Bintu, B. et al. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, eaau1783 Dekker, J. and Misteli, T. (2015) Long-range chromatin interactions. Cold Spring Harb. Perspect. Biol. 7, a019356
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
3
Spotlight
Reshaping Chromatin Architecture around DNA Breaks
tion of discernable nuclear foci. How does the spatial organization of 53BP1 in foci at DSBs contribute to its function in protecting broken ends from degradation?
Pierre Caron1 and Sophie E. Polo1,*
Super-resolution imaging of human cells by 3D structured illumination microscopy (3D-SIM) revealed that 53BP1 and the resection-promoting factor breast cancer type 1 susceptibility protein (BRCA1) spatially organize into defined and mutually exclusive territories on damaged chromatin [3]. This spatial segregation of repair factors reflects their antagonistic functions during the repair of DSBs.
DNA double-strand breaks (DSBs) elicit major chromatin changes. Using superresolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity. DNA double-strand break (DSB) repair invokes distinct repair pathways and is orchestrated by chromatin-associated factors that couple DNA repair events with chromatin changes. The DSB response protein p53 binding protein 1 (53BP1) binds damaged chromatin through the recognition of both DNA damage-induced and cell cycle-regulated histone modifications. 53BP1 accrual is thus favored in pre-replicative chromatin and channels DSB repair to the nonhomologous end-joining (NHEJ) pathway at the expense of homologous recombination (HR) repair by inhibiting the resection of broken DNA ends. Mechanistically, 53BP1 cooperates with its effector replication timing regulatory factor 1 (RIF1), which elicits the assembly of the Shieldin complex protecting broken DNA ends from resection [1] (Figure 1A). Such a scenario implies a role for the 53BP1dependent axis directly at the vicinity of the broken ends, where the competition between NHEJ- and HR-mediated repair occurs. However, 53BP1 accrual on damaged chromatin is not restricted to the broken ends but spreads over megabases [2], leading to the forma-
However, it was still unknown whether the tridimensional organization of chromatin in the nuclear space may control 53BP1 focal accumulation and whether 53BP1 accrual may in turn impact chromatin organization in the vicinity of DSBs. In a recent issue of Nature, Lukas and coworkers explore the reciprocal relationship between chromatin topology and the formation of repair foci in response to DSBs [4]. Using 3D-SIM and 2D-stimulated emission depletion super-resolution microscopy techniques in various human cell lines exposed to ionizing radiation, they reveal that 53BP1 foci consist of several nano-domains that assemble into a circular micro-domain (Figure 1B). This peculiar 53BP1 pattern forms on chromatin as it mirrors the spatial distribution of gH2A.X [5] and of core histones and surrounds a central interchromatin space. Intriguingly, even though the size of 53BP1 foci is reduced in postreplicative conditions [3], circular micro-domains of 53BP1 form similarly in pre- and post-replicative chromatin [4], arguing that this tridimensional organization of repair foci is cell cycle-independent. This observation is consis-
tent with the fact that 53BP1 foci not only foster NHEJ in G1 but more generally prevent mutagenic repair by limiting hyper-resection throughout the cell cycle. Live-cell SIM further reveals sequential steps in 53BP1 accrual, with first 53BP1 spreading around the break to form nano-domains, and subsequent circular arrangement of the nano-domains into a micro-domain. Mechanistically, the formation of 53BP1 micro-domains relies on 53BP1 interaction with RIF1 but not on the Shieldin complex [4]. RIF1 depletion indeed disrupts circular 53BP1 micro-domains, which instead display an elongated shape, and a mutated form of 53BP1 that fails to bind RIF1 also does not organize into micro-domains. Moreover, depletion of RIF1 similarly disrupts the higher-order organization of damaged chromatin as visualized by gH2A.X staining. Together, these findings demonstrate that RIF1 drives the circularization of 53BP1 nano-domains on damaged chromatin. Besides 53BP1 foci arising in response to DSBs, 53BP1 nuclear bodies form in G1 cells on under-replicated DNA from the previous cell cycle and these bodies also contain RIF1 [6]. It is thus tempting to speculate that a similar reshaping of chromatin architecture by RIF1 might sustain the formation of 53BP1 nuclear bodies. Remarkably, although RIF1 binds 53BP1 and organizes 53BP1 foci, RIF1 does not colocalize with 53BP1 nanodomains, but instead accumulates at the boundaries between these domains [4]. Thus, RIF1 appears as the cornerstone that drives higher-order chromatin organization around a DSB by maturing the 53BP1 nano-domains into one micro-domain. Consistent with these findings, a function of RIF1 in shaping the 3D organization of the genome has been described in
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
1
Please cite this article in press as: Caron and Polo, Reshaping Chromatin Architecture around DNA Breaks, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.12.001
Figure 1. A New Perspective on DNA Repair Foci: 3D Reorganization of Chromatin Architecture around DNA Double-Strand Breaks (DSBs). Following DNA damage by ionizing radiation (IR), DSB response proteins form foci in the cell nucleus. (A) Linear view of DSB response protein distribution in the vicinity of a DNA break. p53-binding protein 1 (53BP1, green) binds to histone modifications on damaged chromatin at the vicinity of the DSB and recruits replication timing regulatory factor 1 (RIF1, purple), which elicits the assembly of the Shieldin complex (SHLD, blue). Shieldin protects broken DNA ends from nucleolytic degradation by resection factors (yellow). (B) 3D view of a DSB repair focus: RIF1 shapes chromatin topology around DSBs. The reorganization of DSB-flanking chromatin proceeds in two steps. (1) The spreading of 53BP1 (green) on chromatin occurs over megabases around the DSB and is shaped by chromatin topology with the formation of distinct 53BP1 nano-domains corresponding to chromatin topologically associated domains (TADs). (2) RIF1 accrual (purple) leads to the circularization of 53BP1 nano-domains into one micro-domain. RIF1 builds on a preexisting chromatin topology, defined by cohesins (purple rings), to shape chromatin architecture around DSBs. The resulting higher-order organization into a ring-shaped structure regulates the distribution of repair proteins and constrains pro-resection factors (yellow) into the interchromatin space. This provides a chromatin compartment that is amenable for DSB repair by nonhomologous end-joining factors (NHEJ, orange).
