Brivaëla Moriceau
email: moriceau@univ-brest.fr

Importance of particle formation to reconstructed water column biogenic silica fluxes by

Keywords: Biogenic silica fluxes, dissolution, aggregate, modelling

The particles sinking out of the ocean's surface layer are made up of a mixture of living and dead algal cells, fecal pellets and aggregates, while the parameters used to describe the behaviour of biogenic silica (bSiO 2 ) in today's models are experimentally determined on freely suspended diatoms (FC). In a simple advection-reaction model we combine sinking rates of FC, zooplankton fecal pellets and aggregates with recent measurements of bSiO 2 dissolution to reconstruct theoretical downward profiles of bSiO 2 fluxes at 8 sites in the world ocean. Statistical analyses showed that sinking and dissolution parameters for two pools of bSiO 2 are required to model particle fluxes accurately. The 2-pool model results confirm the importance of seasonality for particle formation and its connection to the percentage of the bSiO 2 production that reaches the seafloor. The depth of bSiO 2 recycling appears to be influenced more by particle formation than by dissolution rates of bSiO 2 or the ballast effect of the dense diatom frustules.

Introduction

Diatoms form the basis of a short food chain that leads to carbon export to higher trophic levels [START_REF] Cushing | A difference in structure between ecosystems in strongly stratified waters and in those that are only weakly stratified[END_REF][START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF] and to deeper waters [START_REF] Buesseler | The decoupling of production and particulate export in the surface ocean[END_REF]. They are an important player in the global carbon cycle because of their participation in an efficient food web and their ability to aggregate [START_REF] Smetacek | Diatoms and the Ocean Carbon Cycle[END_REF]. Because diatoms require silica to build their frustules, their share in total primary production (PP) depends on the availability of the nutrient silicic acid (DSi) in many regions of the world ocean such as the equatorial Pacific [START_REF] Dugdale | The role of a silicate pump in driving new production[END_REF][START_REF] Leynaert | Silicon limitation of biogenic silica production in the Equatorial Pacific[END_REF] or the Southern Ocean [START_REF] Brzezinski | Silicon dynamics within an intense open-ocean diatom bloom in the Pacific Sector of the Southern Ocean[END_REF][START_REF] Pondaven | Modelling the silica pump in the Permanently Open Ocean Zone of the Southern Ocean[END_REF]]. The availability of DSi in surface waters, in particular relative to other nutrients, is linked to the composition of the source waters in upwelling regions and/or the intensity of biogenic silica (bSiO 2 ) recycling in surface waters in other oceanic regions. Depending on the depth of bSiO 2 dissolution, diatom production is affected on different time scale. Shallow depth mineralization immediately influences diatom production and phytoplankton dynamics [START_REF] Officer | The possible importance of silicon in marine eutrophication[END_REF]. Recycling in intermediate waters [START_REF] Sarmiento | Do We Now Understand The Ocean's Biological Pump?[END_REF] and/or deep waters [START_REF] Ragueneau | A review of the Si cycle in the modern ocean: recent progress and missing gaps in the application of biogenic opal as a paleoproductivity proxy[END_REF] affects the meridional distribution of DSi or the redistribution of DSi among oceanic basins, respectively, with implications for intermediate to long time scales.

A global average of 60% of the bSiO 2 produced in surface waters is recycled in the first 100 m [START_REF] Nelson | Production and dissolution of biogenic silica in the ocean: Revised global estimates, comparison with regional data and relationship to biogenic sedimentation[END_REF][START_REF] Tréguer | The silica Balance in the World Ocean: A Reestimate[END_REF]. This global mean hides large regional differences [START_REF] Nelson | Production and dissolution of biogenic silica in the ocean: Revised global estimates, comparison with regional data and relationship to biogenic sedimentation[END_REF][START_REF] Ragueneau | A review of the Si cycle in the modern ocean: recent progress and missing gaps in the application of biogenic opal as a paleoproductivity proxy[END_REF][START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different?[END_REF], which can not be related to any simple trend in production or diatom abundance, nor to any single mechanism known to influence dissolution, such as temperature [START_REF] Lawson | Silica dissolution rates of decomposing phytoplankton assemblages at various temperature[END_REF] or bacterial activity [START_REF] Bidle | Accelerated dissolution of diatom silica by marine bacterial assemblages[END_REF]. The dissolution to production ratio, integrated over the surface layer (∫D:∫P), has been related to the formation of diatom blooms by [START_REF] Brzezinski | The balance between silica production and silica dissolution in the sea: Insights from Monterey Bay, California, applied to the global data set[END_REF]; this ratio is lower during blooms, leaving more room for export and deeper recycling. However, the use of global biogeochemical models to reproduce both DSi and bSiO 2 flux profiles in the global ocean is difficult and does not always agree with in situ measurements [START_REF] Aumont | An ecosystem model of the global ocean including Fe, Si, P colimitations[END_REF]. We hypothesize that the present difficulty in modeling both the bSiO 2 and DSi profiles of the global ocean are due to underestimating the importance of particle formation. Whereas aggregates and fecal pellets of large grazers are major vehicles for export [review in [START_REF] Turner | Zooplankton faecal pellets, marine snow and sinking phytoplankton blooms[END_REF][START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF], the parameters used to represent the fate of PP in global ocean models are still measured on freely suspended diatoms.

To test this hypothesis, we developed a simple advection-reaction model using one and then two different pools of particles. The model combines a series of in vitro bSiO 2 dissolution experiments undertaken on the different constituents of the flux: freely suspended diatoms [compiled in Van Cappellen et al., 2002], zooplankton fecal pellets [Gallinari et al., unpublished;[START_REF] Schultes | The role of mesozooplankton grazing in the biogeochemical cycle of silicon in the Southern Ocean[END_REF] and aggregates [START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF] with data on bSiO 2 production, export and deep fluxes from 8 biogeochemical provinces of the world ocean [described in [START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different?[END_REF]. The bSiO 2 downward fluxes are modeled to fit the downward flux data at the eight sites. This allows us to derive important information about particle formation in the upper ocean. The composition of the bSiO 2 fluxes in terms of large or small particles and, the sinking and specific bSiO 2 dissolution rates of each component help to unravel mechanisms involved in the spatial variability of the intensity and depth of bSiO 2 dissolution.

Approach: from laboratory experiments to a virtual water column

Experimental measurements

The flux of matter sinking through the water column is mainly composed of freely suspended phytoplankton cells, aggregates, and zooplankton fecal pellets [START_REF] Turner | Zooplankton faecal pellets, marine snow and sinking phytoplankton blooms[END_REF][START_REF] Thornton | Diatom aggregation in the sea: mechanisms and ecological implications[END_REF]. Specific bSiO 2 dissolution rates are usually measured experimentally on freely suspended diatoms [compiled by [START_REF] Van Cappellen | Biogenic silica dissolution in the oceans: Reconciling experimental and field-based dissolution rates[END_REF]. Recently, however, specific dissolution rates of bSiO 2 incorporated into aggregates [START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF] and fecal pellets [START_REF] Schultes | The role of mesozooplankton grazing in the biogeochemical cycle of silicon in the Southern Ocean[END_REF]Gallinari, unpublished] have been measured in the laboratory and shown to be about half the rates measured on freely suspended diatoms (Table 1). Including the dissolution rates of all particle types in models should enable us to predict the bSiO 2 sedimentation flux more precisely. Here we consider a model of bSiO 2 flux with depth that includes two types of particles (free diatom cells: FC and large particles: LP). We can keep the model simple with only two particle types by assuming that the sinking rates and specific bSiO 2 dissolution rate of aggregates and fecal pellets are similar as is demonstrated in Table 1. The FC group has the slow sinking rate and fast specific bSiO 2 dissolution rate characteristic of free diatom cells. The LP group has faster sinking rates than the FC and half the specific bSiO 2 dissolution rate. For comparison, the model was first run using only one type of particle with sinking properties and bSiO 2 dissolution rates between those of FC and LP particles as described below.

The data used for this study were collected in eight biogeochemical provinces of the world ocean, for which annual measurements of bSiO 2 production, export and deep fluxes (at 2 depths) are available [data in [START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different?[END_REF]. In this dataset, export fluxes (at 200 m) were derived from models or from sediment trap measurements while deeper water fluxes (1000 and 3000 m depth) were measured using sediment traps. Two sites are located in the Equatorial 2). Four data points can be fitted for each site, providing a total of 32 data points.

Model description

The concentration profiles of bSiO 2 for each particle pool i (LP or FC) can be described by a simple advection-reaction model (Eq. 1):

i 2 i i 2 i i 2 bSiO (T) k z bSiO S t bSiO ⋅ - ∂ ∂ ⋅ - = ∂ ∂ (1)
where t is time (d), z depth (m), S i is the sinking rate (m d -1 ) , and k i (T) is the (temperaturedependent) first-order specific bSiO 2 dissolution rate (d -1 ). Such first-order dissolution of bSiO 2 is often observed in batch experiments and leads to a simple exponential increase in the DSi concentration as a function of time [e.g. [START_REF] Greenwood | Biogenic silica dissolution in seawater -in vitro chemical kinetics[END_REF].

Assuming steady-state ( t bSiO 2 ∂ ∂ i =0), and constant sinking and dissolution rates, the concentration (mmol m -3 ) and flux of bSiO 2 (mmol m -2 d -1 ) at each water column depth (z) are

given by equation 2:

z S k 2 2 i i ) 0 ( bSiO ) ( bSiO ⋅ - ⋅ = e z i i
(2) which in terms of the total fluxes for the 1-particle model and the 2-particle model gives equation 3 and 3'respectively:

z s k 2 bSiO 2 PP ) ( FbSiO ⋅ - ⋅ = e z (3) ] ) α 1 ( α [ PP ) ( FbSiO z s k S k 2 bSiO 2 LP LP FC FC ⋅ - ⋅ - ⋅ - + ⋅ ⋅ = e e z z (3')
where α and (1-α) correspond to bSiO 2 production (PP bSiO2 , in mmol m -2 d -1 ) in the FC and the LP groups in the upper layer, respectively.

