Josefine Schnee
email: josischnee@hotmail.com

François Devred

Eric M Gaigneaux

Alexandre Vimont
email: alexandre.vimont@ensicaen.fr

Assessing the dispersion of supported H3PW12O40 catalysts: No longer a hurdle thanks to in situ IR upon pyridine adsorption

Keywords: FT-IR spectroscopy, In situ monitoring, Pyridine adsorption, Keggin heteropolyacid, Supported catalysts, Dispersion

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pyridine is nowadays widely used as a probe molecule to characterize the acidity of solids (both in terms of number and type/strength of the acid sites). 1,2,3,4 Indeed, it adsorbs onto the acid sites and, depending on whether these are of Lewis or Brönsted type, it forms a different species being detectable/quantifiable through infrared (IR) spectroscopy. 2,3,4 In the case of Brönsted acid sites, a pyridinium cation (PyH + ) is formed, which has its main IR fingerprint band at about 1540 cm -1 (a position being independent of the nature of the counter-anion). 1 The latter band is associated to the vibration of the pyridinium ring in the stretch mode noted "ν19b" (the "b" referring to the symmetry type B2 to 2 which this vibration mode belongs following the group theory, with the pyridinium cation being characterized by a C2v point group of symmetry elements). 1 For conventional solids, the pyridine-IR characterization is limited to the only surface. However, in the case of heteropolyacids (HPAs), standing at the heart of the present work, the situation is different. HPAs are metal-oxygen clusters that are nowadays widely used in acid catalysis. Thanks to their exceptionally strong Brönsted acidity, they generally allow operating chemical reactions under significantly milder conditions than required by conventional acid catalysts (e.g. alumina or zeolites). 5 The most easily available HPAs are those having the Keggin structure. Described by the general formula HnXM12O40, they contain [XM12O40] n-heteropolyanions stabilized by n acidic protons, with X being the heteroatom (often P V , Si IV ) and M being the addenda atom (transition metal, typically Mo VI , W VI ). 6 More precisely, the heteropolyanions are made of a central XO4 tetrahedron, surrounded by twelve MO6 octahedra. They contain three types of oxygen atoms: central (Oa), bridging, and terminal (Ot) ones. The bridging ones are either corner-sharing (Ob) or edge-sharing (Oc). 6,7 HPAs exhibit a so-called "pseudo-liquid" behavior. This means that they absorb polar (and relatively small) molecules such as alcohols, ethers or pyridine into their solid bulk, thus in-between their constitutive heteropolyanions. 8,9,10,11 In the case of pyridine, this leads to the formation of a pyridinium salt. 12,13,14 So, pyridine probes not only the acid sites located at the surface of HPA crystals but also those located within their bulk.

In the case of H3PW12O40, the most acidic Keggin HPA, the pyridinium salt gives rise to an unusual IR fingerprint. Indeed, its ν19b band is split in two (as well as its ν8b band centered at about 1610 cm - 1 ). 1,15,16 The most likely explanation for this phenomenon is the following. 1 In the solid bulk, the pyridinium ring is sterically hindered from rotating around the axis joining the center of the ring to the Ot atom of the heteropolyanion to which the pyridinium cation is bonded. This frustration leads to an in-plane rotation of the pyridinium cation towards another type of oxygen atom, namely an Oc one, through a tunneling effect. So, the pyridinium cation actually switches between two positions differing by the type of oxygen atom of the heteropolyanion to which it is H-bonded. This switch splits the δN + -H energy levels, and, as they present a particularly marked δN + -H character, the ν19b and ν8b ring vibration modes as well. In other words, the frustrated in-plane rotation of the pyridinium ring appears as an additional mode, which is called the tunnelling mode. As a consequence, the IR bands resulting from the ν19b and ν8b modes contain two contributions and are split.

In the present work, we demonstrate for the first time that the above pyridinium IR band splitting can actually be exploited as an analytical tool. We support H3PW12O40 on two different commercial TiO2 supports. The first one is TiO2 "P25" (20% rutile -80% anatase), which is widely used as a support for HPAs in order to increase the accessibility of the individual units. 8,17,18,19 The second one is TiO2 "T805", a hydrophobized version of TiO2 P25, to our knowledge not yet used to support HPAs. The idea of using supports with different hydrophilicities is to obtain supported H3PW12O40 samples with different degrees of dispersion, as recently achieved in reference 20 . If the pyridinium IR band splitting observed for bulk H3PW12O40 really originates from a frustrated rotation of the pyridinium cation due to neighboring Keggin units, its extent in the case of supported H3PW12O40 could possibly depend on whether the Keggin units are well-dispersed or agglomerated. In other words, the extent of the pyridinium IR band splitting could possibly be exploited as a tool to probe the dispersion of supported H3PW12O40, namely a key parameter dictating their catalytic performance (as shown for example in the conversion of methanol to dimethylether 17,20 ). The feasibility of this idea is precisely what we investigate in the present work, by combining in situ Fourier-transform infrared (FT-IR) spectroscopy upon pyridine adsorption with the commonly used 17,20 techniques to assess the dispersion of HPAs, namely X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD).

Experimental

Preparation of the supported H3PW12O40

TiO2 P25 and T805 were purchased from Evonik Industries (AEROXIDE® TiO2 P25 and T805). TiO2 T805 is a fumed titanium dioxide that was treated with octylsilane to achieve a hydrophobic surface (C-content of 2.7 -3.7 wt.% according to Evonik's Product Information Sheet 21 ). H3PW12O40 (hereafter HPW12) was purchased from Sigma-Aldrich in the form of H3PW12O40.xH2O (reagent grade). It was supported on both TiO2 supports by wet impregnation at room temperature. Precisely, the appropriate amounts of HPW12 (corresponding to the nominal loadings/weight fractions provided in Table S1 in the Supporting information) were dissolved in ethanol (AnalaR NORMAPUR®, > 99,8%) and then added dropwise to 1 g of support dispersed also in ethanol. The resulting mixtures were stirred for 2 hours, before the solvent was evaporated in a rotary evaporator at 40 °C. Finally, the samples were dried overnight in a vacuum oven (< 40 Torr) at room temperature.

The real HPW12 loadings were determined through inductively coupled plasma atomic emission spectrometry (ICP-AES) with an ICAP 6500 Thermo Scientific apparatus. The values are provided together with the nominal loadings in Table S1, being expressed both in weight fractions and in "% ML" (and plotted against the nominal loadings on Figure S1). The latter unit means the percentage of the total amount of Keggin units theoretically required to cover the supports with one ideal monolayer (abbreviated here to "ML"). Hereafter, all results are discussed with respect to the real HPW12 loadings, and the latter are expressed in % ML to get rid of the difference in specific surface area of the two TiO2 supports (45 vs. 33 m²/g respectively for P25 vs. T805) when comparing the supported samples in terms of HPW12 dispersion. The specific surface area of the supports was measured with a Micromeritics Tristar Surface Area and Porosity Analyser after having degassed the samples overnight at 150 °C under a vacuum of about 80 mTorr. As reflected by the N2 physisorption isotherms provided on Figure S2 in the Supporting information, both supports are exclusively macroporous. They do not contain micro-or mesopores. The loadings of HPW12 in % ML were calculated by considering that Keggin units are spherical and have a diameter of 1 nm. 22

Assessment of the dispersion in the usual way

XPS was performed with a SSX 100/206 spectrometer from Surface Science Instruments (USA) equipped with a monochromatized and microfocused Al X-ray source (powered with 20 mA and 10 kV). The pressure within the analysis chamber was about 10 -6 Pa. The angle between the normal to the surface and the axis of the input lens of the analyzer was 55°. The zone analyzed was about 1.4 mm² and the pass energy was set at 150 eV for the general spectra and at 50 eV for the elementary spectra. An electron gun set at 8 eV and a nickel grid placed 3 mm above the surface of the samples were used to stabilize the charge. The following sequence of spectra was recorded: general spectrum, C 1s, O 1s, Ti 2p, W 4d, and again C 1s to check the stability of the charge compensation as a function of time and the absence of sample degradation. The elements were quantified by using the sensitivity factors and acquisition parameters provided by the manufacturer, after having set the binding energy scale by fixing the C-(C,H) component of the C 1s peak at 284.8 eV. The W/Ti ratio was plotted as a function of the loading of HPW12 on both TiO2 supports. Indeed, such a plot directly reflects the way how HPW12 interacts with the supports. In general, the higher the W/Ti ratio, the more the Keggin units are dispersed over the surface of the support. In particular, a linear increase of the W/Ti ratio with the HPW12 loading (from the origin of the graph) reflects the formation of an ideal monolayer of Keggin units progressively covering the surface of the support. Any other tendency vs. the loading (i.e. a linear increase with a lower slope than needed to be aligned with the origin of the graph, a horizontal line, or a decrease) reflects the formation of aggregates.

Powder XRD patterns were measured at room temperature with a Kristalloflex Siemens D5000 diffractometer using the copper Kα radiation (λ = 0.15418 nm) and being equipped with a scintillation detector. The X-ray source was operated with a tension of 40 kV and a current of 40 mA. The appearance or not of the diffraction lines observed for pure (thus crystalline) HPW12 on the XRD patterns of the supported samples was used as an indicator of respectively poor or good dispersion of the Keggin units.

In situ FT-IR upon pyridine adsorption-desorption

Samples were pressed into a self-supported disc (2 cm 2 area, 10 mg cm -2 ). The resulting pellets were placed into a homemade IR cell which was equipped with KBr windows and connected to a vacuum line. The latter line served for evacuation steps, thermal treatments and introduction of small doses of pyridine vapor (typically 1 Torr). Thanks to a movable sample holder, the pellets were set alternatively into the IR beam for spectra acquisition at room temperature and into a furnace at the top of the cell for thermal treatments. The spectrometer was a Nicolet Nexus one equipped with a KBr beam splitter and a deuterated-triglycine sulfate (DTGS) detector. The FT-IR spectra were measured at 4 cm -1 resolution, and treated with the Thermo Scientific OMNIC software.

