Lise Dauban

Rémi Montagne

Agnès Thierry

Luciana Lazar-Stefanita

Nathalie Bastié

Olivier Gadal

Axel Cournac

Romain Koszul
email: romain.koszul@pasteur.fr

Frederic Beckouet
email: frederic.beckouet@ibcg.biotoul.fr

Frédéric Beckouët

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners

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

INTRODUCTION

Recent advances in imaging and chromosome conformation capture techniques (3C, Hi-C; [START_REF] Dekker | Capturing chromosome conformation[END_REF]) have unveiled a hierarchy of chromatin structural arrangements [START_REF] Cavalli | Functional implications of genome topology[END_REF][START_REF] Dekker | The 3D genome as moderator of chromosomal communication[END_REF][START_REF] Yu | The Three-Dimensional Organization of Mammalian Genomes[END_REF]. A variety of megabase (Mb) and sub-Mb structures have been proposed to organize the chromosomes of bacteria [START_REF] Le | High-resolution mapping of the spatial organization of a bacterial chromosome[END_REF][START_REF] Lioy | Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins[END_REF][START_REF] Marbouty | Condensin-and Replication-Mediated Bacterial Chromosome Folding and Origin Condensation Revealed by Hi-C and Super-resolution Imaging[END_REF], yeasts [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF][START_REF] Mizuguchi | Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe[END_REF][START_REF] Muller | Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C[END_REF][START_REF] Schalbetter | SMC complexes differentially compact mitotic chromosomes according to genomic context[END_REF] and mammals [START_REF] Dekker | The 3D genome as moderator of chromosomal communication[END_REF][START_REF] Yu | The Three-Dimensional Organization of Mammalian Genomes[END_REF] and are presumably a feature of most if not all organisms. During mitosis, this organization undergoes its most radical change when chromatin is transformed in condensed thread-like structures that facilitate chromosome segregation [START_REF] Liang | Chromosomes progress to metaphase in multiple discrete steps via global compaction/expansion cycles[END_REF].

In many species, the cohesin and condensin SMC protein complexes play important roles in chromosome folding. They consist of a pair of SMC proteins that associate with a Kleisin protein to form a large tripartite ring capable of embracing one or two DNA molecules [START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF]. In addition to mediating sister chromatid cohesion during G2/M phases of the cell cycle [START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF][START_REF] Haering | The cohesin ring concatenates sister DNA molecules[END_REF], cohesin shares with condensin the ability to convert DNA fibers into chromatid thread-like structures through a mechanism that remains poorly understood [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF][START_REF] Paldi | Cohesins functionally associate with CTCF on mammalian chromosome arms[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. SMC rings may organize DNA within chromatids by a DNA loop extrusion process, which consists in capturing small chromatin loops and progressively catalyzing their enlargement into large, megabase-sized structures [START_REF] Fudenberg | Formation of chromosomal domains by loop extrusion[END_REF][START_REF] Gibcus | A pathway for mitotic chromosome formation[END_REF][START_REF] Nasmyth | Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis[END_REF]. The precise mechanisms driving the generation of loops remain however to be fully characterized.

In mammals, condensin-dependent loops represent basic units for the folding of mitotic chromosomes into compact, higher-order chromatin structures. Cohesin-dependent loops, on the other hand, appear to contribute to long-range gene regulation in interphase, and to the segmentation of interphase chromosomes into topologically associating domains (TADs) defined as sub-megabase self-interacting regions. The organization of interphasic chromosomes into self-interacting domains has been proposed to insulate enhancers from non-cognate promoters by blocking cohesin-dependent loop progression when cohesin reaches the insulator protein CTCF bound to DNA [START_REF] Nora | Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization[END_REF]. Interphasic selfinteracting domains are chromosomal features found in many studied living organisms but their dependency on cohesin and CTCF activities is not automatic. Besides mammals, the involvement of cohesin in domain formation has for instance been reported in fission yeast [START_REF] Mizuguchi | Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe[END_REF]. In budding yeast S. cerevisiae however, no clear TADs-like structure nor DNA loops have been identified along interphase chromosomes. Nevertheless, the fact that cohesin is essential to convert the rDNA locus into a thread-like structure [START_REF] Guacci | Chromosome condensation and sister chromatid pairing in budding yeast[END_REF] during mitosis raises the idea that cohesin may also mediate cis-DNA looping along the yeast chromosome. Recently, we and others showed using Hi-C that meiotic and mitotic compaction in budding yeast is dependent on cohesin [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF][START_REF] Muller | Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C[END_REF][START_REF] Schalbetter | SMC complexes differentially compact mitotic chromosomes according to genomic context[END_REF]. Surprisingly, we also found that condensin is dispensable for mitotic chromosome arm compaction. Instead, condensin is required for higher-order chromatin structuring at the rDNA locus during anaphase and for promoting resolution at peri-centromeric regions.

Cohesin is composed of two SMC ATPases (Smc1 and Smc3) and an α-kleisin subunit called Scc1/Rad21. This complex binds to DNA in a dynamic manner throughout the cell cycle. Cohesin loading requires ATP hydrolysis mediated by Scc2/NIPBL, that is thought to result in entrapment of DNA inside the cohesin tripartite ring [START_REF] Chapard | Sister DNA Entrapment between Juxtaposed Smc Heads and Kleisin of the Cohesin Complex[END_REF][START_REF] Ciosk | Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins[END_REF][START_REF] Gligoris | Closing the cohesin ring: structure and function of its Smc3-kleisin interface[END_REF][START_REF] Haering | Molecular architecture of SMC proteins and the yeast cohesin complex[END_REF][START_REF] Hu | ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex[END_REF]. Cohesin may dissociate from DNA by separase-mediated cleavage of Scc1 at anaphase onset [START_REF] Uhlmann | Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1[END_REF] and in a manner that involves a "releasing activity" at other stages of the cell cycle. This separase-independent releasing activity relies on two cohesin-associated regulatory subunits:

Wpl1/Wapl and Pds5 [START_REF] Gandhi | Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase[END_REF][START_REF] Kueng | Wapl controls the dynamic association of cohesin with chromatin[END_REF][START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF]. Pds5 binds within the N-terminal part of Scc1 and recruits Wpl1 [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF] which opens the gate located between Scc1 and Smc3, triggering cohesin dissociation from DNA [START_REF] Beckouët | Releasing activity disengages cohesin's smc3/scc1 interface in a process blocked by acetylation[END_REF][START_REF] Chan | Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation[END_REF][START_REF] Murayama | DNA Entry into and Exit out of the Cohesin Ring by an Interlocking Gate Mechanism[END_REF]. When replication takes place, the releasing activity is repressed by Eco1-mediated acetylation of two conserved lysine residues (K112/113) within the Smc3 ATPase head (Rolef [START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Unal | A molecular determinant for the establishment of sister chromatid cohesion[END_REF]. This results in stabilization of cohesin on DNA and establishment of sister chromatid cohesion. It has been suggested that acetylation of K112/113 is in fact stimulated by both DNA replication fork progression and the Pds5 subunit [START_REF] Beckouët | An Smc3 acetylation cycle is essential for establishment of sister chromatid cohesion[END_REF][START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF][START_REF] Ben-Shahar | Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion[END_REF][START_REF] Vaur | Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction[END_REF].

Smc3 acetylation is then maintained throughout G2 and M phases and only removed after cleavage of Scc1 and subsequent deacetylation by Hos1 [START_REF] Beckouët | An Smc3 acetylation cycle is essential for establishment of sister chromatid cohesion[END_REF].