As temperature is allowed to vary, we use a piecewise approximation to model equation 3. The water column is subdivided into a number of layers (with thickness ∆z) in which temperature is assumed constant. Then, for each fraction, the flux at the bottom (z + ∆z) and top (z) of this layer are related by:

Δz S ki(T) 2i i 2 i ) z ( FbSiO ) Δz z ( FbSiO ⋅ - ⋅ = + e (4)
For the 1-particle model the flux at z = 0 equals the bSiO 2 production (PP bSiO2 ), and for the 2particle model the bSiO 2 flux at each depth is the sum of the LP and FC fluxes.

(z) FbSiO ) ( FbSiO ) ( FbSiO PP ) 1 ( ) 0 ( FbSiO PP ) 0 ( FbSiO LP 2 FC 2 TOT 2 2 bSiO 2LP 2 bSiO 2FC + = ⋅ - = ⋅ = z z α α (5)
In these equations, FbSiO 2TOT (z) is the flux of total bSiO 2 in mmol m -2 d -1 at depth z; PP bSiO2 is the production flux of bSiO 2 in mmol m -2 d -1 ; FbSiO 2 FC (z) and FbSiO 2 LP (z) are the fluxes of bSiO 2 in the FC and LP groups at depth z respectively. k FC (T) and k LP (T) are the specific bSiO 2 dissolution rates in d -1 given in the literature for FC particles (Table 1) and corrected by the factor of 2 less that was measured experimentally for LP particles. Rates were also corrected for temperature, according to equation (6) from [START_REF] Rickert | Dissolution Kinetics of biogenic silica from the water column to the sediments[END_REF]; temperature profiles of the water column at each site were from the NODC (Levitus) World Ocean Atlas 1998 (http://www.cdc.noaa.gov/).

(

) ( )

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - × + = 2 1 app 1 2 z T 1 T 1 R E k ln ) (T k ln (6)
where k 1 is the specific bSiO 2 dissolution rate in d -1 measured in controlled conditions in the laboratory; T 1 is the experimental temperature in degrees Kelvin (286.15 K); k z (T 2 ) is the rate recalculated for the depth z at the in situ temperature T 2 ; and Eapp/R = 7211 °K.

To facilitate comparison among sites, the total flux is normalized to the production flux to obtain a profile of bSiO 2 flux expressed in % of bSiO 2 production.

For both models we have:

100 PP ) ( FbSiO ) ( FbSiO % 2 bSiO 2tot 2 × = z z (7)
which for the 2-particle model becomes:

100 PP ) ( FbSiO ) ( FbSiO ) ( FbSiO % 2 bSiO 2LP 2FC 2 × + = z z z (7')

Parameter determination

As a first step, we applied the 1-pool model to fit the bSiO 2 flux profiles measured in the field. The specific dissolution rate was set to the value given in Table 1 for FC, and corrected for temperature at each site and depth using eq. 6. At each site, the model 1 calculated the bSiO 2 sinking rate required to fit the in situ bSiO 2 flux data. Note that the fit is not improved when the model uses LP specific dissolution rates instead of k FC nor an average of the two values.

In a second step, bSiO 2 specific dissolution rates and the sinking rates for two pools of particles were included in the model (model 2). Specific bSiO 2 dissolution rates of FC and LP

were set to values presented in Table 1 and corrected for in situ temperature at each depth using equation 6. The model 2 calculated a global sinking rate for FC particles (S FC ) for all sites, one LP sinking rate per site and the percent of bSiO 2 from the production which is incorporated into large particles (1-α) or stays freely suspended (α).

Finally in a third step (model 3), the parameter S FC was allowed to vary at each site.

In the three versions of the model, parameters were calculated by minimizing the sum of the square residuals using the Excel Solver and the GRG method (Generalized Reduced Gradient).

These calculations were tested for many initial values because the algorithm used is non linear.

Best fits to the data using the three model versions are shown for each of the eight sites in Fig. 1.

Statistical comparison of the 1-particle and 2-particle models

We used an F-test to compare the three versions of the model. The null hypothesis considers that adding complexity to the model does not significantly decrease the sum of the square residuals between model and observed data (SSR). The calculated F value is obtained using equation ( 8) and compared to the F test critical value.

F = (SSR1-SSR2) (df1-df2) / (SSR2/df2) (8)
SSR1 and SSR2 are the sum of the square residuals between the modeled and the experimental values; df1 and df2 are the degrees of freedom of the different versions of the model calculated as follow.

dfi = E -P -1 ( 9)

With E, the number of observed values the models has to fit (4 data for each of the 8 sites), and P the number of variable parameters used to optimize the fit.

The F test was first applied to compare the 1-particle model (hereafter referred to as model 1) to a 2-particle model using a global S FC (model 2). Next, we compared two versions of the 2-particle model, one using a global S FC (model 2 again) and one using a variable S FC at each site (model 3). When the model considered only one type of particle, it had 23 degrees of freedom. Model 2 used 17 parameters, providing14 degrees of freedom. Finally, model 3 had 7 degrees of freedom. 2) is a good approximation. Note that at the PAP site, the flux at 1000 m is low compared to the flux at 3000 m and can be explained by the presence of swimmers in the trap [START_REF] Lampitt | Material supply to the abyssal seafloor in the Northeast Atlantic[END_REF].

Results

Comparison of the model versions: Importance of particle formation

Model 2, which considered a global FC sinking rate (S FC ), gives more degrees of freedom than model 1, but the use of a global S FC is also an oversimplification. Indeed, for the PAP site in the North East Atlantic Ocean, and for the NACC and APFP sites located in the Pacific sector of the Southern Ocean (North of the Polar Front and on the Polar Front respectively), this approach doesn't fit the data closely. To improve the reconstruction of these bSiO 2 fluxes, the model has to use a S FC higher than the global one. Reconstructions of the Southern Ocean profiles are generally improved by using model 3 with a varying S FC (Fig. 1). The F-test also favors model 3 over model 2 at the 95% confidence level. At the annual scale, FC sinking rates can be classified into two groups: fast S FC in the Southern Ocean and PAP site and slow S FC in the rest of the world. This trend may well reflect the production of large, fast sinking diatoms commonly observed in the Southern Ocean [START_REF] Kemp | The production of giant marine diatoms and their export at oceanic frontal zones: implications for Si and C flux in stratified oceans[END_REF].

Freely suspended diatoms and large particles have such different sinking rates and specific bSiO 2 dissolution rates that parameters of the two groups of particles must be considered to correctly reconstruct bSiO 2 flux profiles. The role and importance of each particle type is confirmed by a sensitivity analysis performed on the profiles using model 3 for the sinking rates of the two particles (shown for POOZ, Fig. 2). Profiles of bSiO 2 fluxes are quite sensitive to variation in sinking rates: in the upper 1000 m the profile is mostly affected by the sinking rate of FC whereas deeper, the profile is more sensitive to variation of the LP sinking rate. The composition of the flux in the upper layer (parameter α) is well constrained, as when the contribution of FC and LP changes by only 5-10 %, the resulting profile of bSiO 2 flux significantly differs from the one measured (Fig. 2c).

Model outputs

For the eight sites, model 3 provides sinking rates ranging from 45 to 605 m d -1 for the LP and from 0.1 to 10.4 m d -1 for the FC (Table 2; Fig. 1), consistent with ranges found in the literature (Table 1). The sinking rates calculated by model 3 confirm the prevalence of aggregates and fecal pellets in the LP group (see values and references in Table1) and the fact that the FC group must be essentially composed of freely suspended diatoms, small and large.

Note that the 2-particles models (models 2 and 3) can not always strictly separate LP and FC using bSiO 2 sinking and specific dissolution rates. For the two sites located in the Southern Ocean (APFP and NACC) and for PAP, model 3 produces faster FC sinking rates and a faster LP sinking rates than those calculated by model 2. The S FC calculated by the model 2 is in these cases lowered by the other sites' S FC . The difference between the results of the two models for APFP, NACC and PAP shows the importance of a third group of particles that is included in LP in model 2 results and in FC in model 3 results. Large diatoms that have been observed in areas like the Southern Ocean, Gulf of California, and North Pacific Gyre [START_REF] Kemp | The "Fall dump" -a new perspective on the role of a "shade flora" in the annual cycle of diatom production and export flux[END_REF][START_REF] Kemp | The production of giant marine diatoms and their export at oceanic frontal zones: implications for Si and C flux in stratified oceans[END_REF] apparently dominate the production of APFP, NACC and PAP. Following the same argument, the very low S LP and S FC calculated by model 3 for APFA and POOZ suggest simultaneous production of small and large diatoms. Therefore, there are really three groups of particles (LP, freely-suspended large cells, and freely-suspended small cells); however, the large, freelysuspended diatoms have been included in the LP group here as the best compromise since the model can only account for two groups of particles.

Also, even if not shown here, giant aggregates with small and even null sinking rates such as those observed by [START_REF] Rinaldi | Mucilages in italian seas: the Adriatic and Thyrrhenian Seas, 1988-1991[END_REF] would certainly be integrated into the FC group of particles.

DISCUSSION

Particle formation: a major factor determining bSiO 2 export and recycling

In their global analysis of bSiO 2 production and dissolution, [START_REF] Brzezinski | The balance between silica production and silica dissolution in the sea: Insights from Monterey Bay, California, applied to the global data set[END_REF] demonstrated that the dissolution to production ratio integrated over the surface layer (∫D:∫P) is low under bloom conditions, leaving ample room for net export. They underlined the importance of considering dissolution when determining export of matter. Using the model results, we compare the importance of the two processes, sinking and dissolution, by calculating particle residence times in the mixed layer at each site with only sinking or only dissolution considered (Table 3). The comparison of these bSiO 2 residence times in surface waters suggests that this global data set of bSiO 2 production and dissolution cannot ignore particle formation. Sinking rates of large particles act on much shorter time scales than dissolution rates, so, rather than having the export determined by the intensity of the recycling, the intensity of export (1-∫D:∫P) is linked to the rate of large particle formation and drives the intensity of shallow-water recycling.