mammalian cells, where RIF1 restricts long-range chromatin interactions between domains having the same replication timing [7]. Functionally, 53BP1 micro-domains constrain the distribution of repair factors, with resection-associated proteins like BRCA1 and replication protein A (RPA) being confined to the central interchromatin space while NHEJ factors colocalize with 53BP1 nano-domains. Loss of the micro-domain structure upon RIF1 depletion leads to an invasion of BRCA1 and RPA within 53BP1 nano-domains [4]. Such spatial segre2
gation of repair factors by 53BP1 micro-domain organization may exert a repressive effect on resection and channel DSB repair to NHEJ. It will be interesting to determine whether 53BP1 liquid phase separation properties [8] may contribute to this compartmentalization of repair factors by reinforcing the higher-order topological arrangement of chromatin at DSBs. The authors further explore the connection between 53BP1 spatial arrangement and chromatin topology by using Cas9 nuclease to introduce a DSB within a topologically associated
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
domain (TAD). Immuno-fluorescence in situ hybridization analyses reveal that each 53BP1 nano-domain actually corresponds to a unique TAD. This strongly suggests that 53BP1 accrual on damaged chromatin follows chromatin topology and that 53BP1 effector RIF1 subsequently elicits the circular arrangement of several TADs flanking a given DSB. The positions of TAD boundaries are cohesin-dependent [9]. Strikingly, perturbation of chromatin topology by cohesin depletion phenocopies the loss of RIF1 in disrupting 53BP1 micro-domains [4]. Thus, RIF1 is not sufficient for shaping chromatin
Please cite this article in press as: Caron and Polo, Reshaping Chromatin Architecture around DNA Breaks, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.12.001
architecture around DSBs. RIF1 builds on a pre-existing chromatin topology, defined by cohesins. While the impact of cohesin depletion on RIF1 loading and on the distribution of resection factors still need to be examined, these findings support the idea that RIF1 stabilizes chromatin topology around DSBs. High-resolution microscopy and chromosome conformation capture techniques unveiled the 3D-organization of the genome and its regulatory function in crucial processes such as transcription [10] or replication [7]. The study by Ochs et al. [4] highlights a novel interplay between chromatin architecture and DNA damage response factors, which shape chromatin topology around DSBs, thus providing a new perspective on repair foci. Future studies, using imaging and orthogonal approaches, may provide further mechanistic insights into the formation and the resolution of these structures,
their functional relevance for the regulation of DSB responses, and their possible long-term and long-range impact on chromatin architecture.
2.
3.
Acknowledgments We thank members of our laboratory for stimulating discussions and the European Research Council (ERC-2018CoG-818625), the French National Research Agency (ANR-18-CE12-001701), and the Fondation ARC (PDF20190509195) for funding.
4. 5.
6.
1Epigenetics
and Cell Fate Centre, UMR7216 CNRS, Universite´ de Paris, F-75013, Paris, France
7.
*Correspondence: [email protected] https://doi.org/10.1016/j.tibs.2019.12.001
8.
ª 2019 Elsevier Ltd. All rights reserved. 9.
References
10.
1. Scully, R. et al. (2019) DNA double-strand break repair-pathway choice in somatic
mammalian cells. Nat. Rev. Mol. Cell Biol. 20, 698–714 Clouaire, T. et al. (2018) Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol. Cell 72, 250–262 Chapman, J.R. et al. (2012) BRCA1associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J. Cell. Sci. 125, 3529– 3534 Ochs, F. et al. (2019) Stabilization of chromatin topology safeguards genome integrity. Nature 574, 571–574 Natale, F. et al. (2017) Identification of the elementary structural units of the DNA damage response. Nat. Commun. 8, 15760 Spies, J. et al. (2019) 53BP1 nuclear bodies enforce replication timing at underreplicated DNA to limit heritable DNA damage. Nat. Cell Biol. 21, 487–497 Foti, R. et al. (2016) Nuclear architecture organized by Rif1 underpins the replication-timing program. Mol. Cell 61, 260–273 Kilic, S. et al. (2019) Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments. EMBO J. 38, e101379 Bintu, B. et al. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, eaau1783 Dekker, J. and Misteli, T. (2015) Long-range chromatin interactions. Cold Spring Harb. Perspect. Biol. 7, a019356
Trends in Biochemical Sciences, Month 2019, Vol. -, No. -
3