In a typical experiment, the sample was first pre-treated for 1 hour under vacuum (until reaching 10 -6 Torr) at 100 °C to evacuate structural water. Indeed, Keggin HPAs are highly hygroscopic. 5 Then, it was exposed to 1 Torr of pyridine (non-deuterated h5-one, 99+% grade from Aldrich), first at room temperature and then at 100 °C, both times for 1 hour. The step at 100 °C aimed at making pyridine diffuse into the bulk of potential HPW12 agglomerates. Finally, during pyridine evacuation (until reaching 10 -6 Torr), the sample was first left at room temperature for 1 hour and then heated up to 100 °C, again for 1 hour (to check the stability of adsorbed pyridine). FT-IR spectra were measured at room temperature during and/or after every step (always after in the case of heating steps because of the setup configuration).

Whatever the sample, before pyridine adsorption, not any absorption band was observed around 1540 cm -1 where appears the ν19b pyridinium ring vibration band of interest here. Upon exposure to pyridine, in the cases where the ν19b band got split, it never did before heating to 100 °C. Moreover, upon evacuation of pyridine, the spectra before and after heating at 100 °C were always the same. For these reasons, the full sequence of spectra is shown here only for some chosen samples (those with the highest HPW12 loading on both supports, as well as pure TiO2 P25 for reference) in the Supporting information (Figure S3). The Results and discussion section focuses on the spectra measured after heating to 100 °C in the evacuation period, as this stage ensures the absence of excess pyridine within the bulk of HPW12. Indeed, such an excess leads to variations of the ν19b band intensity. 13 Regarding the structure of HPW12, it was checked to be of Keggin type as in pure HPW12 for all the supported samples. Indeed, the characteristic bands at 1080 and 980 cm -1 , respectively associated 6 to P-Oa-W and W=Ot vibrations within the Keggin structure, were systematically observed (Figure S4).

Results and discussion

Figure 1 shows the surface atomic ratio of W over Ti as a function of the HPW12 loading on both TiO2 supports (37, 66 and 82% ML on TiO2 P25; 35, 49 and 67% ML on TiO2 T805). On TiO2 P25, the W/Ti ratio systematically increases with the loading. The values at 37 and 66% ML are aligned with the origin of the graph, reflecting that the Keggin units within the two concerned samples are dispersed as an ideal monolayer over the support. Then, from 66 to 82% ML, the W/Ti ratio increases less than predicted by the line passing through the preceding points, reflecting the presence of agglomerated Keggin units at 82% ML. These observations are consistent with the work in reference 20 . On TiO2 T805, the situation is much different. All three supported samples show relatively close W/Ti ratios, however with a decreasing tendency from one loading to the next. Moreover, the W/Ti ratios are all significantly lower (at least two times) than the lowest value observed with TiO2 P25. All this indicates that, on TiO2 T805, from the lowest loading, the Keggin units agglomerate/stack on each other (either starting from the support surface, or forming independent aggregates) instead of forming a well-dispersed layer over the support surface. The same kind of behavior was recently observed for HPW12 supported on hexagonal boron nitride (also hydrophobic). However, in that case, above a threshold loading of 40% ML, the Keggin units tended to penetrate into/disperse within the interlayers of boron nitride, making the W/Ti ratio increase again. 20 Here, on TiO2 T805, the higher the HPW12 loading, the lower the W/Ti ratio, thus the higher the tendency of the Keggin units to interact with each other rather than with the support, and the lower the fraction of Keggin units dispersed over the surface of the support. Figure 2 shows the FT-IR spectra of pyridine adsorbed onto the HPW12/P25 (Figure 2a) and HPW12/T805 (Figure 2b) samples. As a reference, Figure S3 in the Supporting information provides the spectra of pyridine adsorbed onto pure TiO2, measured in the same conditions (the bands being assigned in the related text, according to reference 2 ). On Figure 2, the characteristic ν19b pyridinium ring vibration band due to the protonation of pyridine by HPW12 emerges at around 1540 cm -1 . It is the only band being specific to pyridine interacting with HPW12. All the other bands are either 1) specific to pyridine adsorbed on TiO2 (at 1575-1580 and 1445 cm -1 ), or 2) they appear at a position where a contribution from pyridine adsorbed on TiO2 may not be excluded (as witnessed by the spectra of pyridine adsorbed onto pure TiO2 on Figure S3), or 3) they are specific to the support itself (the band at 1466 cm -1 observed with T805 being associated to the deformation of methyl groups). So, the ν8b pyridinium ring vibration band appearing at around 1610 cm -1 on the spectrum of pure (PyH + )3(PW12O40) 3-shown elsewhere 1 is masked here, and can thus not be considered. From integrating the ν19b pyridinium ring vibration band of the spectra in Figure 2, and applying the Beer-Lambert law with an absorption coefficient of 1.36 µmol -1 cm (according to reference 23 ) and a pellet surface of 2 cm², the number of pyridinium cations formed within the samples was estimated. This number nicely corresponds with the number of protons contained in the samples (Figure 3), which was calculated from the HPW12 loading, knowing that each Keggin unit possesses 3 protons. This correlation confirms that pyridine indeed probes the whole protons within the samples, whatever the HPW12 loading. absorption coefficient of 1.36 µmol -1 cm reported in reference 23 and a pellet surface of 2 cm²) versus their number of protons (calculated from the HPW12 loadings, by considering that each Keggin unit possesses 3 protons).

In the case of the HPW12/P25 samples with 37 and 66% ML of HPW12 (containing well-dispersed Keggin units according to Figure 1), the ν19b pyridinium ring vibration band is not split (Figure 2a). It has a single maximum at 1540 cm -1 . In the case of the HPW12/P25 sample with 82% ML (containing agglomerated Keggin units), the band shows a maximum at 1540 cm -1 , with a slight shoulder at 1530 cm -1 (Figure 2a). Regarding the HPW12/T805 samples (containing more and more poorly dispersed Keggin units with increasing loading), those with 49 and 67% ML both show a split ν19b pyridinium ring vibration band, with the two maxima being located at 1540 and 1530 cm -1 . The sample with 35% ML also shows, next to its maximum at 1540 cm -1 , a shoulder at 1530 cm -1 (Figure 2b). So, as long as the Keggin units are well-dispersed on the support, the ν19b ring vibration band remains non-split. When agglomerated Keggin units are present (either because the loading was too much increased on TiO2 P25, or because, at a given loading, TiO2 T805 was used instead of TiO2 P25), the band is systematically split. More precisely, the splitting occurs to an extent that depends on the fraction of agglomerated Keggin units. Indeed, on TiO2 T805, the higher the loading, in other words the higher the fraction of Keggin units being agglomerated, the more marked the band splitting. More marked means that the intensity at 1530 cm -1 increases relatively to that at 1540 cm -1 (ratio1530/1540 of 0.60, 0.68 and 0.75 respectively at 35, 49 and 67% ML on Figure 4), and that the two maxima get better and better distinguished. It reflects that the fraction of pyridinium cations in the sample which lead to a split band increases. Thus, the higher the fraction of agglomerated Keggin units, the higher the fraction of pyridinium cations leading to a split band. This also appears as comparing the spectra of HPW12/P25 82% ML (Figure 2a) and HPW12/T805 67% ML (Figure 2b). Both show a split band, but the splitting is less marked in the case of the HPW12/P25 sample, although the latter has the highest loading among both. Indeed, according to Figure 1, the Keggin units are still much better dispersed in the HPW12/P25 sample than in the HPW12/T805 one. The fraction of agglomerated Keggin units is lower in the HPW12/P25 sample. So, in light of all these observations, the phenomenon responsible for the band splitting -most likely a tunneling effect associated to a frustrated rotation of the pyridinium cation (see Introduction) -occurs as a direct consequence of the agglomeration of HPW12 on the supports. In other words, the band splitting can be considered as a fingerprint of agglomerated HPW12. Furthermore, by subtracting the spectrum of the pyridinium salt of pure HPW12 from those of pyridine adsorbed onto the supported samples, with a subtraction factor chosen to specifically eliminate the maximum/shoulder at 1530 cm -1 within the ν19b pyridinium band (see an example on Figure 5), it is possible to directly access to the contribution of dispersed HPW12 to the latter band, and thereby to quantitatively estimate the fractions of both dispersed and agglomerated HPW12 within the supported samples (respectively by dividing the area of the band after subtraction by the initial area before subtraction, and by calculating the difference between 100 and the fraction of dispersed HPW12 to get the fraction of agglomerated HPW12, see values in Table 1). This represents a significant improvement compared to XPS which is unable to provide such quantitative information. Moreover, it can be applied to any kind of supported HPW12 sample, whatever the nature of the support. It facilitates the proper selection of samples to be used as catalysts, whatever the reaction. Indeed, as shown in reference 20 , agglomerated Keggin units possess stronger acid sites than dispersed ones. Moreover, a given reaction might occur either only at the surface of the agglomerates or both at their surface and within their bulk. The latter mechanism applies, for example, in the case of the methanolto-dimethylether reaction. 24 So, depending on the requirements in terms of acid strength and on the mechanism of the reaction, samples with a low, intermediate, or high fraction of agglomerated HPW12 should be selected as catalysts, and the innovative methodology reported here allows to easily make the right choice. Table 1. Fractions of dispersed and agglomerated HPW12 within the HPW12/P25 and HPW12/T805 samples determined thanks to the approach shown in Figure 5. The ν19b pyridinium band splitting actually detects agglomerated HPW12 even better than XRD. Indeed, the latter detects here Keggin crystals only for the HPW12/T805 sample with 67% ML, namely the sample with the poorest HPW12 dispersion among all. In all other cases, the XRD patterns only show the diffraction lines of pure TiO2, namely the same for P25 and T805 (see Figure 6 showing the patterns of the HPW12/T805 samples, and Figure S5 in the Supporting information showing the patterns of the pure materials -TiO2 P25 and T805, and HPW12 -and of the HPW12/P25 samples). Indeed, although already agglomerated (but maybe not yet at an advanced state of agglomeration, for example due to the amount of HPW12 introduced in the formulation), Keggin units might not have formed ideal crystals yet, or the latter might still be too small to be detectable through XRD.