The rate at which diatoms are incorporated into large particles will determine how rapidly exported they are as their fast sinking rates leave no time for dissolution to occur in surface waters. In such cases, the dissolution depth is translated downward. The recycling intensity in surface waters depends first on the ability of diatoms to remain as single cells and secondly on factors such as temperature [START_REF] Bidle | Regulation of Oceanic Silicon and Carbon Preservation by Temperature Control on bacteria[END_REF][START_REF] Fujii | Effects of biogenic silica dissolution on silicon cycling and export production[END_REF][START_REF] Lawson | Silica dissolution rates of decomposing phytoplankton assemblages at various temperature[END_REF] and bacterial activity [START_REF] Bidle | Accelerated dissolution of diatom silica by marine bacterial assemblages[END_REF] which control their fate in surface waters.

Thus, we suggest that low ∫D:∫P ratios under bloom conditions [START_REF] Brzezinski | The balance between silica production and silica dissolution in the sea: Insights from Monterey Bay, California, applied to the global data set[END_REF] are caused by a rapid export of bloom diatoms [START_REF] Buesseler | The decoupling of production and particulate export in the surface ocean[END_REF], which extracts bSiO 2 from the surface waters where intense recycling generally occurs under non-bloom conditions.

Recognizing the importance of seasonality to the sinking of particles, we calculated the seasonality index of each site as defined by [START_REF] Berger | Export production: seasonality and intermittency, and paleoceanographic implications[END_REF] (Table 2) as six minus the number of months necessary to generate one half of the annual productivity when the data are ordered from the most productive to the least productive month (the 'production half-time').

Considering productivity, the eight sites could be classified in three groups [START_REF] Berger | Export production: seasonality and intermittency, and paleoceanographic implications[END_REF]: EqPac and BATS have constant productivity (seasonality index close to 0); OSP, APFA, NACC, POOZ, PAP have sinusoidal production (seasonality index between 2 and 3) and APFP has a strong seasonal peak in productivity (seasonality index > 3.5). As shown in Figure 3, sites exhibiting the lowest seasonality (EqPac, BATS) also exhibit the lowest export at 100 m (i.e. low 1-∫D:∫P). Conversely, sites with higher seasonality (APFP, NACC and APFA) display a larger 1-∫D:∫P at 100 m. Even if seasonality is not the only important factor, Figure 3 confirms its major role in particle formation. Indeed grazing and aggregation processes are more abundant at the end of a bloom [START_REF] Buesseler | The decoupling of production and particulate export in the surface ocean[END_REF] and consequently the export of matter out of the surface layer is also increased. To properly model diatom export versus recycling, further research efforts must focus not only on dissolution properties, but perhaps more importantly, on representing particle formation during bloom termination.

Another implication of our model results concerns the ballast theory. Armstrong et al.

[2002] established a correlation between organic carbon and ballast (bSiO 2 , CaCO 3 ) fluxes below the mixing layer. Based on this model, [START_REF] Klaas | Association of sinking organic matter with various types of mineral ballast in the deep sea: Implications for the rain ratio[END_REF] concluded that organic carbon is carried into the deeper waters preferentially by calcium carbonate, which has a higher density (2.71 g cm -3 ) than opal (2.1 g cm -3 ). [START_REF] Passow | Switching perspectives: Do mineral fluxes determine particulate organic carbon fluxes or vice versa?[END_REF] proposed the inverse of this hypothesis, that carbon, in the form of TEP, could transport organic matter and ballast to the deep sea by promoting aggregation. In APFP, OSP and NACC sites (Table 2), the fast S LP suggested the dominance of large diatoms. At these sites, freely suspended diatoms can be exported from the mixed layer (Table 4) due to the low temperature at the surface layer and/or high sinking rates. But at most other sites, even ballasted with a frustule whose density is twice that of organic matter (1.06 g cm -3 ), a single diatom almost completely dissolves in the upper WML. This observation calls for more caution when considering the ballast theory and emphasizes once again the importance of bloom-ending processes to explain sedimentation fluxes. The fact that POC and ballast fluxes are closely linked below the mixed layer [START_REF] Armstrong | A new, mechanistic model for organic carbon fluxes in the ocean based on the quantitative association of POC with ballast minerals[END_REF] is not necessarily related only to the higher density of bSiO 2 and CaCO 3 compared to that of organic matter. The efficiency of sedimentation depends primarily on the incorporation of diatoms into large particles like aggregates or fecal pellets (Fig. 2c) in the mixed layer; the ballasting of these large particles throughout the whole water column plays a secondary role (Fig. 2b).

Implications for the silicate pump

The silicate pump is the process by which bSiO 2 is exported from surface waters with a greater efficiency than PON, driving the system towards DSi limitation. According to [START_REF] Dugdale | The role of a silicate pump in driving new production[END_REF], grazers are the motor of this silicate pump; whereas nitrogen is largely assimilated or recycled by grazers and therefore retained at the surface, bSiO 2 is packaged into fast-sinking fecal pellets and exported from the mixed layer. The silicate pump model was developed initially for the Equatorial Pacific [START_REF] Dugdale | Silicate regulation of new production in the equatorial Pacific upwelling[END_REF]] and later applied to the Southern Ocean [START_REF] Brzezinski | Silicon dynamics within an intense open-ocean diatom bloom in the Pacific Sector of the Southern Ocean[END_REF]. It is based on several assumptions, including that bSiO 2 dissolution in surface waters is negligible [START_REF] Dugdale | The role of a silicate pump in driving new production[END_REF]. Our results clearly contradict this assumption at the EqPac site [START_REF] Dugdale | Silicate regulation of new production in the equatorial Pacific upwelling[END_REF]. EqPac is the site with the shallowest recycling in our model output (Fig. 1), as observed previously in the field [START_REF] Blain | Stocks and fluxes of biogenic silica in the western oligotrophic equatorial Pacific[END_REF]. The EqPac site is well-known for its low export efficiency [START_REF] Buesseler | The decoupling of production and particulate export in the surface ocean[END_REF], possibly related to its small seasonal variability (Table 2, Fig. 3). Consequently, the fraction of bSiO 2 production incorporated into large particles by aggregation/ fecal pellet production is lowest at the EqPac site on an annual basis (Table 2). Our results (Table 4) suggest that the vast majority of the diatoms remain as single cells in surface waters, and 88% dissolve before reaching the base of the mixed layer (on average, 50 m). This extensive dissolution is consistent with the high temperatures (i.e., high dissolution rate) and bacterial activity (i.e., fast removal of protective organic coatings) encountered in these equatorial waters [START_REF] Bidle | Regulation of Oceanic Silicon and Carbon Preservation by Temperature Control on bacteria[END_REF]. It is important to note that these are annual fluxes. Episodic events such as Tropical Instability Waves have been shown to trigger intense diatom concentrations at fronts [START_REF] Yoder | A line in the sea[END_REF], leading to massive sinking of algal material and the formation of large flocs of phytodetritus on the seafloor [START_REF] Smith | Phytodetritus at the abyssal seafloor across 10° of latitude in the central equatorial Pacific[END_REF]. However, although these episodes may be very important for sustaining life in the mesopelagic and at the sediment-water interface [Smith et al., 2002;[START_REF] Smith | Benthic community responses to pulses in pelagic food supply: North Pacific subtropical Gyre[END_REF], or for formation of paleoceanographic records [START_REF] Ragueneau | A review of the Si cycle in the modern ocean: recent progress and missing gaps in the application of biogenic opal as a paleoproductivity proxy[END_REF], our results suggest that they do not contribute appreciably to annual POC and bSiO 2 fluxes in oligotrophic ecosystems. [START_REF] Brzezinski | The balance between silica production and silica dissolution in the sea: Insights from Monterey Bay, California, applied to the global data set[END_REF] have suggested that a measure of the strength of the silicate pump can be obtained by comparing 1-∫D:∫P for Si with the f-ratio for N (representing export intensity for Si and N respectively). When the 1-∫D:∫P values obtained from our model are plotted versus the f-ratio for each site (obtained from [START_REF] Nelson | Vertical budgets for organic carbon and biogenic silica in the pacific sector of the Southern Ocean, 1996-1998[END_REF] for the Southern Ocean and from [START_REF] Falkowski | Phytoplankton and their role in primary, new and export production[END_REF] for the other sites) (Fig. 4), the silica pump appears to be most pronounced at APFP and NACC sites. At the EqPac site, 1-∫D:∫P is close to 0.12, whereas the fratio is between 0.1 and 0.17 [START_REF] Dugdale | Estimating new production in the equatorial Pacific Ocean at 150°W[END_REF]Mc Carthy et al., 1996]. Since 1-∫D:∫P is less than or equal to the f-ratio at this site, it would appear that the silicate pump is weak or nonexistent.

The silicate pump will be most pronounced under the following two conditions: (1) when particle formation is extensive enough to induce rapid export from the mixed layer, and (2) when Si and N cycling are decoupled, so that more Si is exported relative to N. To simplify the model as much as possible, we pooled aggregates and fecal pellets into a single group to reconstruct bSiO 2 fluxes. This simplification may lead to some inaccuracies, as grazing (i.e., fecal pellet production) and aggregation can have very different effects on the silicate pump [see [START_REF] Ragueneau | Si and C interactions in the world ocean: importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF]. While grazers assimilate C and N from diatom material, bSiO 2 passes through the gut quasi-inertly, leading to a five-fold increase in the Si/C ratio as the material is egested [START_REF] Cowie | Digestion and alteration of the biochemical constituents of a diatom (Thalassiosira weissflogii) ingested by an herbivorious copepod (Calanus pacificus)[END_REF][START_REF] Tande | Assimilation efficiency in herbivorous aquatic organismsthe potential of the ratio method using 14 C and biogenic silica as markers[END_REF]. By contrast, diatoms that are incorporated within aggregates rather than ingested by zooplankton would tend to remain alive for a longer time, fixing their Si/C ratio at a relatively constant value [START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF]. Despite this caveat, with our simple model we can at least identify sites with a high flux of fast-sinking particles, and see that 1-∫D:∫P (at 100 m) is highest at sites with the most seasonality (Fig. 3).

Clearly, particle formation at the end of blooms is a key factor controlling the silicate pump.

Further investigation of aggregation dynamics is crucial for understanding temporal and spatial variations in the strength of the silicate pump, as well as the role of diatoms in the biological carbon pump [e.g., [START_REF] Ragueneau | Si and C interactions in the world ocean: importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF].

