**3.1. Evidence of increased mobility in response to DSB**

Most of the studies on chromatin mobility are based on the analysis of MSD curves calculated from the trajectories of fluorescently labeled chromatin loci. Using this approach, the diffusion coefficients reported in the literature varies from 5 × 10−<sup>5</sup> to 10−<sup>3</sup> μm2 /s depending on the organisms, the loci studied, and the type of damage [66, 67]. In several studies, chromatin undergoes confined diffusion [22, 59, 68–71], while others have reported anomalous diffusion [69, 70, 72–75]. So far, no consensus has been reached to describe the nature of DNA motion probably because these studies have been performed using different microscopy techniques and illumination settings. Indeed, multi time-scales observation of chromatin motion revealed that chromatin is driven by different types of diffusion at each time scale [76–78]. As a consequence, the type of diffusion depends on the time scale of observation. While comparing studies on chromatin dynamics, it is thus important to compare studies performed at similar time scales.

Several studies have investigated DNA motion in the context of DNA damage. Since DSB is the most deleterious type of damage in the cell, chromatin dynamics in the context of DNA repair has been mainly investigated in response to DSBs. The changes in chromatin architecture and dynamics following DSB have been studied mostly in yeast, Drosophila and mammalian nuclei. In budding yeast, chromatin mobility has been investigated during the process of HR, when a Rad52 focus is already formed at the damaged locus [23, 25]. In diploid, where a homologous template is available, chromatin mobility is dramatically increased at the damaged site, allowing the damaged locus to explore a nuclear volume 10 times larger [23]. Increased mobility may facilitate homology search; however, haploid yeast cells, where no homologous template is present, also exhibit increased mobility in response to DSBs [25]. Since the two broken ends stay in contact during the process of HR repair [79], the current view is that the two broken ends explore the nuclear space together.

Importantly, only induced DSB associated to Rad52 foci display increased mobility. Indeed, during resection, the early stage of HR, a strong inhibition of chromatin mobility has been reported in yeast, highlighting the importance of the stage of DNA repair in chromatin mobility changes [51]. Finally, different types of damages have very different consequences on DNA mobility. For example, in yeast, spontaneous DSBs occurring during DNA replication exhibit decreased mobility [80]; DSBs induced by a protein-DNA adduct display no change in motion and camptothecin (CPT)-induced Rad52 foci display no increased mobility [25].

In mammalian cells, while several studies have reported increased chromatin mobility upon DNA damage, others fail to observe significant changes. In HeLa cells, after α-particleinduced DSBs, γH2AX foci are more mobile [81, 82]. Similarly, uncapped telomeres exhibit increased mobility in mouse cells, and this movement is dependent on the 53BP1 repair protein [83]. Movement of heterochromatic DSBs toward euchromatin was observed in mouse embryo fibroblasts (MEFs), HeLa cells [84], and Drosophila cells [45]. It was proposed that re-localization of heterochromatic DSBs close to euchromatin regions prevents rearrangements between repetitive DNA sequences present in heterochromatin. Taken together, these studies suggest that chromosome mobility increases significantly in the presence of DSBs. By contrast, in other studies using MEFs [85], HeLa, or U2OS cells [86], DSBs generated by UV laser or γ-irradiation did not significantly alter chromosome mobility. Only energydependent local expansion of chromatin was observed around the initial damaged zone immediately after DNA damage [85]. These contradictory observations in mammalian cells probably result from variation between cell lines, the regions of chromatin damaged, and the type of damage induced. Recent studies suggest that DSBs induced in active genes, naturally enriched in the trimethyl form of histone H3 lysine 36 (H3K36me3), are repaired by HR [87]. These DSBs are susceptible to exhibit increased mobility, while DSBs repaired by Non-Homologous End Joining are rather immobile. Further systematic studies will be necessary to confirm these observations.