Support

Conclusions

For the first time, the present paper shows that the ν19b pyridinium IR band splitting well-known in the case of pure HPW12 actually also appears with supported HPW12, namely systematically in the presence of agglomerated Keggin units. More precisely, the higher the fraction of agglomerated Keggin units on the support, the more marked the band splitting, in other words the higher the fraction of pyridinium cations concerned by a tunneling effect associated in the literature to a frustrated rotation of the pyridinium cation within the solid bulk of HPW12 crystals. Thus, the latter phenomenon occurs as a direct consequence of the agglomeration of HPW12 on the support, what makes the pyridinium band splitting a powerful tool to probe the dispersion of supported HPW12. It is even more powerful than XRD which does not systematically detect Keggin crystals when pyridine-IR detects agglomerates, and it allows quantitatively evaluating the fractions of dispersed and agglomerated Keggin units within the supported samples. As the dispersion in general strongly influences the catalytic performance of HPW12, e.g. by dictating the strength and accessibility of the acid sites, this new approach facilitates the proper selection of samples to be used as catalysts, whatever the reaction, as a function of the reaction requirements.

Supporting information

Nominal and real (ICP-measured) HPW12 loadings (in weight fraction and in % ML) on both TiO2 supports; N2 physisorption isotherms of the pure supports; FT-IR spectra of HPW12/P25, HPW12/T805 and pure TiO2 in particular conditions or spectral regions (with assignment of the bands related to TiO2); XRD patterns of the HPW12/P25 samples and pure materials (TiO2 P25 and T805, and HPW12).

Figure 1 .

 1 Figure 1. XPS-measured W/Ti surface atomic ratio as a function of the loading of HPW12 on TiO2 P25 and T805. The R² applies to the dotted line passing through the first three points relative to TiO2 P25, including the origin.

Figure 2 .

 2 Figure 2. In situ FT-IR spectra of the HPW12/P25 (a) and HPW12/T805 (b) samples after exposure to pyridine, in the region of the ν19b pyridinium ring stretch. They were measured at room temperature during pyridine evacuation out of the IR cell, precisely after 1 hour under vacuum at 100 °C. "Abs." means absorbance, and it is expressed in arbitrary units.

Figure 3 .

 3 Figure 3. Number of pyridinium cations contained in the samples after exposure to pyridine (obtained by integrating the ν19b pyridinium ring vibration band of the spectra in Figure 2, and by applying the Beer-Lambert law with an integrated

Figure 4 .

 4 Figure 4. Ratio of the intensity at 1530 cm -1 over that at 1540 cm -1 within the ν19b pyridinium ring vibration band observed for the HPW12/T805 samples as a function of the HPW12 loading. The IR intensities were all determined according to a baseline defined between 1560.2 and 1500.4 cm -1 .

Figure 5 .

 5 Figure 5. FT-IR spectra after exposure to pyridine of (a) HPW12/T805 67% ML and (b) pure HPW12, in the region of the ν19b pyridinium ring vibration band. Spectrum (c) results from the subtraction "(a) -0.4419 times (b)" to eliminate the maximum/shoulder at 1530 cm -1 , and shows thus the contribution of dispersed HPW12 exclusively.

20

 20

Figure 6 .

 6 Figure 6. XRD patterns of the HPW12/T805 samples. Characteristic peaks of HPW12 crystals are marked with a star.

Acknowledgements

The authors acknowledge Olivier Kallai for having, under the co-supervision of J.S. and E.M.G., contributed to the sample preparation and performed some of the ex situ characterization measurements in the framework of his Master thesis at the Université catholique de Louvain/Faculty of Bioengineering. J.S. acknowledges Prof. Marco Daturi (Normandie Université/Laboratoire Catalyse et Spectrochimie) for the post-doc position, and for his constructive comments on the manuscript.

Author information

Corresponding authors *E-mail addresses: josischnee@hotmail.com, alexander.vimont@ensicaen.fr

Author contributions

J.S. and E.M.G. conceived the project of comparing the dispersion of HPW12 supported on TiO2 P25 vs. T805; J.S. and A.V. conceived the project of investigating in situ FT-IR upon pyridine adsorption as a way to probe the dispersion of supported HPW12; J.S. was in charge of the sample preparation and ex situ characterization (at the Université catholique de Louvain during her PhD thesis), carried out the in situ FT-IR studies (at the Normandie Université/Laboratoire Catalyse et Spectrochimie during her postdoc) and wrote the manuscript; F.D. performed the XPS measurements (at the Université catholique de Louvain).

Conflicts of interest

The authors declare no competing interests.

Abbreviations

HPW12, H3PW12O40 (phosphotungstic acid). % ML, percentage of the amount of Keggin units required to form one ideal monolayer on the support.


V Rzepecki¹

J Reynes¹

V Le Moing¹

P Braquet

V Faucherre¹

L Lohan²

D Morquin¹

A Makinson
email: makinson@chu[-montpellier.fr

Severe HIV-associated aphthous stomatitis treated with etanercept Traitement par étanercept d'une aphtose buccale grave associée au VIH

Keywords: aphthous stomatitis, HIV, etanercept aphtes, VIH, étanercept

1

A 38-year-old homeless man was hospitalized in our infectious disease department for odynophagia and a progressive 26-kg weight loss over the previous two months. His medical history was notable for heroin addiction. Upon arrival fever (38.1°C) and cachexia were observed with a body mass index measured at 16 kg/m². Several deep, painful, round, and well-marked ulcerations were observed on the tongue, palate, and inner cheeks (Figure 1). No genital lesions were observed.

An HIV serology was positive with a complete Western Blot. Plasma HIV-1 viral load was measured at 10 6 copies/ml, and lymphocyte T CD4 count at 5/mm³. EBV, CMV, toxoplasmosis, and B19 parvovirus serologies were consistent with previous immunization. Syphilis serology was negative. Plasma HHV6, CMV, EBV, and leishmaniasis PCR tests were negative. HSV-1 PCR on mouth ulcer sampling was initially positive, and CMV PCR was negative. Treatment with a 7-day course of valaciclovir (1 g thrice daily), followed by a 4-day course of intravenous acyclovir (1 g thrice daily) and a 6-day full-dose course of foscarnet with a 21-day fluconazole therapy (100 mg daily), as well as antiretroviral combination introduction on Day 6 with emtricitabine, tenofovir, and dolutegravir were of no clinical efficacy, despite negative HSV-1 PCR on mouth sample controls. A biopsy procedure revealed deep necrosis in rare muscle fibers, and a polymorph, non-specific inflammation, with no signs of viral cytotoxicity or malignancy. A diagnosis of HIV-related severe aphthous stomatitis was made. Mycobacterium smear and culture were both negative.

A 10-day systemic corticosteroid therapy (oral prednisone, 1 mg/kg), initiated on Day 21 resulted in frank worsening of the aphthous lesions. Oral administration of food, antiretroviral therapy (switch to soluble lamivudine, tenofovir, and etravirine), and cotrimoxazole became impossible, despite intravenous morphine therapy. On Day 35 a jejunostomy procedure was performed. By analogy with reported treatment of severe HIVassociated aphthous lesions with thalidomide [START_REF] Paterson | Thalidomide as treatment of refractory aphthous ulceration related to human immunodeficiency virus infection[END_REF] and given the pathophysiological importance of TNF-α [START_REF] Natah | Immunolocalization of tumor necrosis factor-alpha expressing cells in recurrent aphthous ulcer lesions (RAU)[END_REF], a subcutaneous treatment with the anti-TNF-α etanercept (25 mg twice weekly) was initiated on Day 50. Symptoms and aphthous ulcers improved considerably within three days. On Day 85, after a 35-day course of etanercept, thalidomide was introduced at 100 mg daily. On Day 180 the patient was well with no remaining aphthous ulcers. He remained HIV-viral load undetectable and had increased lymphocyte T CD4 level (189 cells/mm³).

Discussion

Aphthous ulcers are round and well-marked ulcerations, with a hard and clean core, covered with a yellowish pseudo-membrane and surrounded by an erythematous halo. They are painful and can, in severe cases, prevent swallowing and phonation. They are usually found on non-keratinized epithelia of the oropharyngeal, genital, and/or anorectal mucosa [START_REF] Macphail | Recurrent aphthous ulcers in association with HIV infection. Description of ulcer types and analysis of T-lymphocyte subsets[END_REF]. Lesions are made of sub-epithelial mononuclear infiltrates, associated with basal keratinocytes, with neutrophils adjacent to the lesions. TNF-α is central to the pathophysiology [START_REF] Natah | Immunolocalization of tumor necrosis factor-alpha expressing cells in recurrent aphthous ulcer lesions (RAU)[END_REF]. There seems to be an increased incidence and severity of aphthous lesions in HIV-infected patients, particularly in subjects with low lymphocyte T CD4 depletion levels [START_REF] Macphail | Recurrent aphthous ulcers in association with HIV infection. Description of ulcer types and analysis of T-lymphocyte subsets[END_REF]. Persistent and large ulcers may require biopsy to exclude lymphoma, deep fungal or mycobacterial infections, atypical herpes simplex lesions, and cytomegalovirus disease.