Impact on the geochemistry of the seafloor

Our results have important implications for the calibration of bSiO 2 mass accumulation rates (MAR) as a proxy of paleoproductivity [START_REF] Ragueneau | A review of the Si cycle in the modern ocean: recent progress and missing gaps in the application of biogenic opal as a paleoproductivity proxy[END_REF]. Larger opal MAR need not necessarily be ascribed to higher surface productivity. At the POOZ site (Fig. 2), for example, a doubling in the bSiO 2 rain rate can be achieved without any change in surface bSiO 2 production, through either (1) a doubling in the incorporation of bSiO 2 into large particles (i.e., from 20% to 40%), (2) a 3.33-fold increase in the LP sinking rate (from 30 to 100 m d -1 ), or (3) a 2.67-fold decrease in the LP dissolution rate from 0.04 to 0.015 d -1 . These ranges in values are all realistic, and a small variation in one of them can significantly increase the amount of bSiO 2 reaching the seafloor. Furthermore, these 3 factors are not necessarily independent and can therefore have additive effects. For example, incorporation into large particles would most likely increase both the particle sinking rate and decrease the bSiO 2 dissolution rate [START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF]. Clearly, improving our understanding of how siliceous sediments accumulate requires proper consideration of particle formation and biogeochemical cycling in sinking particles. This is crucial not only for determining the depth of bSiO 2 dissolution in the water column, but also the extent of silica burial in sediments [START_REF] Gallinari | The importance of water column processes on the dissolution properties of biogenic silica in deep sea-sediments I. Solubility[END_REF][START_REF] Khalil | Constraining biogenic silica dissolution in marine sediments: a comparison between diagenetic models and experimental dissolution rates[END_REF].

Conclusions and perspectives

In this paper, we have combined sinking and silica dissolution rates [START_REF] Schultes | The role of mesozooplankton grazing in the biogeochemical cycle of silicon in the Southern Ocean[END_REF][START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF] for three types of siliceous particles (diatoms as free cells, incorporated into zooplankton fecal pellets, or into aggregates) to reconstruct theoretical downward profiles of bSiO 2 fluxes. By applying a simple advection-reaction model to 8 sites of the world ocean where annual estimates of bSiO 2 production are available [START_REF] Ragueneau | Si/C decoupling in the world ocean: is the Southern Ocean different?[END_REF], we were able to fit these theoretical profiles to measured bSiO 2 fluxes derived from sediment trap data. Such an approach allowed us to derive the sinking rates of particles and partitioning of bSiO 2 between freely-suspended diatoms and large particles. When diatoms are incorporated into large particles via aggregation or incorporation into fecal pellets, this can impact bSiO 2 recycling and possibly other elements like C and N. Further experimentation would be needed, however, to assess this last point.

The competition between recycling in surface waters and export to depth is controlled by the intensity of particle formation rather than by the extent of dissolution in surface waters. For bSiO 2 incorporated into sinking aggregates and fecal pellets, export is much faster than dissolution. Therefore, the extent of dissolution in surface waters depends primarily on the amount of diatom cells that remain freely suspended and can subsequently be degraded by bacteria [START_REF] Bidle | Accelerated dissolution of diatom silica by marine bacterial assemblages[END_REF]. Thus, even ballasted by its frustule, a diatom would be recycled if not incorporated into fast-sinking aggregates or fecal pellets.

This finding also has implications for the use of opal MAR as a proxy of paleoproductivity, which should account not only for the production signal, but also the extent of incorporation into large particles. Furthermore, just as bSiO 2 preservation depends on the types of particles transporting diatoms toward the seafloor, it likely affects C preservation as well.

Further investigation is needed of the extent of C recycling within aggregates and fecal pellets, as this has major implications for C export and benthic food webs.

Our model demonstrated that dissolution in surface waters is extremely high at the EqPac site, most likely due to the small percentage of bSiO 2 production being incorporated, on an annual basis, into large sinking particles (Table 2). This result does not support the idea of a strong silicate pump in the equatorial Pacific, as previously suggested by [START_REF] Dugdale | The role of a silicate pump in driving new production[END_REF].

Rather, it appears that such a mechanism would be most applicable at highly seasonal sites like NACC or APFP where the fraction of bSiO 2 production that is exported from the surface layer is highest (Fig. 3). At such locations, the efficiency of the silica pump will depend upon the relative fates of Si and N in sinking particles. Since aggregation and incorporation into fecal pellets may have very distinct influences on Si and N decoupling during sinking [START_REF] Ragueneau | Si and C interactions in the world ocean: importance of ecological processes and implications for the role of diatoms in the biological pump[END_REF],

differentiating between these two transport pathways is essential for improving our understanding of the functioning of the silicate pump, the role of diatoms in the biological pump, and the nutritional properties of particles supporting deep-sea food webs. 2). The dashed lines are the fits obtained from the 1-particle model, the dotted lines describe the bSiO 2 fluxes calculated with the 2-particle model that uses a global S FC and the plain lines depict the bSiO 2 profiles obtained from the 2-particle model that uses a variable S FC calculated at each site.

Figure caption

(

  EqPac) and North Pacific (OSP), and two in the North Atlantic Ocean (PAP and BATS). Four are located in the Southern Ocean: one north of the Polar Front in the Pacific sector (NACC), two in the Polar Front Zone of the Atlantic (APFA) and Pacific (APFP) sectors, and one just south of the Polar Front in the Indian sector (POOZ) (see positions in Table

Figure 1

 1 Figure1shows the reconstructed bSiO 2 profiles at the eight sites for the three versions of

Figure 1 :

 1 Figure 1: Best fits (lines) of the three versions of the models and in situ (dots) bSiO 2 flux profiles, for eight sites in the global ocean described in Ragueneau et al. [2002] (positions given in Table2). The dashed lines are the fits obtained from the 1-particle model, the dotted lines describe the bSiO 2 fluxes calculated with the 2-particle model that uses a global S FC and the plain lines depict the bSiO 2 profiles obtained from the 2-particle model that uses a variable S FC calculated at each site.

Figure 2 :

 2 Figure 2: Sensitivity of the model, represented by successive variation of parameters. Fig. 2a sensitivity of the model to the sinking rate of the slow sinking/fast dissolving particle S FC , with S LP set at 51 m d -1 and α at 68%, Fig. 2b, sensitivity of the model to the sinking rate of the fast sinking/slow dissolving particle S LP , with S FC set at 1.4 m d -1 and α at 68%, Fig. 2c, sensitivity of the model to the repartition of the bSiO 2 between the FC group α, and the LP group (1-α), with S LP set at 51 m d -1 and S FC at 1.4 m d -1 . The graphs represent the results of the test for the POOZ site, the full circles and the full lines in each graph represent the in situ measurements of the bSiO 2 fluxes and the results of the model 3.

Figure 3 :

 3 Figure 3: Importance of the seasonality. 1-∫D:∫P at 100 m calculated from the model outputs as a function of the seasonality index [Berger and Wefer 1990]. The site names are given near each corresponding point.

Figure 4 :

 4 Figure 4: 1-∫D:∫P calculated at 100 m with the model 3 versus the f-ratios. The f-ratios are from Nelson et al. [2002] for the Southern Ocean and Falkowski et al. [2003] for the other sites. The names of seven of the eight sites are indicated near the corresponding point. APFA site is not in the graph as no f-ratio is available at this site. The plain line delimits the zones where f-ratio is higher or lower than 1-∫D:∫P at 100 m which gives an indication of the strength of the silica pump.

  and results of the two versions of the 2-particle model calculated to fit the bSiO 2 flux profiles at eight sites of the global ocean (Fig. 1). The depth of the maximum mixed layer (MMLD) and the location of the sites are from Ragueneau et al. [2002]. The seasonality indexes (SI) are calculated following the work of Berger and Wefer [1990]. The last rows depict combinations of sinking rates for FC (S FC ) and LP (S LP ) and percentage of bSiO 2 incorporated into the two groups of particles calculated with each version of the 2

POOZ APFP NACC APFA OSP EqPac BATS PAP

	Latitude	-52	-60	-56.9	-50.1	50	1	28	48.8
	Longitude	62	-170	-170.1	5.8	-145	-139	-67.5	-16.5
	MMLD (m)	259	235	390	180	100	48	100	576
		SI	2.80	3.75	2.80	2.70	2.50	0.33	1.30	3.00
		% PP bSiO2 in FC stage α	80%	70%	40%	20%	69%	88%	83%	65%
	Model 2:	S FC (m d -1 )				2.76
	2-particle
	model	% PP bSiO2
	global S FC	into LP	20%	30%	60%	80%	31%	12%	17%	35%
		(1-α)
		S LP (m d -1 )	470	53	27	47	90	812	92	55
		% PP bSiO2 in FC stage α	68%	81%	78%	17%	64%	88%	83%	85%
	Model 3: 2-particle	S FC (m d -1 )	1.4	4.5	10.3	0.1	2	0.1	0.1	6.7
	model variable S FC	% PP bSiO2 into LP (1-α)	32%	19%	22%	83%	36%	12%	17%	15%
		S LP (m d -1 )	51	452	242	45	62	500	90	605

Table 2 : Environmental characteristics

 2

Acknowledgements. We are grateful to Monique Briand for her technical assistance with graphics. Thanks to Sorcha Ni Longphuirt, Cindy Lee and Lynn Abramson who kindly commented on this paper and corrected it from the English mistakes. The authors acknowledge NODC (Levitus) World Ocean Atlas 1998 for their data provided by the NOAA-CIRES Climate Diagnostics Center, Boulder, Colorado, USA, from their Web site at http://www.cdc.noaa.gov/. This work has been funded by the EU which is greatly acknowledged, partly through the ORFOIS (EVK2-CT2001-00100) project and partly through the Si-WEBS (HPRN-CT-2002-00218) Research Training network of the Marie Curie program. This is contribution # xxx of the IUEM.

