**2. Methods to quantify chromatin dynamics**

### **2.1. Microscopy techniques to visualize chromatin mobility**

During the last 15 years, powerful microscopy techniques have allowed the visualization of chromatin mobility inside living cells. One method to image chromatin dynamics consists in uniformly labeling chromatin, using for example fluorescently tagged histones, or DNA intercalant. Local chromatin movements can then be investigated by FRAP (Fluorescence Recovery After Photo-bleaching) or image correlation methods for example [49]. However, this approach is limited in resolution. In the FRAP approach, the size of the laser spot used to photo-bleach or photo-convert the tagged chromatin or labeled DNA probably encompasses several megabases of DNA wrapped around thousands of nucleosomes. Another common labeling approach uses repeated bacterial sequences (*lac* or *tet* operators) integrated into the genome [50]. These *lacO*/*tetO* arrays are bound by LacI/TetR proteins, which are fused to fluorescent proteins (**Figure 1A**). These arrays are visible by microscopy as distinct spots that can be tracked through time to measure the chromatin dynamics. Importantly, the *lacO*/ LacI and *tetO*-TetR systems offer the possibility to fluorescently tag genomic loci at a defined genomic locus. Another tagging method, consisting of the ParB-INT DNA labeling system, has also been developed to fluorescently mark genomic loci [51]. To increase the resolution and have access the position and the dynamics of individual histones, a uniform chromatin labeling can be performed using photo-activable fluorophors. Such approach allows

the visualization of chromatin organization at 20 nm resolution [52] or the measurement of histones dynamics [53, 54] (**Figure 1B**). However, in contract with the previous approaches (*lacO*, *tetO* arrays or ParB-INT DNA labeling system), a uniform chromatin labeling does not give access to the DNA sequence to which a specific histone is bound to. To combine the visualization of a single region of the genome at super resolution, a recent technique has been developed, named Oligopaint FISH [55]. Overall, these different approaches allow us to access different scales of chromatin organization and dynamics, from several megabases

**Figure 1.** (A) Illustration of the *lacO*/LacI-GFP system to fluorescently mark a specific genomic locus. (B) Singlenucleosome image of a human HeLa cell nucleus expressing H2B-PA-mCherry. Each dot represents single nucleosome [54]. (C) Representative trajectories of fluorescently labeled single nucleosome (50 ms per frame), with permission

Chromatin Dynamics upon DNA Damage http://dx.doi.org/10.5772/intechopen.83559 51

to a single nucleosome.

from [54].

Considerable progress has been recently made to understand the molecular basis of repair pathway choice, pointing toward cell cycle stage and chromatin landscape as key determinants for the choice of the repair pathway. Although chromatin packing may protect the genome against DNA damage [44], multiple studies suggest that DNA repair processes are less efficient in densely packed heterochromatin [45], leading to an accumulation of mutations in these regions [46]. Recent findings also suggest that DSB occurring in transcriptionally active genes displays dedicated repair mechanisms. Indeed, in contrast to the rest of the euchromatic genome (intergenic and inactive genes), damaged active genes are preferentially repaired by HR in G2 cells, thanks to a chromatin modification (H3K36me3) dependent pathway [47], while in G1, they exhibit delayed repair and enhanced clustering [48]. Such results thus highlight the major impact of the chromatin landscape on the DNA

Investigating the nature of DNA diffusion in the context of DNA repair is particularly relevant to understand how cells maintain genome integrity. When a DSB occurs, the two broken ends first need to stay in close proximity, both for NHEJ and HR. Following this first step, chromatin mobility probably differs depending on the repair pathway used. Since HR requires the search for a homologous sequence, many studies investigated chromatin mobility in response to DSB repaired by HR. In the following section, we will present several techniques that have