If cohesin-mediated loops are established by loop extrusion, their lengths should depend on several parameters, notably cohesin residence time on DNA and an extrusion driving force. This prediction is supported by the observation that the length of chromatin loops in mammalian interphasic nuclei increases once cohesin turnover is abolished after inactivation of Wpl1 and Pds5 [START_REF] Haarhuis | Cohesin: building loops, but not compartments[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. However, the mechanism(s) leading to loop enlargement remains a matter of debate. ATP hydrolysis may be the driving force for chromatin loop extrusion, as observed for the purified condensin complex [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF]. Scc2, being essential for cohesin's ATPase activity could thus play a key role in stimulating the translocation process and promoting DNA loop expansion [START_REF] Petela | Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5[END_REF]. Alternatively, transcribing RNA polymerases may promote loop enlargement by pushing cohesin along the DNA [START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF][START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF]. Finally, the frequency of cohesin injection along chromosomes may also influence their structure.

In the present study, we show that mitotic yeast chromosomes are compacted by cohesin dependent loops. By dissecting the roles of cohesin regulatory partners in DNA loop expansion, we also show that the mechanisms regulating loop positioning are highly conserved between yeast and mammals. Moreover, we find that in addition to promoting sister chromatid cohesion Eco1 inhibits the translocation process that extends DNA loops. In the absence of negative regulation, the main barrier to loop expansion is the centromere, which remains physically connected to the microtubule-organizing center (MTOC) during the entire yeast cell cycle.

RESULTS

Yeast mitotic chromosome arms are organized by cohesin-dependent loops

To characterize S. cerevisae mitotic chromosome organization we took advantage of yeast strains in which the cyclosome activator Cdc20 is under the control of the repressible MET3 promoter. To arrest the cells in mitosis, we released them from a G1 alpha-factor arrest by growing them in rich media supplemented with methionine (Figure 1A; Methods). Once arrested in metaphase, genome-wide chromatin contacts were quantified by Hi-C. Normalized contact maps (1 kb resolution) of mitotic chromosomes differed from G1 pattern in several ways (Figure 1B, left panels). First, the broad and thicker diagonal observed in metaphase compared to G1 revealed increased contacts at short/medium range, blurring the boundaries of the small microdomains visible in G1. Second, discrete dots of variable intensities corresponding to enriched contacts between pairs of loci, consistent with DNA loops, were also often visible along mitotic chromosomes (Figure 1B, black arrows). Not all chromosomal regions were covered by a loop signal, but whether this results from the resolution of our experiment or a real feature of chromosome structure remains to be characterized. To better characterize chromosome compaction we computed the contact probability curve Pc(s) for each condition [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF][START_REF] Schalbetter | SMC complexes differentially compact mitotic chromosomes according to genomic context[END_REF] (Figure S1A; Methods).

The derivative of the Pc(s) curve in a log-log space can be used to facilitate the visualization of local differences (Methods). In mice, the maximum of the derivative curve also matches with the average length of extruded loops [START_REF] Gassler | A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture[END_REF]. The same representation in yeast showed a net enrichment in mitosis in the contacts between loci separated by 30 kb or less, compared to G1 (Figure 1C, blue vs. orange curve). It also suggested a loop size in metaphase arrested cells of ~15 kb on average.

To directly test the role of cohesin in the maintenance of these structures, we depleted Scc1 in synchronized cells using an auxin degron strategy (Figure 1A right panel, Methods).

Efficient Scc1 depletion was quantified by a split-dots assay measuring cohesion loss (Figure 1D). Preventing cohesin loading during S phase suppressed establishment of intrachromosomal contacts (Figure S1B) and loop formation (Figure 1B, right panel) and the overall contacts displayed little changes compared to those observed in G1-arrested cells that contain little, if any, Scc1 (Figures S1A). Because of the population average nature of Hi-C, the systematic presence of all these loops in each cell cannot be directly addressed here. However, to further characterize loops and to analyze whether they correspond to contacts between Scc1 enrichment sites [START_REF] Verzijlbergen | Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere[END_REF], we generated pile-up plots. Pile-up plots aim at depicting the average contact signal between pairs of Scc1 enrichment sites [START_REF] Verzijlbergen | Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere[END_REF].

Briefly, for each condition, contacts between all pairs of 80 kb windows, centered on Scc1, and separated by 0 to 20 kb, were aggregated. A randomized set of windows separated by similar distances was also computed. The pile-up plot resulted from the ratio between the Scc1-centered aggregated maps and the randomized set. The same process was applied for pairs of cohesins enrichment sites separated by increasing distances, from 0 kb to 140 kb, with 20 kb steps (Figure 1E, Methods, [START_REF] Gassler | A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture[END_REF][START_REF] Muller | Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C[END_REF]). The color scale of the plots reflects the differences in contacts between the two aggregated maps: the redder, the more contacts are enriched on the maps centered on Scc1 enrichment sites. In metaphase, a strong dot signal appeared at the center of the pile-up, i.e. between pairs of cohesin-binding sites (Figure 1F, Methods), that vanishes over distances larger than 40 kb.

These dots point as enriched contacts between pairs of discrete cohesin binding sites, e.g. loop-like structures. The strength of the dot signal can be assessed by dividing the median value of the central 3x3 square (i.e. the signal value), by the median of the top-right corner square (i.e. the background value). Pile-up plots confirm that the yeast mitotic chromosomes are organized into chromatin loops anchored at cohesin binding sites.

Peaks of cohesin enrichment sites overlap, on average, with convergent transcription sites [START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF][START_REF] Glynn | Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Hu | Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq[END_REF][START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF][START_REF] Paldi | Cohesins functionally associate with CTCF on mammalian chromosome arms[END_REF]. The basis of DNA loops, which involve such cohesin enriched regions, were indeed and unsurprisingly also found to be preferentially positioned within convergent genes (as illustrated in Figure S1C).

Cohesin dependent loops are independent of sister chromatid cohesion

Mitotic loops could arise from the bridging of cohesins involved in sister chromatid cohesion. To test this hypothesis, we generated Hi-C maps of mitotic chromosomes from cells depleted for Cdc45, which reach mitosis without replication [START_REF] Tercero | DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p[END_REF] (Figures 2A and S2A). Contact maps and their associated DNA loops (Figures 2B andS2B), Pc(s) (Figures 2C andS2D) and pile-up plots of mitotic unreplicated and replicated cells showed little, if any, differences (Figure 2D). In addition, DNA loops of unreplicated mitotic chromosomes disappear in the absence of Scc1 (Figures 2B, 2C, 2D and S2C). Altogether, these results

show that sister chromatid cohesion is not necessary for generating Scc1-dependent structures.

To further determine whether cohesin-mediated loops along mitotic chromosomes depend on a mitosis specific activity, we induced cohesin loading on unreplicated chromosomes in G1. An engineered version of Scc1 that cannot be cleaved by separase was induced in G1 arrested cells [START_REF] Uhlmann | Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast[END_REF] (Figures 3A andS3A) and contact maps were generated (Figure 3B). Loading of cohesin on G1 chromosomes led to an accumulation of loops between pairs of Scc1 binding sites (Figures 3B and3C) and a significant increase in intra-chromosomal contacts (Figures 3D,S3B,S3C and S3D). In this condition, loops appeared slightly larger than in mitosis, suggesting that their expansion is constrained in metaphase-arrested cells (Figure 3C). Altogether those results demonstrate that the establishment of cohesin-mediated DNA loops along mitotic chromosomes is independent of sister-chromatid cohesion and of a mitosis specific activity.

Wpl1-mediated releasing activity counteracts loop expansion

In the absence of Wpl1, cohesins cannot be released from DNA. In mammals, Hi-C studies of WAPL-depleted cells revealed an enlargement of cohesin loops along interphase chromosomes which was interpreted as a hint of a loop extrusion process [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF].