 [START_REF] Bienfang | Sinking rate of heterogeneous, temperate phytoplankton populations[END_REF]Mann and Lazier, 1996;[START_REF] Smayda | The sinking and suspension of phytoplankton in the sea[END_REF][START_REF] Turner | Zooplankton faecal pellets, marine snow and sinking phytoplankton blooms[END_REF]0.005-1.3 [START_REF] Van Cappellen | Biogenic silica dissolution in the oceans: Reconciling experimental and field-based dissolution rates[END_REF] 0.044 Aggregates 1-370 [START_REF] Alldredge | In situ settling behaviour of marine snow[END_REF][START_REF] Ridgwell | Modeling the response on the oceanic Si inventory to perturbation, and consequences for atmospheric CO2[END_REF] 0.012-0.056 [START_REF] Moriceau | Evidence for reduced biogenic silica dissolution rates in diatom aggregates[END_REF] Copepod fecal pellets 5-220 [START_REF] Turner | Zooplankton faecal pellets, marine snow and sinking phytoplankton blooms[END_REF][START_REF] Wassmann | Distribution and sedimentation of faecal pellets on the Nordvestbanken shelf, northern Norway[END_REF] 0.003-0.027 [Gallinari et al.,unpubl.;[START_REF] Schultes | The role of mesozooplankton grazing in the biogeochemical cycle of silicon in the Southern Ocean[END_REF] 0.022


Kamila Delaney

Geneviève Almouzni

Transcription-coupled H3.3 recycling: a link with chromatin states. Affiliations

Keywords: H3.3 histone variant, new H3.3, old H3.3, H3.3 recycling, HIRA complex, transcription

Histone variant H3.3 is incorporated into chromatin throughout the cell cycle and even in noncycling cells. This histone variant marks actively transcribed chromatin regions with high nucleosome turnover, as well as silent pericentric and telomeric repetitive regions. In the past few years, significant progress has been made in our understanding of mechanisms involved in the transcription-coupled deposition of H3.3. Here we review how, during transcription, new H3.3 deposition intermingles with the fate of the old H3.3 variant and its recycling. First, we describe pathways enabling the incorporation of newly synthesized vs old H3.3 histones in the context of transcription. We then review the current knowledge concerning differences between these two H3.3 populations, focusing on their PTMs composition. Finally, we discuss the implications of H3.3 recycling for the maintenance of the transcriptional state and underline the emerging importance of H3.3 as a potent epigenetic regulator for both maintaining and switching a transcriptional state.

Introduction

An adult human body is composed of around 37.2 trillion cells, ensuring the provision of the variety of cells that make up all the tissues, yet sharing a common genome [START_REF] Regev | Human Cell Atlas Meeting Participants[END_REF] . The variety of cell types are characterized by different transcriptional programs, thus understanding the mechanisms governing the establishment, maintenance and dynamic changes in these programs is of great scientific interest and therapeutic potential [START_REF] Rajewsky | LifeTime and improving European healthcare through cell-based interceptive medicine[END_REF]. Histone proteins, initially viewed as a monotonous scaffolding system providing compaction of the genetic information in chromatin within the nucleus of the cell, also contribute to a functional versatility of chromatin organization. Indeed, while each nucleosome is composed of two copies of four types of histones, H2A, H2B, H3 and H4 [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF], most of these histones are represented by several variants which, when incorporated into the nucleosome, add variability within the basic module (reviewed in [START_REF] Talbert | Histone variants at a glance[END_REF]). Focusing on the H3 family, the main variants include two replicative histones H3.1 and H3.2, and two non-replicative ones -H3.3 and centromeric CENP-A (Figure 1a). While CENP-A diverges significantly from other H3 variants (Figure 1a), H3.3 differs from its replicative counterparts only by 5 (for H3.1) and 4 (for H3.2) amino acids (Figure 1a). Three of these residues located in the histone fold domain confer a specificity of interaction with the DNA in the nucleosome particle and with unique sets of histone chaperones (Figure 1ab, [START_REF] Keppler | A general method for the covalent labeling of fusion proteins with small molecules in vivo[END_REF][START_REF] Keppler | Labeling of fusion proteins with synthetic fluorophores in live cells[END_REF][START_REF] Evano | Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches[END_REF]). Moreover, a single amino acid Serine31 present uniquely in the N-terminal tail of H3.3 (Figure 1a) plays an important regulatory role [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Martire | The roles of histone variants in fine-tuning chromatin organization and function[END_REF][START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF][START_REF] Scott | Interactions With Histone H3 & Tools to Study Them[END_REF] (discussed further in section 7). The emerging view now defines a chromatin landscape that includes a choice of histone variants along with the post translational modifications (PTMs) and defines distinct chromatin states that correspond to specific cellular types and functions (reviewed in [START_REF] Martire | The roles of histone variants in fine-tuning chromatin organization and function[END_REF]). Recent data uncovered particular histone dynamics during transcription challenging the maintenance of a given state in a cellular lineage. Histones bound to chromatin, when displaced, can get reincorporated. However, this reincorporation relies on a distinct deposition pathway as compared to the one involved in the deposition of newly synthesized H3.3 [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. This finding underlines a capacity to differentiate between the two H3.3 subpopulations (which we refer to as new and old H3.3). In this review, we summarize our current knowledge concerning new and old H3.3 incorporation during transcription. We highlight the key differences between pathways enabling newly synthesized H3.3 deposition and re-incorporation of old H3.3 histones, respectively. We review how PTMs can provide a means to distinguish new and old H3.3. This leads us to discuss how regulating the deposition of old and new histones with distinct PTM signatures could impact chromatin states, either in maintenance or switch of transcriptional programs.

Definition and detection of new and old H3.3 histones

Various approaches have been used to study the fate of histone proteins (summarized in [START_REF] Scott | Interactions With Histone H3 & Tools to Study Them[END_REF]).

Among them, the epitope-tagging of histones with SNAP proved especially insightful in the context of histone deposition because it enables distinguishing newly synthesized and previously existing populations of histones. The SNAP-tagging takes advantage of a mutated DNA repair protein, an O 6 -alkylguanine-DNA alkyltransferase, fused with the protein of interest (Figure 2a). In the cell culture context, the simple addition of a cell-permeable reagent to the media, namely a benzylguanine (BG) derivative, allows for the rapid and irreversible labeling of the SNAP-tagged protein [START_REF] Keppler | A general method for the covalent labeling of fusion proteins with small molecules in vivo[END_REF][START_REF] Keppler | Labeling of fusion proteins with synthetic fluorophores in live cells[END_REF] (a method recently developed also in vivo in mice [START_REF] Evano | Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches[END_REF]). This provides a convenient means to carry out pulse-chase experiments to label distinct populations and access new and old histones (Figure 2bc). The method has been used initially to follow the centromeric H3 variant CENP-A [START_REF] Jansen | Propagation of centromeric chromatin requires exit from mitosis[END_REF]. Stable cell lines expressing tagged versions of other H3 variants provided mechanistic insights into H3 deposition pathways [START_REF] Ray-Gallet | Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit[END_REF][START_REF] Lee | Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors[END_REF] by tracking H3 histones and their dynamics via single cell imaging and super resolution microscopy [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF][START_REF] Clément | High-resolution visualization of H3 variants during replication reveals their controlled recycling[END_REF] (Figure 2d, method detailed in [START_REF] Torné | Imaging Newly Synthesized and Old Histone Variant Dynamics Dependent on Chaperones Using the SNAP-Tag System[END_REF]), as well chromatin immunoprecipitations [START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF] (method detailed in [START_REF] Siwek | time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance[END_REF]). The two distinct old and new H3.3 subpopulations in the context of transcription could be traced recently (Figure 2bc, [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]). Old H3.3 represents histones present in the nucleus before the chase, and new H3.3 the histones synthesized during and after the chase (Figure 2bc). A major fraction of old H3.3 gets evicted from the chromatin co-transcriptionally [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. However, they are either recycled locally or replaced by new H3.3. The recycling and new H3.3 deposition involve distinct pathways / distinct protein partners [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. This opens up questions of how and why this differential recognition occurs and what consequences ensue for the chromatin states.

New H3.3: Mechanisms for newly synthesized H3.3 deposition

Since in this review we wish to focus more specifically on the recycling of H3.3, we will provide only a brief background. For details about the deposition of new H3.3 and the histone chaperones see [START_REF] Ray-Gallet | The Histone H3 Family and Its Deposition Pathways[END_REF].

Histones are small proteins carrying strong positive charges enabling their close association with negatively charged DNA molecules in chromatin. However, these charges may also lead to promiscuous interactions with other proteins or nucleic acids present in the cytoplasm (where they are synthesized), as well as during their trafficking in and out of the nucleus. Thus, when not incorporated into chromatin, histones are constantly escorted by accompanying proteins, histone chaperones ( [START_REF] Laskey | Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA[END_REF], reviewed in [START_REF] Gurard-Levin | Histone Chaperones: Assisting Histone Traffic and Nucleosome Dynamics[END_REF][START_REF] Hammond | Histone chaperone networks shaping chromatin function[END_REF]). Newly synthesized H3 histones are transferred to the nucleus either as monomers or H3-H4 dimers ( [START_REF] Alvarez | Sequential establishment of marks on soluble histones H3 and H4[END_REF], reviewed in [START_REF] Pardal | The histone chaperoning pathway: from ribosome to nucleosome[END_REF]). They first associate with general chaperones, such as heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) [START_REF] Alvarez | Sequential establishment of marks on soluble histones H3 and H4[END_REF]. In the nucleus, newly synthesized H3.3 associates with distinct, major protein complexes, ensuring deposition at specific chromatin regions [START_REF] Elsaesser | HIRA and Daxx constitute two independent histone H3.3containing predeposition complexes[END_REF]. The interaction with chaperones recognizing a distinct motif in the histone fold domain of the H3.3 variant enables the specific H3.3 deposition as opposed to the replication coupled deposition of H3.1 [START_REF] Ray-Gallet | The Histone H3 Family and Its Deposition Pathways[END_REF]. In the first case, an AAIG domain at residues 87-90 is critical for H3.3 recognition, in the second case, it is an SVM motif for H3.1/ H3.2 (Figure 1ab) [START_REF] Lewis | Daxx is an H3.3specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres[END_REF][START_REF] Liu | Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX[END_REF][START_REF] Tang | Identification of an ubinuclein 1 region required for stability and function of the human HIRA/UBN1/CABIN1/ASF1a histone H3.3 chaperone complex[END_REF].