The first-line treatment of moderate to severe aphthous ulcers is a systemic corticosteroid therapy (70% of effectiveness according to a study [START_REF] Femiano | Pilot study on recurrent aphthous stomatitis (RAS): a randomized placebo-controlled trial for the comparative therapeutic effects of systemic prednisone and systemic montelukast in subjects unresponsive to topical therapy[END_REF]). Thalidomide 100 mg daily is considered the most effective second-line treatment of severe cases or in case of treatment failure [START_REF] Paterson | Thalidomide as treatment of refractory aphthous ulceration related to human immunodeficiency virus infection[END_REF], due to its immuno-modulation and anti-TNF-α action. It is mandatory to monitor the hemogram, neurological signs, and to prescribe an effective birth control method. The impossibility to use oral thalidomide in our patient led us to discuss subcutaneous anti-TNF-α therapies. The spectacular efficacy of anti TNF-α in severe aphthous ulcers has been previously described in Behcet's and Crohn's diseases with etanercept [START_REF] Melikoglu | Short-term trial of etanercept in Behcet's disease: a double blind, placebo controlled study[END_REF][START_REF] Hasan | Remission of severe aphthous stomatitis of celiac disease with etanercept[END_REF][START_REF] Robinson | Recalcitrant, recurrent aphthous stomatitis treated with etanercept[END_REF], but only in the general population. The choice of etanercept in our case was justified by its low reported rate of infections in the general population and its shorter half-life in case of adverse events [START_REF] Takeuchi | Pharmacokinetics, efficacy and safety profiles of etanercept monotherapy in Japanese patients with rheumatoid arthritis: review of seven clinical trials[END_REF]. Several cases with safe use of subcutaneous anti-TNF-α in HIV-infected patients presenting with other conditions, including life-threatening immune reconstitution inflammatory syndrome, have also been reported [START_REF] Simone | Treatment of psoriasis with etanercept in immunocompromised patients: Two case reports[END_REF][START_REF] Richaud | Anti-tumor necrosis factor monoclonal antibody for steroid-dependent TB-IRIS in AIDS[END_REF].

The continuous worsening of our patient's lesions prior to treatment initiation and the spectacular improvement of the lesions after introduction of etanercept suggested direct involvement of the drug in the clinical response. To our knowledge this is the first reported case of an HIV-infected patient presenting with severe and refractory aphthous ulcers treated with subcutaneous anti-TNF-α. Further reports could confirm etanercept response in HIV-related aphthous ulcers, particularly in salvage cases.

Figure 1 . 1 .

 11 Figure 1. Mouth aphthous ulcers before (above) and after 15 days of etanercept treatment (below). 1. Aphtes à l'initiation (au-dessus) et après 15 jours de traitement par étanercept (endessous).

Funding

This study has received no funding. Data was generated as part of the routine work in the Department of Infectious Diseases of the University Hospital of Montpellier, France.

Declaration of interests

The authors declare no competing interests.


Thomas Clouaire

Gaëlle Legube
email: gaelle.legube@univ-tlse3.fr

A Snapshot on the Cis Chromatin Response to DNA double-strand breaks

In eukaryotes, detection and repair of DNA Double Strand Breaks (DSBs) operate within chromatin, an incredibly complex structure that tightly packages and regulates DNA metabolism. Chromatin participates in the repair of these lesions at multiple steps, from detection to genomic sequence recovery and chromatin is itself extensively modified during the repair process. In recent years, new methodologies and dedicated techniques have expanded the experimental toolbox, opening up a new era granting the highresolution analysis of chromatin modifications at annotated DSBs in a genome-wide manner. A complex picture is starting to emerge whereby chromatin is altered at various scales around DSBs, in a manner that relates to the repair pathway used, hence defining a "repair histone code". Here we review the recent advances regarding our knowledge of the chromatin landscape induced in cis around DSBs, with an emphasis on histone posttranslational modifications and histone variants.

DSB repair occurs within chromatin

DNA double-strand breaks (DSBs) constitute very dangerous lesions having the potential to produce harmful genetic alterations such as local DNA sequence modifications (insertions/deletions) or more substantial chromosomal rearrangements (e.g. translocations). To faithfully maintain genomic integrity, cells deploy a DNA Damage Response (DDR) comprising specialized machineries able to sense, signal and repair DSBs by Homologous Recombination (HR) or Non-Homologous End Joining (NHEJ) [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation[END_REF] (Box1). DSB can occur upon exposure to a variety of genotoxic agents (ionizing radiations, chemotherapeutic drugs…) and under physiological conditions including developmentally programmed DSBs in lymphocytes or during meiosis but also ubiquitous processes such as replication and transcription. Indeed, DSBs arise more frequently than previously anticipated and their induction rate is not homogeneous along the genome. For instance, transcribed genes (reviewed in [START_REF] Marnef | Transcription-Coupled DNA Double-Strand Break Repair: Active Genes Need Special Care[END_REF]) or chromatin loop anchors [START_REF] Canela | Genome organization drives chromosome fragility[END_REF] appear to represent regions of increased DSB frequency and technological improvements will most likely identify other classes of endogenous DSBprone regions in the future. It is therefore not surprising for DSB repair anomalies to be responsible for many disorders (premature aging, neurodegenerative syndromes) as well as the onset and progression of cancer.

Like any other DNA-related process, DSB repair occurs within a precisely organized and dynamic chromatin environment (Box2). Histone post-translational modifications or variants can alter nucleosome structure and therefore control many properties of the chromatin fiber (such as DNA accessibility, secondary structures, topological constraints, stiffness and mobility…) but can also directly act as recruitment platforms for chromatin reader proteins emphasizing the pivotal function of chromatin during transcription, replication and repair.

Chromatin in DSB repair: New tools, new questions, new answers

During DSB repair, chromatin modifications have the potential to contribute to the accurate execution of repair pathways by regulating DNA end accessibility, synapsis or single-strand DNA generation but also to participate in DSB signaling or the control of DSB mobility within the nucleus. Yet, while the genome-wide sequencing revolution strongly helped to understand the role of chromatin in transcription [START_REF] Zhou | Charting histone modifications and the functional organization of mammalian genomes[END_REF] its detailed contribution to DSB repair has long been impeded by the inability to induce DSBs at defined positions across entire cell populations. This precluded the use of dedicated methods such as ChIP-seq to analyze DSB-induced chromatin changes at high resolution or to compare DSB repair events occurring in different chromatin contexts. The recent development of tools to induce DSBs at annotated positions (Box3), combined with high-throughput sequencing approaches and/or super-resolution microscopy revolutionized our capability to study the nature and function of chromatin at DSBs. These new methods could make it possible to tackle new questions, such as: [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation[END_REF] To what extent the chromatin response to DSB is universal, or in other words how does it change depending on the genomic position of the break, as well as 2) what is the nature of DSB-induced chromatin and how does this chromatin landscape contribute to repair reactions and to the maintenance of genomic integrity? While it is now clearly established that the position of the break, and its associated chromatin features, plays a critical role in its subsequent repair (reviewed in [START_REF] Marnef | Transcription-Coupled DNA Double-Strand Break Repair: Active Genes Need Special Care[END_REF][START_REF] Fortuny | The response to DNA damage in heterochromatin domains[END_REF][START_REF] Van Sluis | Nucleolar reorganization in response to rDNA damage[END_REF]) the precise chromatin landscape induced around DSBs has only recently started to be deciphered.

Repair histone code: Different scale, different functions

Elegant studies, following the recruitment kinetics for many repair factors upon DSB induction at localized microirradiation (for example [START_REF] Aleksandrov | Protein dynamics in complex DNA lesions[END_REF][START_REF] Kochan | Meta-analysis of DNA double-strand break response kinetics[END_REF]) have started to accurately establish the spatiotemporal organization of the DDR. Additionally, the use of phospho-activatable GFP-tagged core histone identified that chromatin is rapidly relaxed following laser microirradiation in a manner that depends on PARP1 and chromatin remodelers (for instance [START_REF] Sellou | The poly(ADP-ribose)dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage[END_REF][START_REF] Smith | CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation[END_REF][START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF][START_REF] Burgess | Activation of DNA damage response signaling by condensed chromatin[END_REF]), followed by a recompaction phase, necessary for full DDR activation [START_REF] Burgess | Activation of DNA damage response signaling by condensed chromatin[END_REF] (Fig. [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation[END_REF]Key Figure). Altogether this revealed that chromatin structure is altered in an ordered manner following DSB (reviewed in [START_REF] Dantuma | Spatiotemporal regulation of posttranslational modifications in the DNA damage response[END_REF]). Yet, the exact molecular nature of in cis chromatin modifications that accounts for this DSB-induced chromatin behavior visualized by microscopy is unknown. Since chromatin is organized at many different scales, it is important to precisely understand how DSBs can reshape this structure at each level, from the nucleosomes adjacent to the broken sites up to entire chromosomal domains and this starts with the molecular characterization of the chromatin landscape induced at DSB. To this aim, a systematic and high-resolution ChIP-Seq mapping of histone modifications induced at multiple DSBs was performed in human cells [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF].

This study took advantage from the DIvA system (see Box3), which allows for the controlled induction of about 100 DSBs, in both transcriptionally active and inactive regions (with exception to heterochromatin), within a short time window of 4 hours in cultured U2OS cells. This revealed a multiscale and complex pattern of induced histone modifications around DSBs, with few large-scale (megabase wide) chromatin changes, as well local (<10kb) alterations [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF] (Fig. 1, Key Figure). Additionally, mapping of repair proteins, such as those involved in HR or NHEJ, allowed assignment of a specific chromatin landscape to a particular repair pathway. These local alterations of the chromatin landscape are both involved in recruiting repair proteins (such as pro or anti-resection factors) and in modulating the nucleosome stability onto DNA, thereby regulating DSB processing. Below we provide a recent update on the nature and (potential) function of the chromatin-induced DSB landscape, with a focus on histone posttranslational modifications occurring during either HR or NHEJ and thereby defining the "repair pathway histone code" (Fig. 2).