To gain insights into the mechanism generating cohesin loops in yeast metaphase chromosomes, we therefore tested whether intra chromosomal contacts, Pc(s) and DNA loops spread to greater distances in Wpl1-depleted cells. Wild-type and wpl1Δ cells were synchronized in metaphase by depleting Cdc20 (Figure S4A) and genome-wide chromatin interactions were quantified by Hi-C (Figure 4A). In Wpl1-depleted cells, the contact map displayed a very different pattern as compared to wild-type, with less short-range contacts overall (but a crispier constriction at the centromere level, clearly separating the two chromosome arms), as well as chromatin loops punctuating the map resulting in a grid-like dotty pattern (Figures 4A andS4B). The former positions of wild-type loops often displayed a weaker signal in the absence of Wpl1 as compared to wild-type cells. On the other hand, contacts between discrete, cohesin enriched sites now appeared enriched over longer distances, as shown by pile-up plots (up to ~130 kb in the absence of Wpl1 compared to ~<40 kb in wild type) (Figures 4B and S4D). The Pc(s) also confirmed the decrease in short-range contacts and the enrichment over longer distances (Figures 4C andS4C).

The resulting grid-like loop pattern was often, but not always, stronger in chromosomal regions proximal to the centromeres. This may be due to high levels of cohesin loading at centromeric regions or due to the fact that the centromeric region represents a roadblock to cohesin progression. Similarly to wt metaphase cells, the basis of DNA loops in Wpl1 depleted cells involve enriched cohesion sites preferentially positioned within convergent genes (Figure S4E). The effects of Wpl1 depletion were further supported by performing the experiment in cells harboring pds5-S81R, an allele abolishing cohesin release even in presence of Wpl1 (Figure S4).

Expansion of DNA loops induced by Wpl1 depletion was not specific to mitosis, as it was also observed in G1-arrested cells expressing the non-cleavable version of Scc1 (Figure S5).

Our data therefore show that similarly to human cells, the size of yeast cohesindependent loops becomes deregulated in the absence of Wpl1, extending over much longer distances, though still involving hotspots of cohesin depositions. It suggests that yeast cohesin organizes mitotic chromosome presumably by loop extrusion, similarly to the mechanism proposed by others to explain establishment of DNA loops by cohesin during mammalian interphase.

Dual roles for Pds5 in DNA looping

As Wpl1 works with Pds5 to mediate "releasing activity" we tested how Pds5 contributes to DNA loop regulation. G1-arrested cells were depleted for Pds5 using the degron system, released into S-phase and arrested in metaphase (Figure S6A). Loss of both Pds5/Eco1-mediated Smc3-K113 acetylation (Figure 5A) and sister chromatid cohesion (Figure 5B) confirmed efficient Pds5 degradation. The contact map of Pds5-depleted cells was generated (Figure 5C) and compared to control (Figures 5C andS6B) and wpl1Δ cells (Figures 5C andS6C). Comparison of maps (Figure 5C) revealed that the absence of Pds5 increased the amount of long-range intra-chromosomal DNA contacts to a much higher extent than in wpl1Δ cells, as confirmed by log ratio between maps (Figures S6B andS6C) and Pc(s) curves (Figures 5E andS6D).

A close inspection of Hi-C map also revealed that, as in metazoan [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF], Pds5 depletion induced a decrease in the number of discrete spots/DNA loops compared to wild-type and wpl1Δ cells (Figure 5C). While in wild-type and wpl1Δ cells, loops bridge discrete pairs of Scc1-enriched sequences, in Pds5-depleted cells loop bases were more loosely defined, with no clear-cut dots in the normalized Hi-C maps (Figure 5C). This was confirmed by pile-up plots showing that in Pds5 depleted cells, DNA loops did not accumulate within those cohesin-rich loci as clearly as in wild-type cells (Figures 5D andS6E).

In metaphase-arrested Pds5-depleted cells, the insulation of peri-centromeric regions from the rest of chromosome arms was reduced. Whereas in wt and Wpl1-depleted cells the immediate left and right centromere-flanking regions appear to form a discrete "domain" (Figure S6F, see pink rectangle), this small structure disappears in the absence of Pds5.

Instead, both regions now became able to engage in long-range contacts with their entire adjacent arm, up to the telomere. In addition, the ratio plot between Pds5-depleted and wildtype mitotic maps also showed a significant loss of contacts between the two arms (Figure S6B). As a result, each chromosome arm appears on the maps as a large self-interacting domain (Figure 5C).

To analyze whether pericentromeric, long-range contacts affect centromere clustering in the absence of Wpl1 or Pds5, we looked at the positioning of the fluorescently labelled Ndc80 kinetochore protein in the nuclei of nocodazole-arrested cells. Whereas in wild-type and Scc1-depleted cells all Ndc80 fluorescent signals appeared as two bright dots, both in wpl1∆ and Pds5-depleted cells this structure was accompanied by multiple, small discrete dots (Figure S6G). Given that the main difference between these different conditions consisted in the size of the loops, we propose that the engagement of each centromeric region in large loops induces a mechanical force promoting centromere de-clustering.

Chromosome contact patterns observed in Pds5-depleted cells suggest that Pds5 regulates loop formation not only via a Wpl1-mediated releasing activity, but also by an unknown, Wpl1-independent mechanism. As Pds5, but not Wpl1, is essential to maintain sister chromatid cohesion, we hypothesized that the increase in DNA loops sizes over longer distances in Pds5-depleted cells could result from loss of cohesion. Indeed, cohesins involved in sister chromatid cohesion may act as physical barriers, or roadblocks, halting the loop expansion process and defining loop basis positions along chromosomes. Inactivation of Pds5 would alleviate those discrete roadblocks, allowing loop expansion to proceed over longer distances, and resulting in an increase in long-range contacts. To test this, we measured the effects of Pds5 or Wpl1 loss on 3D folding of un-replicated (cdc45) chromosomes (Figures 6A, B andC). The depletion of either Wpl1 or Pds5 had the same effect on chromosome structure than that observed in metaphase, with Pds5 inactivation leading to intrachromosomal contacts bridging loci over longer distances than in wpl1Δ cells, as illustrated by log ratio between Hi-C maps (Figure 5F). Moreover, inactivation of Pds5 in Cdc45 depleted cells also induced a decrease in the number of discrete spots/DNA loops compared to wild-type and wpl1Δ cells (Figures 6C andD).

Impact of Pds5 and Wpl1 depletion was also quantified in living cells: fluorescentlylabelled centromeres and a locus positioned 400 kb away on chromosome XV got closer together compared to control cells (Figure 6E), backing the Hi-C results showing increased loop sizes.

Those results therefore demonstrate that Pds5 regulates DNA contacts and suppresses loop expansion through two mechanisms, the recruitment of Wpl1, as well as a Wpl1independent pathway.

Eco1 inhibits loop expansion

Since increase in loop length in Pds5 depleted cells does not only result from the absence of releasing activity, we envisioned that Pds5 recruitment of Eco1 [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF][START_REF] Minamino | Esco1 Acetylates Cohesin via a Mechanism Different from That of Esco2[END_REF][START_REF] Noble | Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism[END_REF][START_REF] Vaur | Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction[END_REF] might regulate a second mechanism required to inhibit DNA loop expansion. We tested the effects of Eco1 depletion using an inducible degron (Figures S7A andS7B; Methods) and showed that loss of Eco1 is sufficient to result in spreading of contacts (Figures 7A,S7C and S7D) and loops (Figures 7A, C andS7F) to longer distances. As the absence of Eco1 promotes dissociation of cohesin from DNA by Wpl1 [START_REF] Chan | Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation[END_REF], these longer loops cannot result from an increase in cohesin residence time on DNA. Therefore, this result shows that in addition to promoting sister chromatid cohesion, Eco1 also inhibits the translocation process extending DNA loops.

Remarkably, loop length (Figure 7A, C and S7F), contact probability (Pc(s)) (Figures 7B andS7B) and centromere clustering defects in Eco1 depleted cells (Figure S7G) were comparable to those observed in Wpl1 depleted cells, despite Eco1 and Wpl1 having opposing effects on releasing.