Concerning the key proteins involved in H3.3 deposition, two major complexes have been identified enabling distinct enrichment at various genomic loci. The death domain associated (DAXX) and alpha-thalassemia/mental retardation, X-linked protein (ATRX) complex [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF] favors H3.3 enrichment at satellite repeats of pericentric and telomeric regions [START_REF] Lewis | Daxx is an H3.3specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres[END_REF][START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF], as well as endogenous retroviral elements (ERVs) [START_REF] Elsässer | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF][START_REF] Jang | Histone H3.3 maintains genome integrity during mammalian development[END_REF] and imprinted loci [START_REF] Voon | ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes[END_REF] (Figure 3 left side).

Interestingly, the interaction of DAXX with a GHKL type ATPase MORC3 can regulate accumulation of H3.3 at ERVs [START_REF] Groh | Morc3 silences endogenous retroviruses by enabling Daxx-mediated histone H3.3 incorporation[END_REF]. Moreover, the histone regulator (HIRA) complex proved critical for new H3.3 incorporation at high nucleosome turnover loci, especially at transcriptional start sites, active gene bodies and promoters [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF][START_REF] Kraushaar | Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3[END_REF][START_REF] Ha | Genome-wide analysis of H3.3 dissociation reveals high nucleosome turnover at distal regulatory regions of embryonic stem cells[END_REF], as well as transcription factor binding sites [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF][START_REF] Jin | H3.3/H2A.Z double variant-containing nucleosomes mark "nucleosome-free regions" of active promoters and other regulatory regions[END_REF], and DNA damage sites [START_REF] Adam | Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA[END_REF] (Figure 3 right side).

Given its capacity to bind naked DNA, the HIRA complex could additionally enable random incorporation onto any region lacking nucleosomes via a gap filling mechanism, thereby maintaining chromatin integrity (Figure 3 right side) [START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF][START_REF] Schneiderman | Nucleosomedepleted chromatin gaps recruit assembly factors for the H3.3 histone variant[END_REF]. Mechanistically, the HIRAmediated new H3.3 deposition requires the trimerization of the HIRA subunit [START_REF] Ray-Gallet | Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit[END_REF]. This trimer associates with two other subunits -calcineurin binding protein 1 (CABIN1) and ubinuclein 1 (UBN1) [START_REF] Ray-Gallet | Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit[END_REF][START_REF] Banumathy | Human UBN1 is an ortholog of yeast Hpc2p and has an essential role in the HIRA/ASF1a chromatin-remodeling pathway in senescent cells[END_REF][START_REF] Tagami | Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis[END_REF]. In addition to these core components, the association with the Anti-Silencing Factor 1 (ASF1) [START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF][START_REF] English | Structural Basis for the Histone Chaperone Activity of Asf1[END_REF] facilitates the transfer of the H3-H4 dimers to UBN1 for deposition onto DNA (Figure 3 right side) [START_REF] Voon | ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes[END_REF][START_REF] Ricketts | A Molecular Prospective for HIRA Complex Assembly and H3.3-Specific Histone Chaperone Function[END_REF][START_REF] Ricketts | Ubinuclein-1 confers histone H3.3-specificbinding by the HIRA histone chaperone complex[END_REF]. ASF1 is present in mammalian cells in the form of two paralogous proteins -ASF1a and ASF1b [START_REF] Silljé | Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases[END_REF], which interact with any of the H3 variants. However, they do show chaperone partner preferences with ASF1a preferentially associated with HIRA, and ASF1b with chromatin assembly factor 1 (CAF1) -a chaperone of replication-dependent H3 variants [START_REF] Tang | Structure of a human ASF1a/HIRA complex and insights into specificity of histone chaperone complex assembly[END_REF]. Recent data suggest that ASF1 can transfer new H3.3 directly to UBN-1, without requiring the interaction with HIRA [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF], although the details of this interaction require further analysis.

Old H3.3: Mechanisms for transcription-coupled recycling of old H3.3

To date, the re-incorporation of previously chromatin-bound histones has been considered mostly in the context of DNA synthesis. It is clear that during the obviously disruptive process of DNA replication, repair and recombination, recycling of parental histones along with new histone deposition can challenge the maintenance of nucleosome density and the chromatin states (reviewed in [START_REF] Groth | Chromatin Challenges during DNA Replication and Repair[END_REF]). While we have learnt a lot about the replicative H3 variants in the DNA synthesis-coupled deposition, we only recently gained insight into the H3.3 variant deposition during S phase [START_REF] Clément | High-resolution visualization of H3 variants during replication reveals their controlled recycling[END_REF][START_REF] Gatto | HIRA-dependent boundaries between H3 variants shape early replication in mammals[END_REF]. Furthermore, H3.3, given its expression throughout the cell cycle, as opposed to replication-coupled expression of H3.1 and H3.2, also offers the possibility to explore the balance between new incorporation and recycling outside S-phase. This aspect is particularly interesting considering histone eviction associated with transcription. In fact, we know relatively little about the histones evicted from chromatin in a transcriptional context; how much is evicted? how? Are they discarded for degradation? Are they recycled back onto chromatin at their original genomic site or elsewhere? Or even simply stored outside of chromatin? Transcription-coupled eviction of the H3 family has been linked to p300-mediated acetylation operating on the H3 N-terminal tail, more specifically H3K14 [START_REF] Luebben | Nucleosome eviction and activated transcription require p300 acetylation of histone H3 lysine 14[END_REF]. The histone chaperone ASF1 itself has also been implicated in the H3 eviction during the transcript elongation by RNA Polymerase II (RNA PolII) [START_REF] Schwabish | Asf1 Mediates Histone Eviction and Deposition during Elongation by RNA Polymerase II[END_REF], possibly as a hand over for histones, in a manner that compares to models proposed in the context of DNA replication [START_REF] Clément | High-resolution visualization of H3 variants during replication reveals their controlled recycling[END_REF]. Concerning histone degradation, the recruitment of the Ring Finger Protein 8 (RNF8) ubiquitin ligase, which in turn marks histones for proteasome-mediated degradation, exploits the specific phosphorylation of H3T11 in the N-terminal tail [START_REF] Xia | RNF8 mediates histone H3 ubiquitylation and promotes glycolysis and tumorigenesis[END_REF]. Finally, to prevent accumulation of excess levels of soluble histones, the histone chaperone NASP directs their degradation via the chaperone-mediated degradation pathway [START_REF] Cook | A Specific Function for the Histone Chaperone NASP to Fine-Tune a Reservoir of Soluble H3-H4 in the Histone Supply Chain[END_REF]. However, how and what proportion of histones gets degraded upon eviction and, more importantly, what happens to the ones that remain in the nucleus, still needs to be defined.

What determines the level of recycling may also depend on the context. A recent report indicates that recycling can potentially increase when the concentration of newly synthesized histones is low [START_REF] Gruszka | Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication[END_REF], suggesting that the level of recycling is not necessarily steady. This may be particularly important to consider in post-mitotic cells that will rely mainly on nonreplicative histones. Importantly, as stated before, the process of transcription results in a massive eviction of H3.3 and a large fraction of this evicted H3.3 is recycled back into chromatin [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. Remarkably, the process requires the involvement of some, but not all, of the components of the typical HIRA chaperone complex [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. More specifically: (i) it does not require the trimerization of the HIRA subunit itself or UBN1 interaction, yet (ii) it does strictly require association with the ASF1 histone chaperone (Figure 4) [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF][START_REF] Ray-Gallet | Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit[END_REF]. Whether this pathway uniquely involves the engagement of HIRA and ASF1 alone, or as part of the typical complex associated with new H3.3 deposition, remains an open question. One could even consider that recycling of histones could be differentially regulated at distinct genomic regions and/or be influenced by the PTM status of the histones. Recent results identifying UBR7 as a novel chaperone interacting specifically with the evicted H3.1-H4 dimers [START_REF] Hogan | UBR7 acts as a histone chaperone for post-nucleosomal histone H3[END_REF] can substantiate the latter possibility. Indeed, recognition of evicted H3.1 by UBR7 depends on the presence of H3K4me3 or H3K9me3 mark on these histones [START_REF] Hogan | UBR7 acts as a histone chaperone for post-nucleosomal histone H3[END_REF].

Schematically, the distinct outcomes of transcriptional-coupled old H3.3 recycling are: (i) precise maintenance of states when recycled back to the same location (Figure 5a), (ii) loose maintenance by staying in the chromatin vicinity as part of the local H3.3-enriched subnuclear region ("conglomerate") (Figure 5b) [START_REF] Hammond | Histone chaperone networks shaping chromatin function[END_REF], or (iii) switch of states when moved ectopically to a different genomic region (Figure 5c). In terms of preserving local chromatin states, the first two scenarios would be more conservative. The latter, however, has the potential to promote a change in the PTMs at the new locus, given the qualitative differences in the PTM landscape between new and old H3.3 (discussed in the following section). Given that HIRA proves crucial for the chromatin states' dynamics, especially in systems relying on robust and coordinated transcriptional regulation (i.e. embryonic development and embryonic stem cells (ESCs) differentiation) [START_REF] Fang | Global H3.3 dynamic deposition defines its bimodal role in cell fate transition[END_REF], it will be important to disentangle how new and old H3.3 histones are channeled through distinct deposition pathways in the nucleus.

PTMs composition as a proxy for distinguishing new and old H3.3 histones

When considering potential functional differences between new and old H3.3, the nature of the PTMs present on their amino acids is revealing. Global analysis of chromatin showed that the bulk H3.3 is enriched in active chromatin marks, including K4 and K79 methylation, as well as K9, K14 and K18 acetylation [START_REF] Mckittrick | Histone H3.3 is enriched in covalent modifications associated with active chromatin[END_REF]. However, to identify newly synthesized histones, which represent only a small fraction of the total histone pool which is usually ignored, it is important to use biochemical fractionation that can separate soluble from chromatin bound pools [START_REF] Martini | Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells[END_REF][START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF].

These fractionation experiments provided a map of the PTMs from histone synthesis to deposition, distinct for each individual histone variant [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF].