Large-scale modifications, regulating the mobility of the fiber within the nucleus?

The first histone modification shown to be specifically induced in cis to DSB was the phosphorylation of the H2AX histone variant on serine 139 also known as γH2AX [START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF]. This modification, mainly catalyzed by kinases such as ATM or ATR, displays the striking behavior to spread over incredibly large domains since it can cover up to 2 megabases of DSBsurrounding chromatin in mammals [START_REF] Iacovoni | High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome[END_REF][START_REF] Caron | Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks[END_REF][START_REF] Savic | Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin[END_REF]. Interestingly, γH2AX signaling may not only rely on modification of pre-existing H2AX but also on de novo deposition of this variant at damaged sites [START_REF] Piquet | The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage[END_REF]. Remarkably, accumulation of ubiquitin conjugates on chromatin after damage [START_REF] Morris | BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair[END_REF] follows a near identical pattern to γH2AX [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF]. Additionally, this study also found a reduction in linker histone H1 on large chromosomal domains (several megabase) upon DSB induction [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF], in agreement with previous findings [START_REF] Sellou | The poly(ADP-ribose)dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage[END_REF][START_REF] Li | Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair[END_REF][START_REF] Strickfaden | Poly(ADPribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation[END_REF]. This large scale H1 eviction may depend on H1 degradation [START_REF] Li | Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair[END_REF], or H1 post-translational modifications (such as acetylation [START_REF] Li | Histone H1 acetylation at lysine 85 regulates chromatin condensation and genome stability upon DNA damage[END_REF] or ubiquitination [START_REF] Thorslund | Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage[END_REF], although controversial [START_REF] Nowsheen | L3MBTL2 orchestrates ubiquitin signalling by dictating the sequential recruitment of RNF8 and RNF168 after DNA damage[END_REF]) that may loosen its interaction with the nucleosomal fiber. Yet, while such extensive and seemingly disproportionate propagation around DSB triggers the formation of cytologically visible foci within the nuclei upon damage, the function of γH2AX -and other associated megabase-scaled modifications -is still unclear. Indeed, H2AX-deleted mice are viable, although sterile, immune-deficient and cancer-prone [START_REF] Scully | Double strand break repair functions of histone H2AX[END_REF] indicating that H2AX phosphorylation upon damage is not absolutely required for DSB repair but may play a more subtle role. A tempting hypothesis is that chromatin modifications on such a large scale contribute to the motion of the damaged chromatin fiber in the nucleus, ensuring DSB mobilization, clustering and/or homology search thereby fine tuning repair events (reviewed [START_REF] Marnef | Organizing DNA repair in the nucleus: DSBs hit the road[END_REF]) (Fig. 3). In support of this hypothesis, in yeast, DSB end mobility depends on the DDR kinase Mec1 and on the presence of its target phosphorylation sites on histone H2A [START_REF] Dion | Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery[END_REF][START_REF] Herbert | Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast[END_REF]. Additionally it was recently reported that the megabase-scaled modifications (γH2AX, H1 removal and ubiquitin accumulation) are much more pronounced at DSB exhibiting movement, i.e. DSBs prone to form large clusters in G1 [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Aymard | Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes[END_REF]. Indeed, though the underlying mechanism remains unclear, this acute chromatin signaling and ability to move and cluster appears to be a key feature of persistent DSB such as DSBs in transcriptionally active genes which are not efficiently repaired in G1 but rather undergo HR repair in S/G2 [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Aymard | Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes[END_REF][START_REF] Aymard | Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks[END_REF]. Large-scale chromatin changes could contribute to DSB motion by a) regulating the stiffness (rigidity) of the chromatin fiber, b) altering its compaction and c) promoting phase separation mechanisms (Fig. 3). Interestingly evidence for all three mechanisms have been reported (see below). Computational models and experimental data suggest that DSB-driven increased chromatin stiffness could translate into enhanced chromatin mobility [START_REF] Herbert | Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast[END_REF][START_REF] Miné-Hattab | Multiscale tracking reveals scale-dependent chromatin dynamics after DNA damage[END_REF]. Yet, how these models fit with the finding that core histone eviction occurs genome-wide following DSB, leading to a generalized increase in chromatin flexibility [START_REF] Hauer | Histone degradation in response to DNA damage enhances chromatin dynamics and recombination rates[END_REF] is unclear. Additionally, the chromatin embedded in γH2AX foci displays a decondensed-like appearance and increased short-range intra-chromosomal contacts [START_REF] Aymard | Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes[END_REF][START_REF] Kruhlak | Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks[END_REF]. This is in line with H1 depletion, which increases DNA accessibility in other contexts (for example [START_REF] Iwasaki | Piwi modulates chromatin accessibility by regulating multiple factors including histone H1 to repress transposons[END_REF]). Finally, liquid-liquid phase separation occurs in irradiation-induced foci [START_REF] Altmeyer | Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)[END_REF]. While such phase separation was shown to be seeded by Poly(ADP-Ribose) Polymerase (PARP) activity, it is tempting to envision that γH2AX, ubiquitin or 53BP1, which decorates entire γH2AX domains in G1 [START_REF] Smith | CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation[END_REF][START_REF] Kruhlak | Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks[END_REF][START_REF] Iwasaki | Piwi modulates chromatin accessibility by regulating multiple factors including histone H1 to repress transposons[END_REF] could further contribute to phase separation. Like its role in the physical interaction of other chromatin compartments [START_REF] Nuebler | Chromatin organization by an interplay of loop extrusion and compartmental segregation[END_REF], phase separation could mediate physical interaction of multiple γH2AX-decorated chromatin domains (i.e. DSB clustering). Whether phase separation cooperates or acts in parallel to other processes promoting DSB mobility, for example involving nucleo-and or cyto-skeletal proteins [START_REF] Aymard | Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes[END_REF][START_REF] Caridi | Nuclear F-actin and myosins drive relocalization of heterochromatic breaks[END_REF][START_REF] Schrank | Nuclear ARP2/3 drives DNA break clustering for homology-directed repair[END_REF][START_REF] Lottersberger | 53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair[END_REF][START_REF] Oshidari | Nuclear microtubule filaments mediate non-linear directional motion of chromatin and promote DNA repair[END_REF] remains to be elucidated.

Hence, large-scale modification of chromatin may not directly participate in the repair reaction itself but could still contribute to the specific dynamic properties of damaged genomic regions.

Local Chromatin Changes during NHEJ repair: Promoting NHEJ repair factors accessibility and restricting resection

Beyond the large scale changes mentioned above, the local chromatin state (<10kb) also experiences severe changes around DSBs. A number of chromatin modifications are reported to foster NHEJ (Fig. 2). Among them, the H3.3 histone variant was found deposited at sites of damage by the chromatin remodeler chromodomain helicase DNA-binding protein 2 (CHD2), where it contributes to efficiently recruit NHEJ core factors (Ku and XRCC4) [START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF]. Similarly, H2A.Z incorporation also potentiates Ku70 loading [START_REF] Xu | Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair[END_REF], although its further removal by ANP32e is required for completion of NHEJ repair [START_REF] Gursoy-Yuzugullu | Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair[END_REF]. H4Y51 was recently reported to be phosphorylated by the Tyrosine kinase receptor TIE2 and contributes to NHEJ [START_REF] Hossain | TIE2mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1[END_REF]. It was also reported that H2BK120 undergoes a monoubiquitin to acetyl transition on ~2-3 kb surrounding DSBs, a conversion that can be catalyzed by the deubiquitinase (DUB) and acetyl transferase (HAT) activities of the SAGA complex in vitro [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF]. Importantly, knock-down of SAGA subunits impaired NHEJ [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF], in agreement with a previous study [START_REF] Ramachandran | The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation[END_REF]. Notably H2BK120ub removal reduces nucleosome occupancy genome-wide [START_REF] Batta | Genome-wide function of H2B ubiquitylation in promoter and genic regions[END_REF], CHD2-mediated H3.3 deposition promotes chromatin expansion [START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF] and H2AZ-containing nucleosomes generally associates with an open chromatin structure (discussed in [START_REF] Billon | Precise deposition of histone H2A.Z in chromatin for genome expression and maintenance[END_REF]). Additionally nucleosome core particles containing both H3.3 and H2AZ were described as particularly unstable [START_REF] Jin | Nucleosome stability mediated by histone variants H3.3 and H2A[END_REF]. Hence several pathways seem to converge towards local nucleosome destabilization in order to promote access to repair machineries. In agreement, evidence suggest that in G1-arrested cells, where DSBs mainly undergo NHEJ, nucleosome removal occurs on a <2kb window around breaks (~8 nucleosomes) [START_REF] Li | Nucleosome disassembly during human nonhomologous end joining followed by concerted HIRA-and CAF-1-dependent reassembly[END_REF] and histone displacement may facilitate the binding of NHEJ factors near the break [START_REF] Goldstein | Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair[END_REF].

The status of H3K36 also seems to play a critical role in NHEJ. In yeast, Set2-mediated H3K36 methylation promotes the use of NHEJ in G1 by antagonizing acetylation on H3K36 or H4 [START_REF] Jha | An RNA polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair[END_REF][START_REF] Pai | A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice[END_REF]. In mammalian cells, H3K36me2 increases at the immediate vicinity of a single I-SceI break (<500bp) where it was shown to contribute to NHEJ [START_REF] Fnu | Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining[END_REF]. Interestingly, genome-wide mapping data at AsiSI-induced DSBs could not recapitulate H3K36me2 accumulation, however, a modest increase of H3K36me3 at NHEJ-repaired DSB was found [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF]. H3K36me3 accumulates on genes bodies during transcription elongation and prevents cryptic transcription initiation by recruiting of Histones Deacetylase (HDAC) (reviewed in [START_REF] Mcdaniel | Shaping the cellular landscape with Set2/SETD2 methylation[END_REF]). Of interest, HDAC1/2 is recruited at sites of damage at very early stage (before ATM) [START_REF] Aleksandrov | Protein dynamics in complex DNA lesions[END_REF] and promote NHEJ [START_REF] Miller | Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining[END_REF]. This raises the possibility that H3K36 trimethylation could contribute in HDAC recruitment at the immediate vicinity of the break during NHEJ repair. One could envision a mechanism whereby histone removal (via SAGA, CHD2 or H2AZ, see above) would promote access to NHEJ factors exactly at the break point whilst H3K36me3-mediated recruitment of HDAC would rather stabilize nucleosome further away to disfavor resection (Fig. 4).