Since Pds5 recruits both Eco1 and Wpl1 on cohesin, we tested whether their coinactivation would mimic the effect of Pds5 depletion. Indeed, depletion of Eco1 in wpl1∆ cells promoted long-range intra-chromosomal contacts and DNA loops nearly identical to those observed in Pds5 depleted cells (Figures 7,S7B,D and E). This suggests that the positioning of loop basis (hence the size of the loops) is regulated through two independent pathways, Eco1 and Wpl1.

DISCUSSION

In the present study, we show that yeast mitotic chromosomes organize into loops along individual chromatids, independently of replication. These loops are enriched in cohesin subunits at their basis. The cohesin binding factors Pds5, Wpl1 and Eco1 affect the sizes and distribution of these loops, with Pds5 regulating loop size via two pathways: the Wpl1-mediated releasing activity and an Eco1-dependent mechanism (Figure 7).

Yeast mitotic chromosomes are organized into cohesin-dependent loops

We show that DNA loops are present along yeast mitotic chromosomes. The bases of these loops are enriched in cohesin. Whether all cohesin deposition sites identified by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) are involved in loop formation in all cells, or whether a loop heterogeneity exists within the population remains to be characterized. Cohesin enrichment sites, and the bases of DNA loops in wt or Wpl1 depleted cells, correspond mostly to site of convergent genes. It has been proposed that accumulation of cohesin at these genomic positions may be driven by transcription [START_REF] Lengronne | Cohesin relocation from sites of chromosomal loading to places of convergent transcription[END_REF]. RNA polymerases along convergent genes may indeed represent boundaries or roadblocks to cohesin-dependent loop expansion, resulting in an accumulation of cohesin-dependent loop basis at sites of convergent transcription. We also noticed that Pds5 is crucial to maintain accumulation of cohesin-dependent loop at those sites. This implies that in absence of Pds5 the loop formation mechanism somehow manages to bypass the roadblocks.

Cohesin binding factors regulate loops expansion

It has been proposed that cohesin could promote chromatin loops formation via a loop extrusion mechanism [START_REF] Fudenberg | Formation of chromosomal domains by loop extrusion[END_REF][START_REF] Goloborodko | Chromosome compaction by active loop extrusion[END_REF][START_REF] Nasmyth | Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis[END_REF]. This model predicts that the length of DNA loops should be dependent on cohesin residence time on DNA. Since Wpl1 inactivation increases cohesin's residence time on DNA, cohesin would extrude longer DNA in Wpl1-depleted cells. Indeed, Hi-C studies in mammals revealed an enlargement of cohesin-mediated loops along chromosomes in WAPL1-depleted cells. Our present study shows that the inactivation of Wpl1-mediated releasing also induces enlargement of DNA loops in S. cerevisiae. By analogy with mammals, we propose that this enlargement of DNA loops is the consequence of an increase of cohesin's residence time on DNA and that cohesin structure yeast mitotic chromosomes through a loop extrusion process.

Alternatively, one can envision another hypothesis whereby the longer range contacts observed in Wpl1-depleted cells result from cohesin loading defects due to Wpl1 depletion [START_REF] Rowland | Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity[END_REF]. Indeed, the inactivation of Wpl1 could reduce the amount of cohesin being loaded on the chromatin, hence decreasing the number of complexes expanding DNA loops present along a chromosome arm. The lower density of cohesin may in turn lead to longer DNA tracks being transformed into loops by the same complex before colliding into another complex, and thus resulting in the formation of longer loops on average. Future works will determine whether loop enlargement in Wpl1-depleted cells is the consequence of cohesin loading defects, of an increase in cohesin's residence time on DNA, or both.

We also analyzed the role of other cohesin binding factors on loop structures and found that Pds5 regulates loop expansion via at least two pathways: the previously described Wpl1-mediated releasing activity [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF] and a novel Eco1-dependent mechanism (Figure 7). Our results point at a role of Eco1 in negatively regulating loop expansion through a mechanism independent of cohesion establishment. In agreement with our result a recent study reported that ESCO1 (human ortholog of Eco1) also inhibits the ability of cohesin to form long DNA loops [START_REF] Wutz | ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL[END_REF].

Competition between Scc2 and Pds5 may regulate DNA loop expansion

One may envision that Eco1-mediated acetylation inhibits translocase activity that expands cohesin-dependent loops. It has been proposed that ATP hydrolysis stimulated by Scc2 could be the driving force for loop expansion. As Pds5 competes with Scc2 for binding the kleisin subunit [START_REF] Petela | Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5[END_REF], a possibility is that Pds5 inhibits the translocation process required to expand DNA loops. Eco1-mediated acetylation may therefore improve Pds5 binding on cohesin and consequently abolish the Scc2-mediated translocation process. This idea is reinforced by in vivo FRAP measurements revealing that Eco1 inactivation increases Pds5 turnover on chromosomes [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF]. Moreover, recent ChIP-seq data show that Eco1 inactivation reduces Pds5 association with DNA while it has no effect on cohesin ring association with DNA [START_REF] Chapard | Sister DNA Entrapment between Juxtaposed Smc Heads and Kleisin of the Cohesin Complex[END_REF].

Competition between Pds5 and Scc2 may also control loop expansion and cohesin positioning in human cells. This hypothesis is supported by the fact that Scc2 does not colocalize with CTCF which halts loop expansion. [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF] showed that the ability of CTCF to block loop extrusion is dependent on PDS5. As discussed by the authors, replacement of Pds5 by Scc2 may be somehow inhibited at CTCF sites, which may consequently abolish or decrease the rate of ATPase hydrolysis required to expand DNA loops [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF].

How does Eco1 regulate competition between Scc2 and Pds5 during establishment and maintenance of cohesin-dependent loops? It may involve Eco1-mediated Smc3 acetylation as this reaction affects how Scc2 promotes loading of cohesin onto DNA [START_REF] Hu | Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq[END_REF].

DNA looping is blocked at centromeres

Our study points out that the yeast centromere is a strong roadblock to cohesindependent loop expansion. The nature of this boundary raises interests beyond the yeast community, as related mechanisms could apply in other species as well. In yeast, kinetochore proteins bound to centromeres are connected through microtubules to the MTOC (spindle pole body or SPB) which itself is embedded in the nuclear envelope. Such a physical bridge could block the progression of incoming cohesin along the chromosome. It is also possible that the huge kinetochore complex bound to centromeres is sufficient to stop DNA loop expansion.

In wild type cells, Wpl1 mediates cohesin release and Eco1 restricts DNA loop progression from chromosome arms to centromeric regions. When those activities are impaired, loops emanating from chromosome arms can extend to centromeres, but no further, resulting in sharp, discrete boundaries as observed on the contact maps. In addition, it is likely that loops also originate from centromeres and spread towards chromosome arms.

Finally, we showed that centromere clustering is impaired in Wpl1, Pds5 or Eco1 depleted cells (Figure S6G and S7G). This suggests that centromere clustering is exposed to opposite forces emanating from microtubules and, presumably, a loop extrusion-like mechanism that promote loop expansion. Inhibiting such loop extruding mechanism in wildtype cells may favor the insulation of clustered centromeres away from chromosome arms, and consequently improve or stabilize microtubule attachment and chromosome segregation.

It remains to be investigated whether similar mechanisms occur in species with larger and epigenetically defined centromeres compared to the budding yeast point centromere, and whether the attachment of microtubules is mandatory to bock DNA looping. for metaphase-arrested cells (cdc20, strain FB133-57B). C) Derivative of the curve plotting contact probability as a function of genomic distance (log scale). D) Pile-up ratio plots of 80 kb windows (2 kb bin) centered on pairs of Scc1-enriched or randomly chosen positions (see Figure 1F). See also Figure S2.