#### **3.2. Local versus global increased mobility**

For anomalous motion with a diffusion coefficient *D*, the MSD formula including the correc-

Most of the studies on chromatin mobility are based on the analysis of MSD curves calculated from the trajectories of fluorescently labeled chromatin loci. Using this approach, the diffusion

isms, the loci studied, and the type of damage [66, 67]. In several studies, chromatin undergoes confined diffusion [22, 59, 68–71], while others have reported anomalous diffusion [69, 70, 72–75]. So far, no consensus has been reached to describe the nature of DNA motion probably because these studies have been performed using different microscopy techniques and illumination settings. Indeed, multi time-scales observation of chromatin motion revealed that chromatin is driven by different types of diffusion at each time scale [76–78]. As a consequence, the type of diffusion depends on the time scale of observation. While comparing studies on chromatin dynamics, it is thus important to compare studies performed at similar time scales. Several studies have investigated DNA motion in the context of DNA damage. Since DSB is the most deleterious type of damage in the cell, chromatin dynamics in the context of DNA repair has been mainly investigated in response to DSBs. The changes in chromatin architecture and dynamics following DSB have been studied mostly in yeast, Drosophila and mammalian nuclei. In budding yeast, chromatin mobility has been investigated during the process of HR, when a Rad52 focus is already formed at the damaged locus [23, 25]. In diploid, where a homologous template is available, chromatin mobility is dramatically increased at the damaged site, allowing the damaged locus to explore a nuclear volume 10 times larger [23]. Increased mobility may facilitate homology search; however, haploid yeast cells, where no homologous template is present, also exhibit increased mobility in response to DSBs [25]. Since the two broken ends stay in contact during the process of HR repair [79], the current view is that the two broken

Importantly, only induced DSB associated to Rad52 foci display increased mobility. Indeed, during resection, the early stage of HR, a strong inhibition of chromatin mobility has been reported in yeast, highlighting the importance of the stage of DNA repair in chromatin mobility changes [51]. Finally, different types of damages have very different consequences on DNA mobility. For example, in yeast, spontaneous DSBs occurring during DNA replication exhibit decreased mobility [80]; DSBs induced by a protein-DNA adduct display no change in motion and campto-

In mammalian cells, while several studies have reported increased chromatin mobility upon DNA damage, others fail to observe significant changes. In HeLa cells, after α-particleinduced DSBs, γH2AX foci are more mobile [81, 82]. Similarly, uncapped telomeres exhibit

thecin (CPT)-induced Rad52 foci display no increased mobility [25].

to 10−<sup>3</sup> μm2

/s depending on the organ-

tions for localization accuracy is described in [65].

54 Chromatin and Epigenetics

**3. Increased mobility in response to DNA damage**

**3.1. Evidence of increased mobility in response to DSB**

coefficients reported in the literature varies from 5 × 10−<sup>5</sup>

ends explore the nuclear space together.

Changes in chromatin conformation have been extensively described around the site of damage but an important question is whether these changes in chromatin mobility also affect the rest of the genome. Interestingly, in budding yeast, increased chromatin mobility is not an intrinsic property of the damaged locus. Indeed, in diploid yeast, after induction of four random DSBs per nucleus by γ-irradiation, undamaged loci explore a 2.4 times larger nuclear volume than in the absence of irradiation [23]. Moreover, the global increased mobility is dose-dependent since upon induction of approximately 20 DSBs, the chromosomes explore almost the entire yeast nucleus [23]. **Figure 3** illustrates the mobility observed for different levels of γ-irradiation in a diploid yeast cell. Global increased mobility is observed in haploid yeast, although it required higher doses of damages and it has been tested with a different type of DSBs (zeocin-induced DSBs) [88].

In mammalian cells, changes in mobility far from a damaged locus are not reported in the literature. Since mammalian nuclei are much larger than the yeast nuclei, but chromatin motion exhibits very similar constrained (~0.5 μm of confined radius), it is likely that global mobility is specific to organisms with small nuclei and is therefore not present in mammalian cells.