Finally, the histone modification landscape also controls NHEJ by attracting anti-resection factors (reviewed in [START_REF] Wilson | Reading chromatin signatures after DNA double-strand breaks[END_REF]) such as 53BP1 (reviewed in [START_REF] Panier | Double-strand break repair: 53BP1 comes into focus[END_REF]), which exerts its activity by recruiting downstream effectors including the recently characterized Shieldin complex [START_REF] Gupta | DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity[END_REF][START_REF] Mirman | 53BP1-RIF1-shieldin counteracts DSB resection through CST-and Polα-dependent fill-in[END_REF][START_REF] Noordermeer | The shieldin complex mediates 53BP1-dependent DNA repair[END_REF][START_REF] Ghezraoui | 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ[END_REF][START_REF] Gao | An OB-fold complex controls the repair pathways for DNA double-strand breaks[END_REF][START_REF] Dev | Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells[END_REF] and the CST/DNA polymerase α that reconstitutes processed DNA ends [START_REF] Mirman | 53BP1-RIF1-shieldin counteracts DSB resection through CST-and Polα-dependent fill-in[END_REF]. 53BP1 displays a strong binding affinity for H4K20me1/2 and for H2A ubiquitinated at Lysine 15 (H2AK15ub), via a tandem Tudor-UDR (Ubiquitin-dependent interacting region) domain [START_REF] Fradet-Turcotte | 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark[END_REF][START_REF] Wilson | The structural basis of modified nucleosome recognition by 53BP1[END_REF]. While RNF168 ensures H2AK15ub accumulation in response to DSB [START_REF] Mattiroli | RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling[END_REF], the majority of nucleosomes are methylated on H4K20, even in absence of damage, outside replication [START_REF] Pesavento | Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle[END_REF][START_REF] Saredi | H4K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22L DNA repair complex[END_REF]. Nevertheless DSB-induced accumulation of H4K20me1/2 (and in some instance me3) has been reported [START_REF] Hartlerode | Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks[END_REF][START_REF] Dulev | SET8 methyltransferase activity during the DNA double-strand break response is required for recruitment of 53BP1[END_REF][START_REF] Pei | MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites[END_REF][START_REF] Tuzon | Concerted activities of distinct H4K20 methyltransferases at DNA double-strand breaks regulate 53BP1 nucleation and NHEJ-directed repair[END_REF][START_REF] Hajdu | Wolf-Hirschhorn syndrome candidate 1 is involved in the cellular response to DNA damage[END_REF][START_REF] Svobodová Kovaříková | H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions[END_REF]. While no changes in H4K20me2 were detected in [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF], NHEJ-repaired DSBs displayed a moderate increase of H4K20me1 on 2-3kb in these ChIP-seq assays. Hence, pre-existing or de novo deposited H4K20me1/2 may allow, in G1, initial recruitment of 53BP1, which would be further stabilized by DSB-induced H2AK15 ubiquitination (Fig. 4).

Local Chromatin changes promoting Homologous Recombination

Many histone modifications were shown to be involved in promoting HR (Fig. 2). ChIP-seq mapping revealed significant changes at the vicinity of DSB repaired by HR. This includes macroH2A deposition, in agreement with previous reports [START_REF] Khurana | A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance[END_REF][START_REF] Xu | The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1[END_REF], and a decrease in H2BK120ub, H2A.Z, H3K4me3, H3K79me2 and H4K12ac [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Gong | Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair[END_REF]. Importantly, most of these modifications were previously shown to be interdependent since macroH2A regulates H2BK120 acetylation while H2BK120ub and H4 acetylation stimulates both H3K4/H3K79 methylation and H2A.Z stabilization onto chromatin (see for example [START_REF] Chen | MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBPmediated H2B acetylation[END_REF][START_REF] Mcginty | Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation[END_REF][START_REF] Altaf | Interplay of chromatin modifiers on a short basic patch of histone H4 tail defines the boundary of telomeric heterochromatin[END_REF][START_REF] Segala | Monoubiquitination of Histone H2B Blocks Eviction of Histone Variant H2A.Z from Inducible Enhancers[END_REF][START_REF] Zhu | Monoubiquitination of human histone H2B: the factors involved and their roles in HOX gene regulation[END_REF]). Of note, this work [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF] could neither recapitulate the increase of H2B ubiquitination, nor the requirement of the SAGA H2B DUB activity in counteracting HR seen by others [START_REF] Evangelista | Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair[END_REF][START_REF] Zheng | Bre1-dependent H2B ubiquitination promotes homologous recombination by stimulating histone eviction at DNA breaks[END_REF][START_REF] Moyal | Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks[END_REF][START_REF] Nakamura | Regulation of homologous recombination by RNF20-dependent H2B ubiquitination[END_REF]. The reasons for such inconsistencies are yet unclear and call for further investigations, but may relate to the nature and position of induced breaks and/or more likely to kinetics issues (see Box 3 and Outstanding Questions).

The contribution of this specific nucleosome composition to HR remains to be elucidated. Indeed, a better characterization of these DSB-induced chromatin modifications across the cell cycle together with functional investigation of their involvement in the execution of accurate HR repair still need to be performed (see Outstanding Questions). Yet evidence suggests that the chromatin landscape can regulate HR by a) controlling the initiation and extent of resection, b) regulating the assembly of RAD51 filament downstream of resection and c) regulating the chromatin reconstitution of newly synthetized DNA (see below).

• Chromatin as a pro-resection factor recruitment platform.

Chromatin features have been shown to favor recruitment of pro-resection activities [START_REF] Wilson | Reading chromatin signatures after DNA double-strand breaks[END_REF] (Fig. 5, right side of the break). Pre-existing SETD2-dependent, H3K36me3 can be recognized by the PWWP domain of LEDGF, resulting in CtIP recruitment and stimulation of resection at DSBs in active genes, where H3K36me3 is usually present [START_REF] Aymard | Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks[END_REF][START_REF] Pfister | SETD2dependent histone H3K36 trimethylation is required for homologous recombination repair and genome stability[END_REF][START_REF] Carvalho | SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint[END_REF][START_REF] Daugaard | LEDGF (p75) promotes DNA-end resection and homologous recombination[END_REF]. Furthermore, DSB-induced H3K9me2 favors recruitment of BRCA1 and its partner BARD1 [START_REF] Khurana | A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance[END_REF][START_REF] Wu | Interaction of BARD1 and HP1 is required for BRCA1 retention at sites of DNA damage[END_REF]. BRCA1 recruitment to damaged sites is also enhanced by the DNA-and H2A/H2AX-binding protein ZMYM3 [START_REF] Leung | ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair[END_REF].

Finally, recruitment of the anti-resection factor 53BP1 is also downregulated as cells progress to S and G2 phases. Dilution of H4K20me2 on newly replicated chromatin triggers eviction of 53BP1 [START_REF] Pellegrino | Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin[END_REF] and in G2, the Histone acetyltransferase complex TIP60 can compete with 53BP1 for H4K20me2 binding, promoting H2AK15 and H4K16 acetylation which will antagonize 53BP1 binding, promote BRCA1 recruitment and favor resection [START_REF] Tang | Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination[END_REF][START_REF] Jacquet | The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation[END_REF].

• Relieving the nucleosomal barrier to resection

Pre-existing and DSB-established chromatin state can also directly control the generation of ssDNA. The presence of nucleosomes inhibits resection in vitro [START_REF] Adkins | Nucleosome dynamics regulates DNA processing[END_REF]. Moreover a recent genome-wide analysis of resection end points around Spo11 DSB hotspots during meiosis clearly demonstrated that, in yeast, resection frequently terminates at nucleosomes [START_REF] Mimitou | A global view of meiotic doublestrand break end resection[END_REF] providing the first experimental proof that nucleosomes somehow represent barriers to exonucleases processivity in vivo. Yet, Exo1 can efficiently progress through nucleosomes in vivo, since the resection rate fits with the one observed on naked DNA [START_REF] Mimitou | A global view of meiotic doublestrand break end resection[END_REF]. This suggests that histone modifications and ATP-dependent chromatin remodelers ensure nucleosome destabilization/remodeling in order to overcome this otherwise natural barrier. Indeed a plethora of chromatin modifying activities were found to control resection, both in yeast and mammals, including chromatin remodelers (SWI/SNF, INO80, SMARCAD1), H2A.Z exchange factors and histone modifying enzymes (extensively and recently reviewed in [START_REF] Rother | DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance[END_REF][START_REF] Chiu | Bromodomain proteins: repairing DNA damage within chromatin[END_REF][START_REF] Gursoy-Yuzugullu | Patching broken DNA: nucleosome dynamics and the repair of DNA breaks[END_REF][100]). In that context, a specific HR nucleosome post-translational signature (Fig. 2) may allow to bypass the inherent anti-resection properties of nucleosomes by decreasing its stability onto DNA or/and by acting as a recruitment platform for ATP-dependent remodelers, as shown for example for BRCA1/BARD1-mediated H2AK125/127/129 ubiquitination [101]. This would translate into partial or total nucleosome disassembly around DSBs (Fig. 5a, left side of the break), as repeatedly documented in both yeast and mammalian cells [START_REF] Li | Nucleosome disassembly during human nonhomologous end joining followed by concerted HIRA-and CAF-1-dependent reassembly[END_REF][START_REF] Goldstein | Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair[END_REF][102][103][104][105][106][107]].