STAR METHODS

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Romain Koszul (romain.koszul@pasteur.fr)

EXPERIMENTAL MODEL AND SUBJECT DETAILS Yeast Strains and Plasmids

All strains are derivatives of W303. Strains and plasmids are listed in Key Resources Table and in Supplementary Table S2 Media and growth conditions Strain yLD118-1a (MET3-CDC20) was grown overnight at 30°C in 150ml of synthetic complete medium deprived of methionine (SC-Met) (SC: 0.67% yeast nitrogen base without amino acids (Difco)), supplemented with a mix of amino-acids, uracil and adenine, 2% glucose) to reach 4,2 x 10 8 cells. To induce metaphase arrest, cells were arrested in G1 for 2h30 by addition of alpha-factor (Antibody-online, ABIN399114) every 30min (1μg/ml final), washed 3 times and released in rich medium (YPD: 1% bacto peptone (Difco), 1% bacto yeast extract (Difco) and 2% glucose) supplemented with methionine (2mM final). 2h latter cells were fixed for Hi-C. Strains yLD127-20b, yLD121-1a, FB133-57B, FB133-20C, FB133-49B, FB133-1D were processed as described above except auxin addition (Sigma-Aldrich, I3750) (1mM final) to the media 1h after starting alpha-factor treatment. Cells were released from G1 in YPD supplemented with methionine and auxin (1mM final). Strains FB08-5C, FB08-6A, FB09-4A, FB09-9C were grown overnight in 300ml of YP medium supplemented with raffinose 2% (Sigma-Aldrich, R0250) to reach 8,4 x 10 8 cells. Expression of Scc1(R180D,268D)-HA was induced 1h after starting alpha-factor treatment by addition of newly-made galactose (Sigma-Aldrich, G0750) (2% final) to the cultures. Cells were fixed for Hi-C after 2h30 in alpha-factor. Strains FB154, FB149-11B, FB148-3C, FB156-5a (CDC45-AID) were grown overnight in 150ml YPD to reach 4,2 x 10 8 cells. G1 arrest and auxin addition were conducted as previously, (except auxin concentration, 2mM final), cells were released in YPD media supplemented with auxin (2mM final) for 80min and fixed for Hi-C.

METHOD DETAILS

Flow cytometry

About 2,8 x 10 6 were fixed in ethanol 70% and stored at -20°C. Cells were the pelleted, washed and incubated overnight in Tris-HCl 50mM pH 7,5 complemented with RNase A (10 mg/ml; Sigma-Aldrich) at 37°C. Cells were pelleted, resuspended 400µl of 1,0mg/ml propidium iodide (Fisher, P3566) in 50mM Tris pH 7,4, NaCl, MgCl2 and incubated for 1 h at room temperature. Flow cytometry was performed on a CyFlow® ML Analyzer (Partec) and data were analyzed using FloMax software.

Microscopy

Strains FB124, yLD126-38b, yLD126-36c were grown overnight in SC-Met at 30°C. The next day cells were diluted in fresh media. Exponentially growing cells were arrested in G1 with alpha-factor treatment, induced with auxin (1mM final), washed and arrested in metaphase as described above. Strains yLD162-13a, yLD162-2b, yLD163-22a were grown overnight in YP-raffinose at 30°C. The next day, after dilution in fresh media, exponentially growing cells were arrested in G1 with alpha-factor treatment and while being induced with auxin (1mM final). Expression of non-degradable Sic1 was induced 30min before release by addition of galactose (2% final) to the media. Cells were washed and released in YP supplemented with raffinose and galactose for 120min. Cells were placed on 2% agarose pads made of synthetic complete medium plus glucose. Live cell imaging was performed under a spinning disk confocal system (Nipkow Revolution, Andor Technology) with an EM chargecoupled device (CCD) camera (DU 888; Andor Technology) mounted on an inverted microscope (IX-81; Olympus) featuring a CSU22 confocal spinning disk unit (Yokogawa Corporation of America). Image acquisition was done at 30°C. 41 Z-stacking images with 0.25μm intervals were acquired by using IQ2 software with 200ms exposure time. Were used:

100× objective lens (Plan-Apochromat, 1.4 NA, oil immersion; Olympus) and single laser lines for excitation, diode pumped solid state lasers (DPSSL). GFP fluorescence was excited at 488 nm (50 mW; Coherent) and mCherry fluorescence at 561 nm (50 mW; Cobolt jive).

Green and red fluorescence were collected using a bi-bandpass emission filter (Em01-R488/568-15; Semrock). Pixel were 65nm in size.

Acetylation assays

A pellet from 10 7 cells was frozen in liquid nitrogen and stored at -20°C overnight. The cell pellet was resuspended in 100µl H20, 20μl trichloroacetic acid (Sigma-Aldrich, T8657) and broken with glass beads at 4°C. Precipitated proteins were resuspended in Laemmly buffer/ Tris HCl pH 8,0 and extracted by cycles of 5min heating at 80°C-5min vortexing at 4°C.

Eluates were analyzed by SDS-PAGE followed by western blotting with antibodies anti-V5 tag (VWR, MEDMMM-0168-P), anti-Pgk1 (Invitrogen, 459250) and anti-Smc3-K113Ac [START_REF] Beckouët | An Smc3 acetylation cycle is essential for establishment of sister chromatid cohesion[END_REF].

Hi-C libraries

Hi-C was performed as described [START_REF] Lazar-Stefanita | Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle[END_REF], except cells were disrupted using a Precellys apparatus (Bertin Instruments) instead of processed through zymolyase treatment. Aliquots of 1-3 x 10 9 cells in 150 ml YPD/synthetic medium were fixed in 3% formaldehyde (Sigma, F8775) for 20 min at room temperature and quenched with 25 ml glycine 2.5 M for 20 min at 4°C. Cross-linked cells were recovered through centrifugation, washed with YPD and a 150 mg pellet was stored at -80°C. Hi-C DNA libraries were 500 bp sheared using CovarisS220 apparatus, and the biotin-labeled fragments were selectively captured by Dynabeads Myone Streptavidin C1 (Invitrogen). The resulting libraries were used as template for the llumina amplification by PE-PCR primers and paired-end sequenced on a NextSeq500 Illumina platform. All Hi-C libraries are listed in Supplementary Table 1.

QUANTIFICATION AND STATISTICAL ANALYSIS

Processing of the reads and generation of contact maps

Pairs of reads were aligned independently using Bowtie2 in its most sensitive mode against the latest S. cerevisiae W303 reference genome (GCA_002163515.1), corrected for a chromosomal inversion on chromosome 16 revealed by the Hi-C data. Alignment was done using an iterative procedure and each uniquely mapped read was assigned to a restriction fragment. Uncuts, loops and religation events were filtered as described [START_REF] Cournac | Normalization of a chromosomal contact map[END_REF]. Contact matrices were built with resolutions of 2 or 20kb (bin sizes) and normalized using the sequential component procedure [START_REF] Cournac | Normalization of a chromosomal contact map[END_REF]. Log-ratios were generated by dividing 2 normalized contact maps, with the same resolution, by one another and then computing the log2 of the resulting matrix.

Computation of the contact probability as a function of genomic distance

Contact probability as a function of genomic distance Pc(s) was determined as described [START_REF] Muller | Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C[END_REF]. Intra-chromosomal pairs of reads were selected and partitioned by chromosome arms. Pairs oriented towards different directions or separated by less than 1.5 kb were discarded. For each chromosome, the remaining pairs were log-binned as a function of their genomic distance s using the formula: bin = [log1. 1(s)]. The number of read pairs in each bin was counted and weighed by the bin size 1.1(1+bin), as well as the difference between the length of the chromosome and the genomic distance.