Importantly, most of these studies investigated chromatin mobility at one specific time scale. However, when studying the diffusion of a specific locus, the time scale at which data are collected reflects a specific spatial scale of the exploration studied. From nucleosomes to fiber, the different scales of chromatin organization might exhibit different diffusion behaviors. Using fast microscopy, a recent study investigated DNA mobility at several time scales, up to 1000 times faster than previously observed [65]. These experiments revealed that DNA motion following DNA damage is more complex than what had been previously described. Chromatin dynamics therefore appears to be scale-dependent: in response to DNA damage,

These studies revealed that several genes involved in DSBs repair and chromatin remodeling are involved in chromatin mobility changes. In diploid yeast, Rad51, the central protein of HR, is required for chromate in mobility [23], as well as for increased chromatin rigidity [65]. Several checkpoint proteins acting earlier than Rad51 nucleoprotein filament formation are also essential for increasing the mobility of the damaged locus. For example, Rad9, a protein containing a BRCT domain roughly equivalent to human MDC1, BRCA1, and 53BP1, is also required for increased mobility at the damaged site in haploid yeast [25]. MEC1 and SML1, but not RAD53 and Tel1, are essential for increasing the mobility of the damaged locus [23, 25, 66]. Interestingly, the activation of the checkpoint protein Mec1 at a specific locus is sufficient to promote increased mobility, even in the absence of physical DSB [88]. Finally, in mouse cells, increased chromosome movements are associated with uncapped telomeres, and this movement is dependent on the 53BP1

Chromatin Dynamics upon DNA Damage http://dx.doi.org/10.5772/intechopen.83559 57

Another study proposed that centromere and telomere release following DSBs are at the origin of chromatin changes in mobility. Strecker et al. found that a combined disruption of telomeres and centromeres can reproduce chromatin mobility observed after a DSB [96]; they identified the Mec1-dependent phosphorylation of Cep3, a kinetochore component, as

More recently, it has been shown that molecular motors play an important role in DSBs mobility [93–95]. For example, in fly in human genomes, heterochromatin constitutes about 30% of the genome [97], and "safe" repair of heterochromatic DSBs by homologous recombination relies on the relocalization of repair foci to the nuclear periphery. During this process, nuclear actin filaments form at repair sites to drive heterochromatin DSBs at the periphery and disassemble after relocalization [93]. Actin filaments act in concert with Smc5/6, Arp2/3, Arp2/3 activators Scar and Wash, nuclear myosins Myo1A, Myo1B, and MyoV. Interestingly, in U2OS cells, ARP2/3-mediated actin polymerization enhances DSBs motion during homologous recombination, increasing the clustering of repair foci [94]. In budding yeast, DNA-damaged induced nuclear microtubule filaments (DIMs) form in response to endogenous or exogenous DNA damage [95]. These DIM filaments, formed at repair sites, reach the nuclear periphery to dive irreparable DSBs and disassemble after relocalization. Such DSBs motion is mediated by the Rad9 DNA damage response mediator and the Kar3 kinesin motor. Another model implicating microtubules has been proposed by Lawrimore et al. to explain the global increased mobility observed in yeast in response to DNA damage: in their model, microtubules would be responsible for a global chromatin shake-up that would be essential for global increase

In these different examples, DSBs mobility is promoted by molecular motors, and DSBs exhibit a complex motion including mixture of directive and Brownian motions [93] or nonlinear directive motion [95]. Overall, these recent studies revealed the essential role of molecular motors in DSBs mobility to drive heterochromatic or irreparable DSBs to the nuclear periphery or in clustering of multiple DSBs. Importantly, these mechanisms are conserved through several organisms (human, *Drosophila melanogaster*, *Xenopus laevis*, and

an essential player in global increased chromatin mobility on DSBs.

repair protein [83].

mobility on DSBs [98].

budding yeast).