Newly synthesized histone H4 shows a conserved dual K5, K12 acetylation pattern, unique characteristic of the new H4 fraction [START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF]. In the case of the H3 family, analysis carried out in HeLa cells showed that both H3.1 and H3.3 in the soluble fraction are largely unmodifiedamong examined residues on average about 80-100% of histones carry no detectable modifications [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF]. Among the rare detected modifications, the only methylation observed corresponds to lysine 9, more specifically H3K9me1 (present on both H3.1 and H3.3) and H3K9me2, present specifically on the H3.3 variant (Figure 6 left side) [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF]. Since H3K9 trimethylation is important for heterochromatin, this observation is particularly interesting in the context of DAXX/ATRX-mediated heterochromatic deposition of H3.3. It is possible that the small fraction of H3.3 methylated on lysine 9 is being targeted to pericentric and telomeric regions, especially given that H3K9me1 is a preferential substrate of Suv39 to reach trimethylation [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF]. Interestingly, newly synthesized H3.3-H4 dimers can be sequestered for a later deposition into PML bodies in a DAXX-dependent manner [START_REF] Delbarre | DAXX-dependent supply of soluble (H3.3-H4) dimers to PML bodies pending deposition into chromatin[END_REF], and it is tempting to envisage that H3K9 methylated H3.3 could be the one preferentially recognized in this process.

Other than marks on K9, lysine 14, 18 and 23 show low levels of acetylation on newly synthesized H3.3 (with 20-30% of the residues carrying the modification) (Figure 6 left side) [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF]. These modifications, associated with differentiation and tissue-specific gene activation [START_REF] Regadas | A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation[END_REF][START_REF] Luo | H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling[END_REF][START_REF] Klein | Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation[END_REF], reflect the euchromatic profile of bulk H3.3 [START_REF] Mckittrick | Histone H3.3 is enriched in covalent modifications associated with active chromatin[END_REF][START_REF] Hake | Expression patterns and post-translational modifications associated with mammalian histone H3 variants[END_REF]. Notably, some of the marks on newly synthesized H3.3 are imposed sequentially and can be mutually exclusive -such as heterochromatic H3K9me1 and euchromatic H3K14K18ac [START_REF] Alvarez | Sequential establishment of marks on soluble histones H3 and H4[END_REF]. Thus, several subpopulations exist that could possibly preferentially engage with distinct chaperones. In summary, the newly synthesized H3.3 population is largely unmodified and acquires most of the PTMs after the chromatin incorporation, at least in the systems studied to date.

In contrast to newly synthesized H3.3, old, chromatin-bound H3.3 is massively enriched in methylation-based active marks, mostly H3K4me3 (present on over 70% of the old histones), as well as methylation of lysine 36 and 79 (Figure 6 right side) [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF]. It also shows some acetylation on residues K9, K14 and K23 -all common markers of the active transcriptional state (Figure 6 right side) [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF].

Therefore, while new histones are mostly unmodified, the old, chromatin bound H3.3 is extensively methylated, especially at lysine 4. In light of the recognition of post-nucleosomal H3.1 via direct binding to H3K4me3 by UBR7 [START_REF] Hogan | UBR7 acts as a histone chaperone for post-nucleosomal histone H3[END_REF], it is possible that a similar mechanism exists to specifically recognize and deposit the old H3.3. Thus, the striking differences in methylation levels between new and old H3.3 provide not only a reliable means to distinguish them experimentally but also a potential mechanism to sort out which histones to incorporate, sequester or degrade (Figure 5).

Histone H3.3 distribution and local modification landscapes

Within chromatin, the density of H3.3 shows a pattern averaging one H3.3 nucleosome for every three H3.1 nucleosomes. This dispersed nature is not mirrored by the PTMs present on the histones in shared genomic locus -biochemical analysis of short oligonucleosome arrays showed that if an array contains H3.3 nucleosomes, the active PTMs (e.g. H3K4me3 and H3K9ac) are similarly enriched on all H3 histones within that array, and not only the H3.3 [START_REF] Loyola | PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state[END_REF].

The shared local PTM status of both H3.3 and H3.1 indicates it is the modification landscape, rather than the histone variant composition, that has a dominant role in controlling the chromatin state. In support of the central role of the PTMs, direct targeting of an H3K4me3 writer, PRDM9 methyltransferase, to the promoter region can effectively re-initiate silenced gene expression [START_REF] Cano-Rodriguez | Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner[END_REF]. This suggests that the introduction of specific PTMs, either by recruiting appropriate chromatin modifiers or perhaps by incorporating already modified histones, can indeed be sufficient for a transcriptional switch. Given that local inheritance and spreading of repressive marks, specifically H3K27me3, from parental histones was previously put forward in the context of replication (reviewed in [START_REF] Escobar | Parental nucleosome segregation and the inheritance of cellular identity[END_REF]), it is tempting to speculate that the observed dispersed nature of H3.3 may reflect its function in promoting/seeding and propagating of open chromatin-related PTMs outside of S-phase. If transcription-coupled recycling of methylated H3.3 indeed serves the purpose of introducing and/or reinforcing the active state of the locus, even a small number of such histones would suffice, thanks to the read-write mechanism-based spreading of marks such as H3K4me3 (reviewed in [START_REF] Hyun | Writing, erasing and reading histone lysine methylations[END_REF]). This would explain (i) why the cell retains a fraction of evicted H3.3 and (ii) evolved pathways allowing for specific recognition and deposition of these histones back onto the chromatin. Interestingly, ESCs carrying an alanine substitution of lysine 4 in H3.3 lose the capacity to differentiate, while they can still self renew [START_REF] Gehre | Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy[END_REF]. These cells lose H3.3 occupancy at ESCs-specific enhancers, thereby disrupting their transcriptional state [START_REF] Gehre | Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy[END_REF]. Therefore, in addition to the classical AIG recognition motif for H3.3 chaperones, some regulatory elements in the N-terminal tail may require a proper K4 site for accurate deposition and/or maintenance of H3.3 in specific chromatin regions. It will be exciting to uncover and understand the mechanisms of modified H3.3 recognition and their role in chromatin states maintenance.

7.

H3.3S31Phos -a specific H3.3 chromatin mark

Remarkably, it is possible to distinguish identical PTMs present in the context of distinct H3 variants in chromatin. This was first exemplified with the candidate tumor suppressor protein ZMYND11 that recognizes trimethylation of lysine 36 (H3.3K36me3) specifically on H3.3 to impact transcript elongation and mRNA processing [START_REF] Wen | ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression[END_REF][START_REF] Guo | BS69/ZMYND11 Reads and Connects Histone H3.3 Lysine 36 Trimethylation-Decorated Chromatin to Regulated Pre-mRNA Processing[END_REF]. This recognition can function thanks to an inherent unique feature of H3.3 -the presence of a serine at the amino acid 31 that can be modified by phosphorylation [START_REF] Hake | Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes[END_REF]. Interestingly, H3.3S31Phos, initially described as metaphase-specific and important for proper chromosome condensation and protecting centromeres from euchromatinisation [START_REF] Hake | Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes[END_REF], also links to transcriptional program control [START_REF] Armache | Histone H3.3 phosphorylation amplifies stimulation-induced transcription[END_REF][START_REF] Martire | Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation[END_REF]. In the context of murine macrophages, upon stimuli, the levels of H3.3S31Phos increase and result in rapid transcriptional activation [START_REF] Armache | Histone H3.3 phosphorylation amplifies stimulation-induced transcription[END_REF]. Mechanistically, in this case, the transcriptional boost is mediated by ZMYND11 ejection initiated by H3.3S31 phosphorylation, followed by binding and increased activity of SETD2 methyltransferase on the H3.3K36 locus [START_REF] Armache | Histone H3.3 phosphorylation amplifies stimulation-induced transcription[END_REF]. In mESCs, H3.3S31Phos acts as a nucleosomal cofactor of p300 acetyltransferase to help regulate enhancer function and transcriptional reprogramming during differentiation [START_REF] Martire | Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation[END_REF].

Additionally, the mark proved essential for early Xenopus embryonic development during gastrulation, further stressing its role in transcriptional reprogramming [START_REF] Sitbon | Histone variant H3.3 residue S31 is essential for Xenopus gastrulation regardless of the deposition pathway[END_REF]. Most of these regulatory functions of H3.3S31Phos link to the modification status of neighboring amino acids (e.g. H3K36me3, or H3K27Ac/me), and future work should address the nature and extent of all possible cross-talks. Importantly, whether the phosphorylation of H3.3S31 is retained upon eviction from the chromatin and in turn could influence a target locus when recycled back to the chromatin remains to be explored. In stimulated macrophages the H3.3S31Phos is enriched strictly over gene bodies of genes responding to the bacterial lipopolysaccharide (LPS) [START_REF] Armache | Histone H3.3 phosphorylation amplifies stimulation-induced transcription[END_REF].

Importantly, this enrichment is dependent on IkappaB kinase (IKK) association with elongating RNA Pol II [START_REF] Armache | Histone H3.3 phosphorylation amplifies stimulation-induced transcription[END_REF], demonstrating that the phosphorylation occurs co-transcriptionally, rather than being brought by already modified histones. How is the kinase recruited to its target sites?

Is there a dedicated chaperone recognizing the phosphorylation specifically? Since similarly to other histone phosphorylations [START_REF] Josefowicz | Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription[END_REF], H3.3S31Phos has the potential to trigger rapid cellular responses it will be important to further explore its function as a promising candidate for a transcriptional programs' modulator.

Opened questions, outlook and future perspectives

We have come to appreciate the existence of co-transcriptional dynamics between deposition of new and recycling of old H3.3. The question that ensues now is not only to understand the mechanism but also functional consequences of this interplay for the physiology of a whole organism. Lessons from H3.3-deficient organisms point to a high importance of this histone variant across multicellular species (recently reviewed in [START_REF] Franklin | Cell Fate Decisions in the Wake of Histone H3 Deposition[END_REF]). In C. elegans, the H3.3 requirement only stands out upon heat shock to ensure an appropriate transcriptional activation and the timely firing of replication origins [START_REF] Delaney | Differential Expression of Histone H3.3 Genes and Their Role in Modulating Temperature Stress Response in Caenorhabditis elegans[END_REF][START_REF] Strobino | Loss of histone H3.3 results in DNA replication defects and altered origin dynamics in C. elegans[END_REF]. In flies, H3.3 is mainly critical for male germline development [START_REF] Sakai | Transcriptional and developmental functions of the H3.3 histone variant in Drosophila[END_REF][START_REF] Hödl | Transcription in the absence of histone H3.3[END_REF], while in Xenopus a specific requirement during gastrulation could be revealed [START_REF] Szenker | A developmental requirement for HIRAdependent H3.3 deposition revealed at gastrulation in Xenopus[END_REF]. Finally, mice lacking H3.3 show phenotypes ranging from infertility to lethality and embryonic development defects, depending on the number of H3.3 genes lost [START_REF] Tang | Contribution of the two genes encoding histone variant h3.3 to viability and fertility in mice[END_REF][START_REF] Couldrey | A retroviral Gene Trap Insertion into the Histone 3.3A Gene Causes Partial Neonatal Lethality, Stunted Growth, Neuromuscular Deficits and Male Sub-fertility in Transgenic Mice[END_REF][START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. While many of these defects can be linked with aberrant or failed transcriptional programs at a specific time, the loss of H3. The HIRA protein itself is clearly important for both new deposition and recycling of H3.3 [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF].