Identification of cohesin binding-sites and generation of pile-up plots

Data from [START_REF] Verzijlbergen | Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere[END_REF] were used to generate Scc1 ChIP-Seq profiles with a 2kb resolution. Bins with a signal over 1.5 were labelled as cohesin binding sites (CBS). CBS were determined for wild type and Pds5-AID [START_REF] Petela | Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5[END_REF] strains. All possible pairs of CBS within chromosomal arms were determined and partitioned according to their genomic distance. In 2kb resolution contact maps, windows surrounding these positions were extracted and averaged. The resulting observed signal was divided by the expected signal, generated by averaging the windows around random positions having the same genomic distance as the pairs of CBS. For each window, undercovered bins were defined as bins with a total number of reads under median (number of reads / bin) -SD and excluded of the averaging operations to reduce noise. The strength of the central (loop) signal was assessed by dividing the median value of the central 3x3 square (i.e. the signal value), by the median of the top-right corner square (i.e. the background value). As a consequence, the more "loop signal", the higher the score (> 1). Since loops will deplete longer range contacts, a strong loop signal will be accompanied at longer distances by a weak one (< 1). No loops will result in a signal ~1. See [START_REF] Flyamer | Coolpup.py: versatile pile-up analysis of Hi-C data[END_REF], for a similar approach.

  Therefore, DNA loops and local contact enrichment observed along mitotic chromosomes are cohesin-dependent.

  However, additional investigations are necessary to determine whether Smc3 acetylation is involved in regulation of DNA loops. Deciphering how Eco1 influences DNA loop expansion may provide new perspectives to understand how human Eco1 homologues (ESCO1 and ESCO2) regulate gene transcription or how they deficiency induces the developmental disorder called Roberts syndrome.

Figure 1 .

 1 Figure 1. Mitotic yeast chromosomes are organized as cohesin-dependent loops and domains.

Figure 2 .

 2 Figure 2. Cohesin-dependent loops and domains on mitotic yeast chromosomes are

Figure 3 .

 3 Figure 3. Expression of Scc1 in G1-arrested cells induces DNA looping. A) Left panel:

Figure 4 .

 4 Figure 4. Wpl1 restricts DNA loop expansion. A) Contact maps of a part of chromosome IV

Figure 5 .

 5 Figure 5. A Wpl1-independent pathway regulates cohesin-dependent loops during mitosis. A)

Figure 6 .

 6 Figure 6. Effect of Pds5 or Wpl1 loss on the 3D folding of un-replicated mitotic

Figure 7 .

 7 Figure 7. Eco1 counteracts loop expansion. A) Hi-C maps (1 kb bin) for strains FB133-57B

Acknowledgements

We thank T.U. Tanaka and K. Nasmyth for sharing strains and K. Shiahige for the Smc3 antibody. We thank Aurèle Piazza, Luis Aragon, Julien Mozziconacci, Christophe Chapard, Olivier Cuvier, Tony Marchal, and OG and RK teams for discussions and comments on the manuscript. LD was supported by ARC fellowships. This research was supported by funding to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 260822).

Data Availability

Sample description and raw sequences are accessible on SRA database through the following accession number: PRJNA528616.

DATA AND CODE AVAILABILITY

Sample description and raw sequences are accessible on SRA database through the following accession number: PRJNA528616.

Codes and functions used to generate the figures from the raw data are available online (https://github.com/koszullab/hicstuff).

Imaging data are available on Mendeley (https://data.mendeley.com/datasets) under the DOI: 10.17632/pgwsf28v2d.

Author contributions

LD, RK and FB designed research. LD and AT performed the experiments, with help from LLS who initiated the study. RM analyzed the data, with contributions from AC. All authors interpreted the data. LD, RK, FB wrote the manuscript, with contributions from RM.

Declaration of interest

The authors declare no competing interests.

Pds5 loop expansion

Pds5

Eco1 through expansion block through release


MD Alessio Stivala
email: drstivala@gmail.com

MD Baptiste Bertrand

MD Neil Ouar

MD Marc Revol

MD Michael Atlan

MD Sarra Cristofari

Lower rates of lipofilling sessions in latissimus dorsi flap breast reconstruction with initial higher volume transfer by preservation of subfascial fat: A 3D camera-assisted volumetric case series

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

Introduction

Breast reconstruction techniques are becoming more and more reliable, precise and aesthetically harmonious.

The role of adipose tissue has become essential in breast reconstruction. Since 1977 when liposuction was invented by Yves-Gérard lllouz, until the first publications of Coleman in 1995 [START_REF] Coleman | Long-term survival of fat transplants: controlled demonstrations[END_REF] , the adipose tissue grafting has improved significantly. It is nowadays widely used in breast reconstruction, as an independent technique or in association with implants or with pedicled / free flaps.

Breast reconstruction with latissimus dorsi flap leads to long-term volume loss, this due by muscle wasting. This complication must be well preconized in order to compensate by a secondary addition of adipose tissue. On the one hand, the adipose graft has limited viability with a resorption rate which rates from 30 to 80% 2 . Moreover, there is no evidence in literature evaluating the durability of fat grafting in the muscular tissue.

In plastic surgery, volume restoration is a crucial goal to achieve, particularly after reconstruction.

Although the surgeon's experience is fundamental, objective measurements are increasingly considered as a major surgical criterion.

Our hypothesis is that LDF reconstruction with preservation of subfascial fat can achieve a higher volume in a one-time procedure if compared to a classic LDF harvest.

Three-dimensional imaging is an invaluable tool for obtaining precise and objective calculation of breast volume. Given its unique ability to critically analyze differences in size and shape, this tool is an essential component of preoperative planning in both reconstructive and aesthetic breast surgery, achieving breast symmetrization and satisfactory outcomes.

Materials and Methods

Study design

In this interventional study, all female patients requiring an immediate or delayed reconstruction with a latissimus dorsi flap were included in our center between January 2016 and May 2017.

The ethical principles outlined in the Declaration of Helsinki were respected by all authors. All patients signed an informed consent-form specifically designed for this study, authorizing the use of photographs. Research was approved by the local institutional review board. This study was conducted according to the STROBE and STARD guidelines.

The power of the study has been calculated before patient selection (Fig. 1). A first-step evaluation was made at first day after surgery (day 1), 3 and 6 months postoperatively.

Two different senior surgeons of the same

Secondly, each group underwent to lipofilling procedures in order to achieve a final volume similar to the contralateral breast according to patient's satisfaction. A self-assessment satisfaction questionnaire was developed. Six items assessed both general (4 items) and aesthetic (2 items) satisfaction:

1. I would definitively choose to have the type of breast reconstruction I had.

2. The volume of my breasts is the same. A second-step evaluation had the aim to measure volumes at zero and three months after each fat grafting session (Fig. 1).

The primary endpoint of the study was to quantify the volume resorption in LDF reconstructions with preservation of subfascial fat. Volume comparison with simple LDF was performed to find any significant resorption rate of the subfascial fat.

The secondary endpoint was to determine which of the two techniques gives the most satisfactory result in less surgical reinterventions. All characteristics were registered (number of patients, age, average BMI, type of breast reconstructions, number of supplemental lipofilling sessions). Postoperative complications and their length were also recorded.

Exclusion criteria such as obesity (BMI >30), diabetes, active smokers or patients who have undergone radiotherapy were applied.

Breast cancer patients have already undergone to several medical and surgical procedures. Obtaining an optimal result with the fewest number of interventions and the shortest recovery time was our aim.

Patient preparation

Before breast reconstruction, a preoperative assessment was made during consultation: medical history, any antecedents, the state of local tissues, the condition of the contralateral breast, and any available donor sites.

Particular attention is given to the amount of tissue obtainable in the dorsal region by pinching the latero-dorsal pad to gauge the thickness of the adipose layers. It is crucial to compare the mass available with that which will be needed to achieve a suitable breast size.

The patient was also informed of the prospect of dorsal seromas and of their treatment, possibly by repeated puncture, during the postoperative phase.