**Figure 3.** Examples of the dynamics of *URA3* loci (chromosome V) in budding yeast as a function of the number of doublestrand breaks (DSBs) in the nucleus. The lines indicate a 2D projection of the trajectories of the two *URA3* loci taken at between 10- and 30-s time intervals for approximately 15 min. (A) In the absence of DSBs, the two homologous loci are distant and explore only 3% of the nuclear volume. (B) In the presence of one to four DSBs induced on chromosomes III, the mobility of the *URA3* loci increases and each locus can explore 11% of the nuclear volume. (C) After about 20 random γ-irradiation-induced DSBs per nucleus (200 Gy), *URA3* loci explore almost the entire nuclear volume and their trajectories overlap. The scale is 1 μm. These three examples illustrate that more DSBs in the nucleus induce greater DNA mobility, thereby increasing the probability of collisions between loci (with permission from [66]).

chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales, this effect being stronger at the damaged site. Such a pattern of dynamics is consistent with a global chromatin stiffness that has been proposed to arise in response to DNA damage [65, 89, 90]. These results underline the importance of performing multiscale tracking to fully understand the complex dynamics of chromatin at each scale.

#### **3.3. DSBs clustering**

In addition to the increased chromatin mobility, several studies indicate that multiple DSBs cluster together into a single "repair center." Clustering of unrelated DSBs might be a consequence of increased mobility, although such clusters of DSBs might promote translocations and their functions is not clear. In yeast, several DNA lesions collapse together into the same repair focus, suggesting that these multiple DSBs are driven to a shared location, in so-called "repair centers" or "repair factories" [91]. In mammalian cells, clusters of radiation-induced foci have been observed in many independent studies [45, 81, 85, 86]. DSB clusters could be formed by collisions and fusion of several DSBs. One interesting hypothesis is that repair foci have a higher viscosity than the rest of the nucleus, due to their high concentration of repair proteins around the damaged site. It has been proposed that several repair foci act as dynamic liquid droplets that are able to fuse together when they collide [92].

#### **3.4. Factors controlling local and global increased mobility**

To investigate the mechanism of increased chromatin mobility in response to DSBs, several studies have tested chromatin mobility in mutant cells [23, 25, 65, 81, 83, 88, 90, 93–95]. These studies revealed that several genes involved in DSBs repair and chromatin remodeling are involved in chromatin mobility changes. In diploid yeast, Rad51, the central protein of HR, is required for chromate in mobility [23], as well as for increased chromatin rigidity [65]. Several checkpoint proteins acting earlier than Rad51 nucleoprotein filament formation are also essential for increasing the mobility of the damaged locus. For example, Rad9, a protein containing a BRCT domain roughly equivalent to human MDC1, BRCA1, and 53BP1, is also required for increased mobility at the damaged site in haploid yeast [25]. MEC1 and SML1, but not RAD53 and Tel1, are essential for increasing the mobility of the damaged locus [23, 25, 66]. Interestingly, the activation of the checkpoint protein Mec1 at a specific locus is sufficient to promote increased mobility, even in the absence of physical DSB [88]. Finally, in mouse cells, increased chromosome movements are associated with uncapped telomeres, and this movement is dependent on the 53BP1 repair protein [83].

Another study proposed that centromere and telomere release following DSBs are at the origin of chromatin changes in mobility. Strecker et al. found that a combined disruption of telomeres and centromeres can reproduce chromatin mobility observed after a DSB [96]; they identified the Mec1-dependent phosphorylation of Cep3, a kinetochore component, as an essential player in global increased chromatin mobility on DSBs.

chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales, this effect being stronger at the damaged site. Such a pattern of dynamics is consistent with a global chromatin stiffness that has been proposed to arise in response to DNA damage [65, 89, 90]. These results underline the importance of performing multiscale