• ssDNA nucleosomes during homologous recombination?

Evidence suggests that nucleosomes could also reassemble onto ssDNA in order to favour downstream recombinational events. A recent study suggested that histone chaperones ASF1 and CAF-1 can assemble nucleosomes on resected DNA, promoting the recruitment of MMS22L-TONSL [108], previously shown to specifically recognizes newly incorporated histone H4 unmethylated on lysine 20 [START_REF] Saredi | H4K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22L DNA repair complex[END_REF] and to promote RAD51 nucleofilament assembly [109][110][111]. Remarkably, nucleosomes can form in vitro on ssDNA [112] and ChIP analyses at DSB, especially in yeast, show rather moderate histone loss on efficiently resected regions [START_REF] Zheng | Bre1-dependent H2B ubiquitination promotes homologous recombination by stimulating histone eviction at DNA breaks[END_REF]108]. Similarly, H3 was only mildly depleted in human cells at HR-repaired AsiSI-induced DSBs, despite strong RAD51 binding and efficient resection [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Aymard | Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks[END_REF]113,114]. Furthermore, several chromatin-modifying activities promote RAD51 nucleofilament assembly downstream of resection. This includes INO80, Anp32E, p400, H2AZ removal and in yeast, Bre1-dependant H2BK123ub [106,115,116] suggesting that newly assembled nucleosomes on ssDNA requires remodeling to sustain efficient loading of RAD51. Taken together these data indicate that a specific nucleosomal state assembled onto ssDNA could regulates RAD51filament formation (Fig. 5b). Yet, a dedicated methodology that would formally prove the existence of nucleosomes assembled in single-stranded DNA in vivo is still awaited.

• Chromatin re-assembly during recombination-dependent DNA synthesis

Following resection and homology search, the canonical HR reaction proceeds with templated-DNA synthesis restoring an undamaged sequence at the broken locus. Yet, it is unclear if chromatin is reassembled during repair synthesis, similarly to normal DNA replication, or only after the resolution of recombination intermediates. It was recently shown that the chromatin remodeler ATRX, together with the histone chaperone DAXX and PCNA, mediates the incorporation of H3.3 during repair-dependent DNA synthesis in G2 [117] highlighting a coupling between repair synthesis and chromatin assembly (Fig. 5c). Maybe more surprisingly, ATRX-dependent chromatin assembly can stimulate long-range DNA synthesis and sister chromatid exchanges thereby favoring a particular form of HR leading to longer gene conversion tracts and potential crossovers. Further studies will be required to better understand 1) how chromatin assembly participates in these late stages of HR and 2) how accurate epigenetic information can be faithfully restored upon HR completion, since DNA damage have been linked with long term epigenetic defects [118].

Transient heterochromatin assembly in cis to DSB?

Several pieces of evidence also suggests that repressive histone marks and transient heterochromatinization could take place in cis to DSB. Both heterochromatin proteins (HP1, KAP1, Suv39h1 and Polycomb proteins) and histones marks associated with heterochromatin (H3K9me2, H3K9me3, H3K27me3 and macroH2A) accumulate around DSBs [START_REF] Fnu | Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining[END_REF][START_REF] Khurana | A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance[END_REF][START_REF] Xu | The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1[END_REF]119-122] but this was not consistent across all studies [123-126]. While a recent genome-wide ChIP-seq study confirmed macroH2A deposition on a 6-10kb window around DSBs, no changes in H3K9me2/3 were reported [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF] suggesting that some of these events may only occur at specific subsets of DSBs or that such events are too transient to be captured by ChIP-seq (see Box 3 and Outstanding Questions).

The function of this transient heterochromatin state is not fully established but H3K9 methylation, HP1 and macroH2A may help to promote resection and assemble BRCA1/ RAD51 at sites of breaks [START_REF] Khurana | A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance[END_REF][START_REF] Wu | Interaction of BARD1 and HP1 is required for BRCA1 retention at sites of DNA damage[END_REF]120,122,127]. In agreement, HP1 recruitment precedes those of BARD1, RPA and RAD51 at sites of micro-irradiation [START_REF] Aleksandrov | Protein dynamics in complex DNA lesions[END_REF]. Importantly, after an initial chromatin decondensation, macroH2A promotes re-condensation [START_REF] Khurana | A macrohistone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance[END_REF], suggesting that heterochromatin marks deposition indeed modifies chromatin structure around DSBs.

H3K9me3, via HP1 plays a pivotal role in heterochromatin domain formation through phase separation [128,129]. Hence, local (<10kb) and transient heterochromatinization could participate in DSB mobility and facilitate homology search, in agreement with the reported function of HP1/macroH2A in HDR. In addition, this heterochromatic state could help to turn off transcription at damaged genes (see below).

Transcriptional repression related changes

It has been convincingly demonstrated that the induction of a lesion in or near a transcribed unit will result in transcriptional silencing locally, in cis to the DSB (reviewed in [START_REF] Marnef | Transcription-Coupled DNA Double-Strand Break Repair: Active Genes Need Special Care[END_REF]130]). This does not solely arise from the mere presence of the lesion itself and likely involves several different mechanisms including DNAPK-, ATM-and PARP1-mediated control of RNA Pol II [119,[131][132][133][134][135]. This DSB-induced transcriptional silencing was also proposed to depend on Polycomb Group proteins-mediated H2AK119 ubiquitination and H3K27 methylation [131-134, 136] and on the NURD (Nucleososme remodeling and histone deacetylase) complex [START_REF] Gong | Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair[END_REF]137]. NURD is recruited to DSB at transcribed genes by the bromodomain protein ZMYND8, which recognizes acetylated H4, though the precise mechanism is not fully resolved [137].

Furthermore, NURD-mediated transcriptional silencing was shown to be necessary for the completion of HR [START_REF] Gong | Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair[END_REF]137] suggesting that it may represent a crucial step in DSB repair.

Transcription inhibition may also depend on a reduction in activating histone marks. Indeed it was found that DSB induction leads to a decrease in many histone modifications associated with active transcription (H3K4me3, H3K79me2, H2AZ, H4ac [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Gong | Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair[END_REF]), and H3K4me3 drop was shown to be required to mediate NURD recruitment and subsequent transcription silencing [START_REF] Gong | Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair[END_REF]. Further experiments are required to distinguish modifications solely required to ensure accurate transcriptional silencing from DSB-induced chromatin features directly participating in the execution of HR at transcriptionally active genes (see Outstanding Questions). Of note, several studies have reported that DSB can also lead to RNA pol II-dependent de novo RNA synthesis (reviewed in [130])) but the contribution of chromatin structure in this process remains to be elucidated.

Concluding Remarks

Recent methodological developments have provided us with a unique opportunity to apprehend in great details how and why chromatin is modified following DNA double-strand breaks and to understand how specialized chromatin pathways are set up depending on where the lesion occurs. Systematic mapping of the expanding number of chromatin features potentially involved in DNA repair (for example [138]) at precisely annotated DSB, combined with advanced proteomics, genome editing and super-resolution microscopy, will undoubtedly provide new and exciting results . Yet, key challenges will need to be tackled in the near future (see 'Outstanding questions'). For example, a kinetically resolved map of DSB-induced chromatin modifications, integrated with orderly recruitment of DSB repair proteins, will significantly increase our understanding of the spatio-temporal organization of DSB repair.

Furthermore, the advent of single-cell methods will allow to characterize the extent of cell-tocell variability in the response to DSBs. In this regard, the integration of advanced imaging using state-of-the-art locus labeling technologies [139] with the extent of details provided from single cell next-generation sequencing methods will undeniably continue to revolutionize our understanding of DSB repair in the context of organized chromosomes.

Box 1: DSB repair pathways

The two main DSB repair mechanisms are Non Homologous End joining (NHEJ) and Homologous Recombination (HR). NHEJ functions by direct ligation of the two broken ends, requiring no or minimal processing of the DNA ends. This pathway operates throughout the cell cycle and key NHEJ factors include Ku, DNA-PKcs, XRCC4, XLF, PAXX and DNA ligase IV (recently reviewed in [140]). During HR, the undamaged copy of the broken chromosome will be used to initiate DNA synthesis and restore the nucleotide sequence (reviewed in [141]).

For this, 5' to 3' nucleolytic degradation of the broken end, called resection, will be initiated by the MRN complex (MRE11-RAD50-NBS1) and CtIP and is further extended by EXO1, DNA2 and accessory factors such as the helicase BLM. RPA will bind newly generated singlestranded DNA to stabilize it. Subsequently, BRCA2 will stimulate the loading of the RAD51 recombinase to resected DNA, which results in a nucleofilament that will find the homologous sequence to initiate DNA synthesis. Under particular circumstances like HR deficiencies, alternative pathways such as Single-Strand Annealing (SSA) or MMEJ (Microhomology-Mediated End joining) can be mobilized, at a cost for genome integrity since these pathways tend to be highly mutagenic [140]. The initiation and extent of resection is a key aspect governing DSB repair pathway choice since it is a requirement for HR (and alternative mutagenic pathways) and will inhibit the execution of NHEJ. Key DSB repair factors such as 53BP1 and BRCA1 play very important roles in controlling resection.