Surgical Technique

The line of the cutaneous paddle was made in the posterolateral thoracic region, in a crescent shape, opening upward. The paddle length was between 22 and 25 cm. The harvest of the autologous latissimus dorsi flap was made in the lateral supine position.

The cutaneous paddle was then incised and detached on all sides by taking in all the fatty layer over the muscle's surface including its anterior border, the iliac crest and the scapula (fatty zones from 1 to 5 by Delay et al. [START_REF] Delay | Autologous latissimus breast reconstruction: a 3-year clinical experience with 100 patients[END_REF] ).

The fat over the muscle is vascularized by the perforators coming from the muscle itself. The thickness of fat varies according to the dorsal adiposity of the patients. The detachment of the flap proceeded downward and then upward. It is crucial to preserve the subdermal plexus blood supply of the dorsal skin (at least 1 cm thick), which becomes the main source of vascularization once the perforators of the latissimus dorsi muscle have been removed.

Secondly, the patient was put in a supine, half-sitting position in order to achieve a correct placement of the flap.

In immediate breast reconstruction, the paddle was positioned into the mastectomy scar. In case of skin-sparing mastectomy, the de-epithelialized flap was put in the subcutaneous layer in place of the breast gland, leaving only an areolar cutaneous disc corresponding to the future areola. In delayed breast reconstruction, cutaneous paddle positioning is dependent on residual thoracic tissues and can be achieved in different ways:

• inserting the cutaneous paddle above the inframammary fold of the new breast;

• reopening the mastectomy scar and positioning the cutaneous paddle in this site;

• combining the de-epithelialized flap with an abdominal advancement flap.

When a satisfactory arrangement of the tissues was achieved, the flap was fixed on itself by using a 2-0 resorbable suture. Closure was performed on two layers after a drain placement. Bandages were put in place leaving a surveillance window over the cutaneous paddle.

The two techniques used in this study differed to each other by the presence or not of the fatty zone two over the harvested muscle.

Lipofilling procedure

Fat tissue was harvested (after at least 6-months post LDF procedure) from the abdominal, flank and thighs fatty areas under general anesthesia. The fat was treated by centrifugation at 3000 rotations per minute for 30 seconds and then purified. The fat tissue was then transferred to the reconstructed breast using a two-millimeter transfer cannula injected in a three-dimensional pattern in order to bring volume to the reconstructed breast.

Post-operative measurements

The material used for our work consists of an iPad mini 2 ® , characterized by a 5-megapixel camera with an aperture of f/2.4 and a Structure-Sensor camera by Occipital ® . Each patient received a 3D measurement in immediate postoperative and then after three and six-months follow-up. The patient was positioned in the middle of a room, without dressing and jewelry, the feet were 35 cm apart, the hands were placed on the hips. The patient had to maintain the position for the duration of the measurement without moving. A decimeter was fixed on the patient's chest, which made it possible to have an objective measurement unit for numerical calibration. The camera has to turn around the patient at a distance of 1,50 meters in order to capture all patient's details. After complete acquisition, the software generates a file in format ".obj" ready to be sent via mail or stored in the hard drive of the device.

Post-production analysis

Three dimensional acquisitions were analyzed using Geomagic Studio ® 12 software (Geomagic Solutions, Morrisville, NC, USA). Two different engineers made the 3D analysis. The principle was based on the superposition of volumes. The subtraction of the different volumes analyzed for the same patient made it possible to obtain the volume variation between the two acquisitions. For each patient, the immediate postoperative image was compared with images at three and 6 months after.

During post-production, all artifacts were eliminated from the original acquisition and the 3D mold was "cleaned" cutting-off elements not useful to the analysis (Fig. 2).

Once all modeling has finished, scan accuracy was checked by using the "Deviation Analysis tool".

The result is a color map on the mesh object showing the surface difference between the two models superimposed (Fig. 3). A blind evaluation of the outcomes (3D analysis and volume rate) was performed by two engineers during the whole study.

Statistics

Data were analyzed in order to show any significant differences between the findings collected.

GraphPad Prism ® (version 7.0 GraphPad Software, San Diego California USA) was used for the statistical analysis. A p-value < 0.05 was considered significant. T-student test was used for volume resorption comparison and Kruskal-Wallis non-parametric test was used to compare BMI modifications during follow-up.

Results

The prospective study included 30 reconstructed breasts (29 patients). The repartition of the procedures, and additional lipofilling sessions are resumed in figure 1.

There were nine cases of delayed breast reconstruction and six cases of immediate reconstruction.

There was only a case of bilateral breast reconstruction, so 29 patients were included in the study.

The average age was 56 years (ranging from 36 to 65 years) and the BMI had an average of 23,3 kg/m 2 in LDF sec. Delay 3 group and 22,8 kg/m 2 in simple LDF group (Tab.1).

The volumetric study focused on each reconstructed breast after LDF and, secondly, after fat grafting.

The average LDF "with subfascial fat" volume at "time zero" (immediate postoperative) was 526 cc (from 350 to 746). After a three-months follow-up an average resorption rate of 9,1% was found (mean volume of 479 cc, SD of differences 16,2). Immediate postoperative "simple" LDF mean volume was 547 cc (range 340 to 732). Analysis after three months revealed an average intramuscular resorption rate of 8,5% (SD of differences 16,2) (Tab.2).

Moreover, the LDF+subfascial fat group at postoperative six months was 466 cc with a resorption rate of 2,9% (SD of differences 9,7). The average "simple" LDF volume at postoperative six months was 491 cc demonstrating a supplemental resorption rate of 2,1% (SD of differences 7,8). (Tab.3).

Comparison at three and six months revealed no significant difference between the two groups (P value of 0,8 and 0,4 respectively).

Six months after LDF reconstruction, all patients in both groups underwent a fat grafting procedure.

The fat tissue harvested for the second part of the study had an average of 420 cc (from 280 to 560) and the fat volume obtained after centrifugation was 194 cc (from 90 to 300) (Tab.4).

A satisfactory result was achieved if volumetric 3D assessment of the reconstructed breast was comparable to the contralateral one and confirmed by patient's expectations at post-operative six months and after every lipofilling session.

After a three-months follow-up, twelve patients (LDF+subfascial fat group) had a satisfying result (80%) (SD of differences 14,8) compared to six patients (40%) belonging to the simple LDF group (SD of differences 16). Volume resorption rate was of 19,3% in both groups with no statistical differences between each group (P value of 0,72) (Tab.5).

A supplemental lipofilling was needed in those breasts with an unsatisfactory volume (three patients in LDF+subfascial fat group and nine in simple LDF group). At this point, volume resorption slightly increased up to 23,5% demonstrating any statistical difference between the two groups (P value of 0,4 and SD of differences 14). (Tab.6).

Discussion

Limitations of the study can be attributed to fat harvesting technique used and variations in fat quality.

If the harvested fat is very oily (after centrifugation) the resorption can be more important, rising up to 50% [START_REF] Ho Quoc | Breast asymmetry and pectus excavatum improvement with fat grafting[END_REF][START_REF] Ho Quoc | Breast reconstruction after mastectomy[END_REF] . Each surgeon slightly varies in fat harvest and preparation but accordingly to many authors there is no significant difference on the adipocyte survival [START_REF] Coleman | Fat grafting to the breast revisited: safety and efficacy[END_REF][START_REF] Smith | Autologous human fat grafting: effect of harvesting and preparation techniques on adipocyte graft survival[END_REF] even if integrated with plateletrich plasma [START_REF] Salgarello | Breast fat grafting with platelet-rich plasma: a comparative clinical study and current state of the art[END_REF] .

In order to obtain a scientifically-precise result we had to tackle with several problems encountered during acquisition and post-production analysis.