**Figure 3.** Examples of the dynamics of *URA3* loci (chromosome V) in budding yeast as a function of the number of doublestrand breaks (DSBs) in the nucleus. The lines indicate a 2D projection of the trajectories of the two *URA3* loci taken at between 10- and 30-s time intervals for approximately 15 min. (A) In the absence of DSBs, the two homologous loci are distant and explore only 3% of the nuclear volume. (B) In the presence of one to four DSBs induced on chromosomes III, the mobility of the *URA3* loci increases and each locus can explore 11% of the nuclear volume. (C) After about 20 random γ-irradiation-induced DSBs per nucleus (200 Gy), *URA3* loci explore almost the entire nuclear volume and their trajectories overlap. The scale is 1 μm. These three examples illustrate that more DSBs in the nucleus induce greater DNA

In addition to the increased chromatin mobility, several studies indicate that multiple DSBs cluster together into a single "repair center." Clustering of unrelated DSBs might be a consequence of increased mobility, although such clusters of DSBs might promote translocations and their functions is not clear. In yeast, several DNA lesions collapse together into the same repair focus, suggesting that these multiple DSBs are driven to a shared location, in so-called "repair centers" or "repair factories" [91]. In mammalian cells, clusters of radiation-induced foci have been observed in many independent studies [45, 81, 85, 86]. DSB clusters could be formed by collisions and fusion of several DSBs. One interesting hypothesis is that repair foci have a higher viscosity than the rest of the nucleus, due to their high concentration of repair proteins around the damaged site. It has been proposed that several repair foci act as dynamic liquid droplets that are able to fuse together when

To investigate the mechanism of increased chromatin mobility in response to DSBs, several studies have tested chromatin mobility in mutant cells [23, 25, 65, 81, 83, 88, 90, 93–95].

tracking to fully understand the complex dynamics of chromatin at each scale.

mobility, thereby increasing the probability of collisions between loci (with permission from [66]).

**3.4. Factors controlling local and global increased mobility**

**3.3. DSBs clustering**

56 Chromatin and Epigenetics

they collide [92].

More recently, it has been shown that molecular motors play an important role in DSBs mobility [93–95]. For example, in fly in human genomes, heterochromatin constitutes about 30% of the genome [97], and "safe" repair of heterochromatic DSBs by homologous recombination relies on the relocalization of repair foci to the nuclear periphery. During this process, nuclear actin filaments form at repair sites to drive heterochromatin DSBs at the periphery and disassemble after relocalization [93]. Actin filaments act in concert with Smc5/6, Arp2/3, Arp2/3 activators Scar and Wash, nuclear myosins Myo1A, Myo1B, and MyoV. Interestingly, in U2OS cells, ARP2/3-mediated actin polymerization enhances DSBs motion during homologous recombination, increasing the clustering of repair foci [94]. In budding yeast, DNA-damaged induced nuclear microtubule filaments (DIMs) form in response to endogenous or exogenous DNA damage [95]. These DIM filaments, formed at repair sites, reach the nuclear periphery to dive irreparable DSBs and disassemble after relocalization. Such DSBs motion is mediated by the Rad9 DNA damage response mediator and the Kar3 kinesin motor. Another model implicating microtubules has been proposed by Lawrimore et al. to explain the global increased mobility observed in yeast in response to DNA damage: in their model, microtubules would be responsible for a global chromatin shake-up that would be essential for global increase mobility on DSBs [98].

In these different examples, DSBs mobility is promoted by molecular motors, and DSBs exhibit a complex motion including mixture of directive and Brownian motions [93] or nonlinear directive motion [95]. Overall, these recent studies revealed the essential role of molecular motors in DSBs mobility to drive heterochromatic or irreparable DSBs to the nuclear periphery or in clustering of multiple DSBs. Importantly, these mechanisms are conserved through several organisms (human, *Drosophila melanogaster*, *Xenopus laevis*, and budding yeast).