Box 2: Chromatin is organized at many different levels

The primary level of chromatin organization consists in DNA winding around octamers of core histones (H2A, H2B, H3 and H4) forming arrays of nucleosomes, usually conceptualized as "beads on a string". In living cells, this basic chromatin fiber further folds and adopts a range of configurations and compaction states at many different scales, providing the foundation for the 3D organization of chromosomes in the nuclear space. Modifications at the nucleosomal level include DNA methylation, histone post-translational modifications (such as phosphorylation, acetylation, methylation, ubiquitination…) or incorporation of histone variants [142,143]. Nucleosomes themselves are also very dynamic in their positioning, occupancy and turnover. Furthermore, linker histones such as H1 can directly interact with the nucleosome and participate in the folding of nucleosome arrays. These chromatin marks do not occur randomly on the genome but follow a stereotyped pattern depending on the function of the underlying locus [START_REF] Zhou | Charting histone modifications and the functional organization of mammalian genomes[END_REF]. Indeed, specific chromatin features associate and characterize promoters, active gene bodies, regulatory elements (enhancers, domain boundaries), Polycombrepressed domains and heterochromatin (pericentromeres, subtelomeric regions, laminaassociated domains or nucleolus-associated domains). Furthermore, recent data suggest that genome folding in 3D is governed by both the segregation of active or inactive chromatin into compartmental domains as well as the formation of long-range chromatin loops by CTCF and cohesin, leading to the formation of topologically associating domains or TADs [144].

Box 3: The Pros and Cons of prevalent DSB induction methods

Common DSB-induction methods include ionizing radiations (γ-or X-rays), cross-linking agents (mitomycin C, cisplatin), radiomimetic compounds (bleomycin, neocarzinostatin), toposiomerase inhibitors (camptothecin, etoposide), replication inhibitors (hydroxyurea, aphidicolin), chemicals (such as hydrogen peroxyde) and localized laser microirradiation (damaging only a stripe in the nucleus). Since no new DSBs should occur after treatment, repair events are synchronous and can be kinetically resolved. Yet, while very potent and in many cases clinically relevant, these methods suffer drawbacks limiting their use in high-resolution chromatin studies. First, most of these methods induce DSB randomly throughout the genome, with the exception of topoisomerase poisons and replicative stress inducers, which produce DSB within "hotspots" (ie. respectively at sites of topoisomerase binding, or at sites where replication forks collapse). However even in the latter cases, where position of the DSBs are known, it is still unsure how often breakage occurs across the cell population, which will effectively dilute any high resolution signal obtained at the population level. Furthermore, most methods induce other types of damage (oxidized bases, abasic sites, single-strand DNA breaks) or may only effectively trigger DSBs in some cells in the population (for example in S phase).

A variety of sequence-specific DSBs induction systems have been developed over the years. This includes the HO endonuclease in yeast, TALE nucleases, Zinc Finger nucleases (e.g FokI), CRISPR/Cas9, homing endonucleases (I-SceI or I-PpoI) and restriction enzymes such as AsiSI (reviewed in [145]). These tools allow induction of DSBs at known positions on the genome, and importantly in a homogenous fashion in the cell population, making them amenable for high-resolution studies such as ChIP-seq. Furthermore, some systems (including AsiSI, I-PpoI or CRISPR/Cas9) allow to induce multiple DSBs scattered across the genome allowing to compare repair at various endogenous loci, while HO (unless used at its original Mat locus) or I-SceI require the enzyme recognition site to be artificially introduced in the genome. By design, the main disadvantage of these methods stems from the ability of nuclease to recut properly repaired sites, impairing efficient kinetic deconvolution of repair reactions, in sharp contrast with localized laser micro-irradiations, which can resolve events occurring a few seconds apart. Instead, the cell population represents a mixture of events ranging from early cleavage to late repair, which may impede the efficient detection of transient events. Lastly it is important to stress that these annotated DSBs induction systems may not all be equivalent. For example, AsiSI is unable to create DSB in heterochromatin (being inhibited by DNA methylation) and Cas9-induced breaks display an inherent propensity to be repaired by mutagenic pathways [146].

Glossary:

Chromatin reader: A protein with the ability to recognize specific modification on a nucleosome, most often a post-translational modification state, usually using a dedicated protein domain. Specific reader domains include for example Bromo-, PHD-, SANT, Chromoor BRCT domains ChIP-seq: Chromatin immunoprecipitation followed by next-generation sequencing, a method to assess the localization of a protein, or its post-translationally modified form, over the entire genome.

DIvA system: DSB Induced via AsiSI, a system for controlled induction of about 100 DSB in the human genome based on the controlled nuclear translocation of the restriction enzyme AsiSI which will subsequently cleave endogenous recognition sites in the genome (GCGATCGC).

DSB induced using AsiSI are restricted to the non CpG methylated fraction of the genome (the enzyme is inhibited by CpG methylation) so no cuts are induced in heterochromatin.

Histone variants: Non canonical variant of core histones, usually encoded by different genes and incorporated by specific complexes. Prominent histone variants include H2AZ or H2AX (for H2A) and H3.3 or CENP-A for H3 Phase separation: A physicochemical process resulting in intracellular compartmentalization by the formation of membrane-less compartments (or condensates). Liquid-liquid phase separation, or liquid demixing, leads to the formation of phase-separated liquid droplets with a denser liquid phase (with similarities to droplets of oil in water). Phase separation events can be seeded by multivalent interactions between DNA, RNA or proteins. This process has been involved in the formation of heterochromatin, the nucleolus or ionizing radiation-induced foci.

Chromatin remodelers: Enzymes, usually part of large protein complexes, using the energy of ATP hydrolysis to alter the structure of nucleosomes or nucleosomal arrays. Remodelers can evict or slide entire octamers or promote the incorporation or eviction of histones from the nucleosome.

SAGA complex: Spt-Ada-Gcn5 acetyltransferase complex, a multiprotein chromatin modifying complex with both histone acetyl transferase (HAT) and deubiquitinase (DUB) activities.

I-SceI break :

A DNA double-strand beak induced using the homing endonuclease I-SceI. Its large recognition site (18bp) is usually not found in the host genome and needs to be introduced by transgenic method, allowing for precise control of the position of the DSB.

AsiSI-induced DSB: DSBs induced using the restriction enzyme AsiSI, the key feature in the DIvA system (see Box3). Few large-scale, megabase-sized chromatin modifications occur in cis to DSBs, including the phosphorylation of the H2AX histone variant (γH2AX), ubiquitin chains accumulation and linker histone H1 removal. While their exact function still need to be deciphered, a tempting hypothesis is that they regulate the overall behavior of the damaged chromosomal domain, such as its mobility within the nuclear space. Widespread chromatin changes could alter physical properties of the chromatin fiber such as its rigidity (or stiffness), its compaction status, or/and its ability to phase separate from its local environment. These newly acquired physical properties would translate into a modified ability to move in the nucleus, hence fine-tuning downstream repair outputs. However, a detailed molecular and structural description of a γH2AX focus is still awaited. The use of super resolution microscopy as well as sequencingbased technologies allowing the characterization of chromatin composition and conformation (ChIP-seq, 4C-seq, Hi-C) will surely help to characterize γH2AX foci structures in better details, including the position of looping factors (such as cohesin, in yellow), nucleosomes density or chromatin loops which could alter the rigidity and compaction status of the fiber.

Pink and grey circles indicate γH2AX modified and unmodified nucleosomes respectively. H3.3 incorporation, H2BK120 deubiquitination and the complex cycle of H2AZ exchange will tend to locally destabilize nucleosomes, contributing to recruitment of NHEJ factors near the break. Other modifications will tend to create an environment that is not favorable for resection.

For example, H3K36 methylation may reduce accessibility away from the break by potentially recruiting HDACs, generally associated with chromatin condensation. Local methylation of H4K20, ubiquitination of H2AK15 and the absence of H4K16ac will favor the recruitment of 53BP1, a factor able to actively inhibit resection.

Figure 1 :

 1 Figure 1: Chromatin during DSB repair Upon DSB detection, both the initial chromatin context and the induced chromatin landscape play a central role in the repair process. How DSB repair varies across the genome, to deal with DSB induced in different chromatin context (thanks to specific recruitment of reader proteins, due to specific chromatin impediments or as a consequence of the particular physical position of the break in the nucleus) has been reviewed elsewhere (see main text). In this review we rather focus on the chromatin changes induced by DSBs (Panel A, central grey box), with a strong emphasis on the spatial distribution of histone modifications around DSB and their potential function during HR and NHEJ. However, since ChIP and ChIP-seq (allowing for a good characterization of the spatial distribution) only provide very low temporal resolution, it is unclear how these chromatin changes relate to the dynamic chromatin behavior observed by microscopy at sites of localised micro-irradiation (Panel B, also reviewed elsewhere, see main text). Describing the DDR response and chromatin changes at a high spatio-temporal resolution remains one of the greatest challenge for future research in the field (see Outstanding questions).

Figure 2 :

 2 Figure 2: A DSB repair histone code Recent studies revealed that nucleosomes exhibit a specific composition and post-translational signature during repair by NHEJ (left) and HR (right) repair. Yet, whether, how and when these histone modifications, individually or collectively, are actually required for repair still remains to be determined. The cross on top of a histone modification indicates the disappearance of this particular modification upon damage

Figure 3 :

 3 Figure 3: A function for large-scale chromatin modifications in DSB mobility?

Figure 4 :

 4 Figure 4: Proposed roles for chromatin modifications in NHEJ repair.

Figure 5 :

 5 Figure 5: The various impacts of chromatin modifications upon HR repair a) Several histone posttranslational modifications will facilitate resection via distinct mechanisms. On one hand (right side of the break), pre-existing or/and DSB-induced histone modifications will recruit pro-resection factors containing reader modules. For instance, H3K36me3 favors the recruitment of CtIP via the adaptor protein LEDGF, H3K9me2 can be recognized by BRCA1/BARD1 and Tip60-mediated acetylation of H2AK15 and H4K16

Acknowledgements

We apologize to our colleagues whose works could not be included in this review owing to space limitations. The GL laboratory is funded by grants from the European Research Council (ERC-2014-CoG 647344), Agence Nationale pour la Recherche (ANR-14-CE10-0002-01and ANR-13-BSV8-0013), the Institut National contre le Cancer (INCA), and the Ligue Nationale contre le Cancer (LNCC). TC is an INSERM researcher.

Competing interests

The authors have no competing interest to declare.