In systems studied to date, HIRA activity was linked to the control of cell fate either to maintain stemness or to enable differentiation, which may appear conflicting. Indeed, in C2C12 myoblasts, HIRA is required for both further differentiation into myotubes [START_REF] Yang | Myogenic transcriptional activation of MyoD mediated by replication-independent histone deposition[END_REF] and conversion into osteoblasts [START_REF] Song | The role of histone chaperones in osteoblastic differentiation of C2C12 myoblasts[END_REF]. Conversely, muscle stem cells in adult mice depend on HIRA to maintain their self-renewal capacity rather than to enable their differentiation [START_REF] Esteves De Lima | HIRA stabilizes skeletal muscle lineage identity[END_REF]. Conflicting dependencies are also reported in the context of hematopoiesis -in some, but not all mouse lineages, HIRA proved critical for transcriptional reprogramming [START_REF] Chen | HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis[END_REF][START_REF] Murdaugh | The histone H3.3 chaperone HIRA restrains erythroid-biased differentiation of adult hematopoietic stem cells[END_REF]. Finally, in hematopoietic transdifferentiation and neuronal differentiation of mESCs, HIRA also proved critical -at early stages, to protect paternal lineage identity, and later on for the successful cell fate change [START_REF] Fang | Global H3.3 dynamic deposition defines its bimodal role in cell fate transition[END_REF]. This seemingly dual and potentially opposing role of HIRA, whereby it can contribute to both maintenance and switch in chromatin state, is a puzzle. A possible explanation may relate to different chromatin outcomes when considering deposition of new H3.3 vs recycling old H3.3, especially in light of the different marks that they may maintain or lose. A way to test a key role for deposition could take advantage of the fact that HIRA trimerization is strictly required for the new deposition but dispensable for the recycling.

Furthermore, another striking difference between the transcription-coupled mechanisms of new and old H3.3 deposition directly relates to the distinct involvement of partners of HIRA, namely ASF1 and UBN1 proteins. Indispensable for incorporation of old H3.3, ASF1 is not strictly required for the new deposition [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. Consistently, ASF1 is also important for histone recycling during replication and associates with evicted histones during replication stress [START_REF] Jeronimo | Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications[END_REF][START_REF] Groth | Regulation of Replication Fork Progression Through Histone Supply and Demand[END_REF][START_REF] Jasencakova | Replication Stress Interferes with Histone Recycling and Predeposition Marking of New Histones[END_REF].

Moreover, ASF1 is necessary for the reprogramming of adult cells into iPCSs [START_REF] Gonzalez-Muñoz | Histone chaperone ASF1A is required for maintenance of pluripotency and cellular reprogramming[END_REF], as well as mESCs differentiation, where it facilitates transcriptional activation of differentiation genes [START_REF] Gao | Asf1a resolves bivalent chromatin domains for the induction of lineage-specific genes during mouse embryonic stem cell differentiation[END_REF]. In the case of UBN1, required only for the incorporation of new H3.3 [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF], it is interesting to note that its activity is important for H3.3 deposition at promoters of developmental genes and successful neural differentiation of mESCs [START_REF] Xiong | UBN1/2 of HIRA complex is responsible for recognition and deposition of H3.3 at cisregulatory elements of genes in mouse ES cells[END_REF]. How the particular implications of ASF1 and UBN1 in new and old H3.3 deposition relate to these phenotypes will be an exciting area to explore in order to uncover the details of these dependencies.

In this review, we discussed the ability of the cell to distinguish between newly synthesized (new) and previously chromatin-bound (old) H3.3 in the context of transcription. We propose that the functional significance of such distinction may lie with fundamental differences in the PTMs landscapes detected at these histone subpopulations, in essence putting forward an extension of the "H3 barcode hypothesis" [START_REF] Hake | Histone H3 variants and their potential role in indexing mammalian genomes: The "H3 barcode hypothesis[END_REF]. Enrichment of H3K4me3 on old histones vs largely unmodified status of new H3. Refining and exploring this idea by identifying more interactors and revealing detailed dynamics of old and new H3.3 on chromatin will allow for a deeper understanding of the function of differential H3.3 deposition and its impact on chromatin plasticity in the context of cell identity [START_REF] Yadav | Chromatin plasticity: A versatile landscape that underlies cell fate and identity[END_REF]. Triton extraction is performed to eliminate the soluble fraction and visualize chromatin associated/incorporated histones. c) Image visualization of H3.3 histones; ("P -Pulse"), nolabeling control ("QP -Quench-Pulse"), new histones ("QCP -Quench/Chase/Pulse") and old histones ("PC -Pulse/Chase"). Scale bar=10μm. Bottom panel: Cell-by-cell signal intensity is quantified and average is normalized to "Pulse" levels. "Quench/Pulse" leads to dramatically reduced TMR intensity as no histones should be available for labeling after the "Quench" step (this serves as a control for quenching efficiency). The addition of a "Chase" period before the "Pulse" ("Quench-Chase-Pulse") leads to increased TMR intensity, as newly synthesized histones are available for labeling. The "Pulse-Chase" exhibits reduced signal intensity compared to the "Pulse" as only old retained histones remain labeled. De novo deposition of H3.3 (i.e. deposition of newly synthesized histones) occurs both in heterochromatic and euchromatic contexts. In heterochromatic regions, especially repeats present at telomeres, pericentric chromatin and endogenous retroviral sequences, as well as imprinted loci, H3.3 enrichment relies on the DAXX/ATRX complex and is linked to proper heterochromatin formation [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Liu | Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX[END_REF][START_REF] Jang | Histone H3.3 maintains genome integrity during mammalian development[END_REF][START_REF] Voon | ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes[END_REF][START_REF] Groh | Morc3 silences endogenous retroviruses by enabling Daxx-mediated histone H3.3 incorporation[END_REF][START_REF] Hoelper | Structural and mechanistic insights into ATRX-dependent and -independent functions of the histone chaperone DAXX[END_REF]. In actively transcribed regions and nucleosomal gaps, new H3.3 deposition relies on a replication-independent process linked to the HIRA chaperone complex [START_REF] Ahmad | The Histone Variant H3.3 Marks Active Chromatin by Replication-Independent Nucleosome Assembly[END_REF][START_REF] Ray-Gallet | HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis[END_REF]. The core HIRA complex consists of a trimer of HIRA associated with CABIN1 and UBN1 proteins [START_REF] Ray-Gallet | Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit[END_REF][START_REF] Banumathy | Human UBN1 is an ortholog of yeast Hpc2p and has an essential role in the HIRA/ASF1a chromatin-remodeling pathway in senescent cells[END_REF][START_REF] Rai | Human CABIN1 Is a Functional Member of the Human HIRA/UBN1/ASF1a Histone H3.3 Chaperone Complex▿[END_REF]. While specific stoichiometry of these interactions remains to be determined, dimerization of UBN1 in vitro could indicate the joint deposition of two dimers of H3.3-H4 [START_REF] Ricketts | The HIRA histone chaperone complex subunit UBN1 harbors H3/H4-and DNA-binding activity[END_REF]. Besides these core members, the HIRA complex also associates with the ASF1 protein, especially ASF1a [START_REF] Drané | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF][START_REF] Abascal | Subfunctionalization via adaptive evolution influenced by genomic context: the case of histone chaperones ASF1a and ASF1b[END_REF]. Of note, the strict association with HIRA is dispensable since ASF1 can also deliver newly synthesized H3.3 directly to UBN1 for deposition without the need to associate with HIRA (dashed arrow) [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF]. The schemes are based on [START_REF] Mendiratta | Histone supply: Multitiered regulation ensures chromatin dynamics throughout the cell cycle[END_REF] and [START_REF] Torné | Two HIRA-dependent pathways mediate H3.3 de novo deposition and recycling during transcription[END_REF].

  3 function experiments cannot address what would be the outcome of failed deposition or recycling during transcription. To unravel this issue, the question could thus be considered by exploring the cellular consequences of the disruption of specific components of the HIRA complex with the capacity to disentangle H3.3 recycling from new deposition.

  3 may be leveraged by the cell to fine-tune chromatin states. By recycling old H3.3 the cell has the potential to aid transcriptional regulation of key genes -maintain the active transcription by being re-deposited back into the same locus, or facilitate a switch in transcriptional state by incorporating it into regions previously silent.

Figure 1 .

 1 Figure 1. Sequences of major H3 variants and corresponding nucleosomal core particles

Figure 2 .

 2 Figure 2. SNAP-tagging technology to follow histone dynamics

3 (

 3 (d) Example of data obtained using SNAP-tagging technology: Representative STORM images of global H3.TMR, orange) and replicated DNA (EdU, green) in HeLa H3.3-SNAP. EdU labeling allows the selection of cells outside S phase, in early S phase and mid/late S phase. The color gradient corresponds to the z range. Scale bar=5 μm. Insets represent enlarged images of selected area where scale bars=600 nm.

Figure 3 .

 3 Figure 3. De novo deposition pathways of histone variant H3.3

Figure 4 .

 4 Figure 4. Old H3.3 recycling in the context of transcription

Figure 5 .

 5 Figure 5. H3.3 recycling and its consequences -a model

Figure 6 .

 6 Figure 6. Major PTMs present on new and old H3.3 histones

Acknowledgments

We thank Dominique Ray-Gallet and Sebastien Lemaire for critical reading of the manuscript. This work was supported by the ERC-2015-ADG-694694 'ChromADICT' and the Ligue Nationale contre le Cancer (Equipe labellisée Ligue). KD is supported by the EMBO Long Term Postdoctoral Fellowship.