### **3.5. Modifications of chromatin compaction in response to DSB and perspectives**

In addition to chromatin mobility, many studies investigated the modulation of the chromatin compaction state both at a specific damaged site and throughout the genome. Several studies showed a chromatin decondensation visible at the micrometer scale accessible by conventional light microscopy [85, 99]. In the recent studies, super resolution imaging of a *lacO* array before and after damage allows the visualization of chromatin decompaction at the damaged site in haploid yeast [90, 100]. In mammalian cells, most of the studies report chromatin decondensation at the damaged site. However, it has been shown that following this initial fast decondensation, the damaged chromatin area slowly recondenses to reach higher compaction levels than before damage induction [101]. In addition, both chromatin expansion and compaction occur at the same time but in different regions of the chromatin near the DSBs [49]. Overall, chromatin changes in compaction are tuned in space and time upon DSBs, but the precise role of each step remains to be elucidated.

explores the nuclear space. Following DSBs, chromatin mobility is dramatically altered in budding yeast both at the site of DNA damage and genome wide. In mammalian cells, DSBs mobility is strongly influenced by cell cycle stage, chromatin state, and repair pathway choice. For example, while DSBs induced in pericentric heterochromatin during the S and G2 phases of the cell cycle are more mobile and relocate to the nuclear periphery, DSBs generated during the G1 phase remain stable. Changes of chromatin mobility upon DNA damage is an intriguing phenomenon, and over the last five years, several views to explain it have been proposed in the literature. They can be grouped into two classes: (1) increase in chromatin motion is due to intrinsic chromatin modifications which require chromatin remodelers, kinases, and repair proteins involved in the DNA response machinery; (2) increase in chromatin motion due to changes of external mechanical constraints that maintain chromatin and to the action of molecular motors. It is likely that both mechanisms act in concert to drive differently damaged chromatin depending on the type of damages, the chromatin state, or the cell cycle.

Chromatin Dynamics upon DNA Damage http://dx.doi.org/10.5772/intechopen.83559 59

Several questions remain open: how changes in chromatin dynamics alter its organization at the scale of TADs? Is there a global change in mechanical properties of the chromatin upon DSBs, such as an increase in chromatin rigidity? In the future, it will be an exciting challenge to investigate changes in chromatin organization and dynamics upon DNA damage combining different approaches from live cell microscopy, super resolution imaging, and Hi-C.

**Acknowledgements**

**Author details**

United States

**References**

This work was funded by the ANR-12-PDOC-0035-01.

\*Address all correspondence to: judith.mine@curie.fr

1 Institut Curie, PSL Research University, CNRS, UMR3664, Paris, France

3 Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley,

[1] Robinson PJ et al. EM measurements define the dimensions of the "30-nm" chromatin fiber: Evidence for a compact, interdigitated structure. Proceedings of the National

Academy of Sciences of the United States of America. 2006;**103**(17):6506-6511

2 Institut Curie, Sorbonne Université, CNRS, UMR3664, Paris, France

Judith Miné-Hattab1,2\* and Xavier Darzacq3

An interesting way to interpret chromatin changes in dynamics and compaction upon DSBs is to think in terms of mechanical properties of chromatin, such as chromatin stiffness (or persistence length). As illustrated in **Figure 4**, the persistence length of a polymer is a mechanical property that quantifies its stiffness. The persistence length is the length over which correlations in the direction of the tangent are lost.

Changes in chromatin persistence length following DNA damage have been discussed, however, with contradictories interpretations. While some studies suggest that chromatin is more flexible following DSBs [100, 102, 103], other results indicate that chromatin is globally stiffer upon DSBs [65, 90]. One proposed explanation for chromatin stiffening upon DSBs could be the presence of negative charges due to H2A S129 phosphorylation [90]. Further studies will be required to solve to this open question and more generally to understand the physical mechanisms underlying the modifications of chromatin dynamics in response to DNA damage.

**Figure 4.** Illustration of the persistence of a polymer, where Lp is the polymer persistence length and L is the polymer length.