During the last 15 years, powerful microscopy techniques have allowed the visualization of chromatin mobility inside living cells. One method to image chromatin dynamics consists in uniformly labeling chromatin, using for example fluorescently tagged histones, or DNA intercalant. Local chromatin movements can then be investigated by FRAP (Fluorescence Recovery After Photo-bleaching) or image correlation methods for example [49]. However, this approach is limited in resolution. In the FRAP approach, the size of the laser spot used to photo-bleach or photo-convert the tagged chromatin or labeled DNA probably encompasses several megabases of DNA wrapped around thousands of nucleosomes. Another common labeling approach uses repeated bacterial sequences (*lac* or *tet* operators) integrated into the genome [50]. These *lacO*/*tetO* arrays are bound by LacI/TetR proteins, which are fused to fluorescent proteins (**Figure 1A**). These arrays are visible by microscopy as distinct spots that can be tracked through time to measure the chromatin dynamics. Importantly, the *lacO*/ LacI and *tetO*-TetR systems offer the possibility to fluorescently tag genomic loci at a defined genomic locus. Another tagging method, consisting of the ParB-INT DNA labeling system, has also been developed to fluorescently mark genomic loci [51]. To increase the resolution and have access the position and the dynamics of individual histones, a uniform chromatin labeling can be performed using photo-activable fluorophors. Such approach allows

been used in the literature to investigate chromatin mobility.

**2.1. Microscopy techniques to visualize chromatin mobility**

**2. Methods to quantify chromatin dynamics**

repair processes.

50 Chromatin and Epigenetics

**Figure 1.** (A) Illustration of the *lacO*/LacI-GFP system to fluorescently mark a specific genomic locus. (B) Singlenucleosome image of a human HeLa cell nucleus expressing H2B-PA-mCherry. Each dot represents single nucleosome [54]. (C) Representative trajectories of fluorescently labeled single nucleosome (50 ms per frame), with permission from [54].

the visualization of chromatin organization at 20 nm resolution [52] or the measurement of histones dynamics [53, 54] (**Figure 1B**). However, in contract with the previous approaches (*lacO*, *tetO* arrays or ParB-INT DNA labeling system), a uniform chromatin labeling does not give access to the DNA sequence to which a specific histone is bound to. To combine the visualization of a single region of the genome at super resolution, a recent technique has been developed, named Oligopaint FISH [55]. Overall, these different approaches allow us to access different scales of chromatin organization and dynamics, from several megabases to a single nucleosome.

#### **2.2. Quantification of chromatin loci mobility**

Several theoretical studies have shown that the mode of diffusion of a moving object drastically changes the way it explores the available space. The time to reach a specific target can dramatically change depending on the way a particle samples its surrounding environment [56]. To quantify the mobility of a chromatin locus marked using a lacO/LacI system, the most common method consists in measuring its position (*x, y, z*) over time and calculating its mean square displacement (MSD) (Eq. (1)) [57]. The MSD curve represents the amount of space a locus has explored in the nucleus (**Figure 2A**).

$$MSD(\mathbf{n} \cdot \Delta \mathbf{t}) = \frac{1}{N-n} \sum\_{j=0}^{N-1-n} \left[ (\mathbf{x}\_{\neq n} - \mathbf{x}\_j)^2 + (\mathbf{y}\_{\neq n} - \mathbf{y}\_j)^2 + (\mathbf{z}\_{\neq n} - \mathbf{z}\_j)^2 \right] \tag{1}$$

types of sub-diffusive motion have been observed. When a chromosomal locus is confined inside a sub-volume of the nucleus, the motion is called *confined sub-diffusion* and the MSD exhibits a plateau. Confined motion has been observed and quantified in living cells, when chromatin motion is examined during several minutes [23, 59, 60]. In that case, the MSD curves

When the force or structure that restricts the motion is not a simple confinement but is modulated in time and space with scaling properties, the motion is called *anomalous sub-diffusion* [58, 61]. In this case, sub-diffusive loci are constrained, but unlike confined loci, they can diffuse without boundary and thus reach further targets if given enough time. For sub-diffusive motion, the MSD exhibits a power law (*MSD* ~ *At<sup>α</sup>*), where *α*, the anomalous exponent, is smaller than 1. The anomalous exponent *α* is linked to the degree of recurrence of DNA exploration, that is, the number of times a DNA locus reiteratively scans neighboring regions before reaching a distant position [62]. When *α* is small, the locus explores recurrently the same environment for a long time, while a large α indicates that the locus is able to explore new environments often. The anomalous diffusion coefficient *A* represents the amplitude of DNA motion; it is proportional to the diffusion coefficient only in the case of normal diffusion (when *α* = 1), which is rarely observed in biological systems [58]. Finally, a moving particle

moves in a directive manner toward a target, and the motion is called directive.

movement of a single photon emitter, the localization accuracy can be divided into:

The MSD is a standard statistical tool that describes a set of trajectories of similar objects. However, numerous artifacts perturb this statistic. The localization accuracy can have a strong impact on the MSD curve, even computed on simple Brownian motion [63]. Considering the

i. The error in the determination of the accurate particle position due to convolution with the point spread function (PSF) and the finite number of photons. This error is more important for short acquisition times since the number of photons collected is small. ii. The error due to the movement of the particle during the camera acquisition. This error is more important with higher exposure times and is sometimes referred to as "motion blur."

For 2D Brownian motion with a diffusion coefficient D, Michalet computed the formula of the

2 (1 + *Dt* \_\_\_*<sup>E</sup> s*0 <sup>2</sup> ) <sup>−</sup> \_\_4

the *motion blur* of the particle along its path during acquisition. Since the localization accuracy

blur term therefore converges to a fixed value as the exposure time increases. The term \_\_4

accounts for the correlation between successive displacements due to the exposure overlap.

<sup>3</sup> *DtE* (2)

is the variance of the PSF; *t*

<sup>2</sup> <sup>∝</sup> \_\_1 *t E*

2 (<sup>1</sup> <sup>+</sup> *Dt* \_\_\_*<sup>E</sup> s* 0

*E* is

<sup>3</sup> *DtE*

<sup>2</sup> ) accounts for

. The motion

0 2

converged MSD including the corrections for localization accuracy (see Eq. (2)) [64]:

is the localization accuracy of an immobile particle; *s*

The term 4*Dt* is the theoretical MSD for simple Brownian motion. The term *σ*<sup>0</sup>

is inversely proportional to the number of collected photons, we have *σ*<sup>0</sup>

*MSD*(*t*) = 4*Dt* + *σ*<sup>0</sup>

the exposure time of the camera.

where *σ*<sup>0</sup> 2

*σ*0 2 ), where *L* is the plateau of the MSD curve (proportional

Chromatin Dynamics upon DNA Damage http://dx.doi.org/10.5772/intechopen.83559 53

can be fitted by *MSD* ∝ *L*2(1 − *e* <sup>−</sup>4*Dt*/*L*<sup>2</sup>

to the radius of confinement) and *D* is the diffusion coefficient.

The shape of MSD curves then reveals the nature of DNA motion. Four main types of motion have been described in the literature (**Figure 2B**): confined motion, anomalous sub-diffusion, Brownian motion, and directive motion.

The simplest type of motion is Brownian diffusion: when a particle freely diffuses, its MSD curve is linear with time and its motion is called "Brownian." However, in living cells, DNA motion is often slower than Brownian diffusion and is called "sub-diffusive" [58]. Several

**Figure 2.** (A) Illustration of trajectories: from the left to the right, points are represented spaced by 1.Δt, 2.Δt and 3.Δt and (B) mean square displacement for normal anomalous and confined diffusion, with a representation of the corresponding trajectories.

types of sub-diffusive motion have been observed. When a chromosomal locus is confined inside a sub-volume of the nucleus, the motion is called *confined sub-diffusion* and the MSD exhibits a plateau. Confined motion has been observed and quantified in living cells, when chromatin motion is examined during several minutes [23, 59, 60]. In that case, the MSD curves can be fitted by *MSD* ∝ *L*2(1 − *e* <sup>−</sup>4*Dt*/*L*<sup>2</sup> ), where *L* is the plateau of the MSD curve (proportional to the radius of confinement) and *D* is the diffusion coefficient.

**2.2. Quantification of chromatin loci mobility**

52 Chromatin and Epigenetics

locus has explored in the nucleus (**Figure 2A**).

*MSD*(*<sup>n</sup>* <sup>⋅</sup> <sup>Δ</sup>*t*) <sup>=</sup> \_\_\_\_ <sup>1</sup>

Brownian motion, and directive motion.

trajectories.

Several theoretical studies have shown that the mode of diffusion of a moving object drastically changes the way it explores the available space. The time to reach a specific target can dramatically change depending on the way a particle samples its surrounding environment [56]. To quantify the mobility of a chromatin locus marked using a lacO/LacI system, the most common method consists in measuring its position (*x, y, z*) over time and calculating its mean square displacement (MSD) (Eq. (1)) [57]. The MSD curve represents the amount of space a

> [(*xi*+*<sup>n</sup>* − *xi* )

The shape of MSD curves then reveals the nature of DNA motion. Four main types of motion have been described in the literature (**Figure 2B**): confined motion, anomalous sub-diffusion,

The simplest type of motion is Brownian diffusion: when a particle freely diffuses, its MSD curve is linear with time and its motion is called "Brownian." However, in living cells, DNA motion is often slower than Brownian diffusion and is called "sub-diffusive" [58]. Several

**Figure 2.** (A) Illustration of trajectories: from the left to the right, points are represented spaced by 1.Δt, 2.Δt and 3.Δt and (B) mean square displacement for normal anomalous and confined diffusion, with a representation of the corresponding

<sup>2</sup> + (*yi*+*<sup>n</sup>* − *yi*

)

<sup>2</sup> + (*zi*+*<sup>n</sup>* − *zi*

) 2

] (1)

*<sup>N</sup>* <sup>−</sup> *<sup>n</sup>* <sup>∑</sup> *i*=0 *N*−1−*n* When the force or structure that restricts the motion is not a simple confinement but is modulated in time and space with scaling properties, the motion is called *anomalous sub-diffusion* [58, 61]. In this case, sub-diffusive loci are constrained, but unlike confined loci, they can diffuse without boundary and thus reach further targets if given enough time. For sub-diffusive motion, the MSD exhibits a power law (*MSD* ~ *At<sup>α</sup>*), where *α*, the anomalous exponent, is smaller than 1. The anomalous exponent *α* is linked to the degree of recurrence of DNA exploration, that is, the number of times a DNA locus reiteratively scans neighboring regions before reaching a distant position [62]. When *α* is small, the locus explores recurrently the same environment for a long time, while a large α indicates that the locus is able to explore new environments often. The anomalous diffusion coefficient *A* represents the amplitude of DNA motion; it is proportional to the diffusion coefficient only in the case of normal diffusion (when *α* = 1), which is rarely observed in biological systems [58]. Finally, a moving particle moves in a directive manner toward a target, and the motion is called directive.

The MSD is a standard statistical tool that describes a set of trajectories of similar objects. However, numerous artifacts perturb this statistic. The localization accuracy can have a strong impact on the MSD curve, even computed on simple Brownian motion [63]. Considering the movement of a single photon emitter, the localization accuracy can be divided into:


For 2D Brownian motion with a diffusion coefficient D, Michalet computed the formula of the converged MSD including the corrections for localization accuracy (see Eq. (2)) [64]:

$$MSD(t) = 4Dt + \sigma\_0^2 \left(1 + \frac{Dt\_\varepsilon}{s\_0^2}\right) - \frac{4}{3}Dt\_\varepsilon \tag{2}$$

where *σ*<sup>0</sup> 2 is the localization accuracy of an immobile particle; *s* 0 2 is the variance of the PSF; *t E* is the exposure time of the camera.

The term 4*Dt* is the theoretical MSD for simple Brownian motion. The term *σ*<sup>0</sup> 2 (<sup>1</sup> <sup>+</sup> *Dt* \_\_\_*<sup>E</sup> s* 0 <sup>2</sup> ) accounts for the *motion blur* of the particle along its path during acquisition. Since the localization accuracy *σ*0 2 is inversely proportional to the number of collected photons, we have *σ*<sup>0</sup> <sup>2</sup> <sup>∝</sup> \_\_1 *t E* . The motion blur term therefore converges to a fixed value as the exposure time increases. The term \_\_4 <sup>3</sup> *DtE* accounts for the correlation between successive displacements due to the exposure overlap.

For anomalous motion with a diffusion coefficient *D*, the MSD formula including the corrections for localization accuracy is described in [65].

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

Chromatin Dynamics upon DNA Damage http://dx.doi.org/10.5772/intechopen.83559 55

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

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,

to confirm these observations.

**3.2. Local versus global increased mobility**

type of DSBs (zeocin-induced DSBs) [88].