Limitations in the acquisition phase are the difficulty of scanning large ptotic breasts hiding the inframammary fold and light conditions. For this reason, is crucial the need of performing the digitalization through different heights by inclining the device by multiple angles in a large and bright room in order to reduce the number of "pixel-holes".

Moreover, volume variations are correlated with breathing according to Liu et al. [START_REF] Chunjun | Does Respiration Influence Breast Volumetric Change Measurement with the Three-Dimensional Scanning Technique?[END_REF] . We applied a position protocol requiring the patient to hold her breath at the end of normal exhalation during 5 seconds. The acquisition was then paused until another inhalation and exhalation was done.

The need to standardize breast surface measurements represented an important objective.

Acquisitions were performed several months after each other and patient's morphology could vary.

In terms of surface, breast area had to correspond at every superimposition in order to give the exact discrepancy of volume.

Tepper et al. [START_REF] Tepper | Mammometrics: the standardization of aesthetic and reconstructive breast surgery[END_REF] established fixed planes and points to perform objective breast measurements thus called "mammometrics".

The standardized landmarks helped us to clearly identify the breast region over a two-dimensional plane but the posterior chest-wall hidden below the breast cannot be acquired by the camera.

Many techniques have been proposed in order to overcome this problem [START_REF] Chunjun | Measuring Volumetric Change After Augmentation Mammaplasty Using a Three-Dimensional Scanning Technique: An Innovative Method Aesth[END_REF] . We preferred to modify the traditional method introduced by Kovacs et al. [START_REF] Kovacs | New aspects of breast volume measurement using 3dimensional surface imaging[END_REF] . First, the anatomic border of the breast is delineated. A mark is made from 10 cm below the sternal notch along the middle of the sternum to the offshoot of the medial breast fold and caudal to the inframammary fold (IMF) up to the lateral offshoot of the breast fold. The posterior side of the frontal axillary fold and the postero-lateral offshoot of the pectoral muscle up to 5 cm below the clavicle forms the lateral delimitation. The cranial demarcation is placed 10 cm caudal and parallel to the clavicle. The simulated chest wall template is constructed via ''curvature-based filling'' function after the breast region inside the anatomic border is removed. Breast volume is then computed by Boolean operation of superimposition of the scan containing the breast with the chest wall created (Fig. 2).

Data interpretation let us better understand volume variations depending on the reconstructive technique chosen.

Resorption volume percentage in simple LGD after three (8,5%) and six months (2,2%) is comparable to LGD and subfascial fat (9% and 2,9%) without any statistically significant difference. The advantage is to immediately obtain more volume by including the fatty layer in one surgical step. As the percentage of volume variation is similar in both groups we assume that the volume loss is mainly due to muscular atrophy.

Fat graft resorption is a difficult aspect to evaluate in an objective way. Several clinical evaluations reporting important volume variations (up to 80%) have been published in literature [START_REF] Ho Quoc | Volumetric evaluation of fat resorption after breast lipofilling[END_REF] . EL Fadl et al. [START_REF] El Fadl | Lipomodelling of the breast: a review[END_REF] showed a volume variation rate ranging from 40 to 60% after fat grafting. Contrastingly, Delay et al.

demonstrated through a CT study an average resorption rate of 21,5% [START_REF] Ho Quoc | Volumetric evaluation of fat resorption after breast lipofilling[END_REF] . A possible evolution to the average 30% volume loss can be taken into account, this due to the edema remission.

The resorption rate of latissimus dorsi flap fat grafting is comparable to those found in Delay et al.

study [START_REF] Ho Quoc | Volumetric evaluation of fat resorption after breast lipofilling[END_REF] . This confirms that the muscle is an optimal receiving tissue for autologous breast reconstruction. BMI measurements were considered in order to avoid an important bias given to weight variations. These data remain sufficiently stable without significant differences (Tab.7).

A detailed analysis of postoperative complications, especially for lymphocele drainages, demonstrates that there is not a significant difference in number of punctures between the two groups this, defining a similar risk in both techniques.

The real gain is given by the reduced number of fat graft interventions (Tab.1). The achievement of a supplemental immediate volume during LDF reconstruction avoids additional interventions, contributing to the patient's physical and psychological well-being.

Conclusions

This study shows no difference in volume retention at follow up between the two techniques. The gain of an immediate and stable fatty layer in LDF + subfascial fat technique leads to breast volume improvement in one surgical step.

Breast reconstruction with LDF and subfascial fat is a reliable solution which conducts to an optimal result with a reduced number of surgical reinterventions (lipofilling sessions) and follow-up.

This study provides a valuable achievement for breast surgery giving us the capability to evaluate the future resorption in order to obtain a satisfying breast result.

Figure legends

  Department performed all the interventions. Each surgeon equally performed the 2 different techniques compared in this study: 1. a latissimus dorsi flap as classically described (simple LDF); 2. a latissimus dorsi flap with preservation of fat extensions as described by Delay et al 3 (LDF + subfascial fat).

3 .

 3 The size and shape of my breasts are the same.4. My reconstructed breast(s) feel soft to the touch. 5. Overall, I am satisfied with my reconstruction.6. I would recommend this type of reconstructive procedure to a friend.The questionnaire used a 5-point Leikert scale with 1 indicating high satisfaction and 5 indicating low satisfaction. Only responses of 1 or 2 for all items in each subscale were rated as "satisfied." The questionnaire was distributed 3 months postoperatively after lipofilling sessions.

Fig. 1 Fig. 2 Fig. 3 Fig. 4

 1234 Fig. 1 Power calculation of the study Fig. 2 Study design algorithm Fig. 3 Volumetric extraction technique in post-production analysis Fig. 4 Deviation analysis showing no volume modifications (green) and volume reduction (blue) between two 3D models

Table 1 .

 1 Comparison between the two techniques

	Tables legends
	Tab. 2 Comparative volumes in LDF reconstruction at 3-months post-op
	Tab. 3 Comparative volumes in LDF reconstruction at 6-months post-op
	Tab. 4 Volume of fat graft injected
	Tab. 5 Comparative volumes in first fat grafting at 3-months post-op
	Tab. 6 Comparative volumes in second fat grafting at 3 months post-op

Table 7 .

 7 BMI analysis

Table 1 . Comparison between the two techniques LDF sec. Delay

 1

			Simple LDF	Significance level
		(15 breasts)	(15 breasts)	(P)
	Age
	Range (years)	36-65		-
	Type of reconstruction
	Immediate	6	4	-
	Delayed	9	11	-
	BMI (Kg/m 2 )
	Average	23,3	22,8	0,77
	Range	19-30	18-32
	Complications
	Cutaneous necrosis	1 (6,6%)	0	-
	Scar disunion	3 (20%)	2 (13,3%)	-
	Lymphocele	15 (100%)	14 (93,3%)	-
	N° of lymphocele drainages
	Mean weeks	7	6	0,07
	Range	5-9 weeks	4-7 weeks	-
	Follow-up duration
	(months)
	Mean	13,2	14,8	0,13
	Range	12-18	6-48	-
	N° of lipofilling sessions
	mean	1,2	1,6	0,02 *

Second fat graft infiltration (cc)

		LGD + subfascial fat volume (cc)	Volume resorption
	PPN	Post FIRST Lipofilling (immediate)	3-months post lipofilling	cc		% compared to volume injected
	1	415	392	23		19
	2	552	527	25		16,6
	3	536	488	48		20
	4	723	682	41		19,5
	5	610	558	52		20
	6	697	661	36		20
	7	743	723	20		18
	8	605	555	50		17,8
	9	754	736	18		18
	10	664	643	21		17
	11	708	652	56		26,6
	12	658	606	52		17,3
	13	727	672	55		18,9
	14	683	656	27		20,7
	15	724	705	19		19
	Mean	653	617	36,2	19,3%
		Simple LGD volume (cc)	Volume resorption
	PPN	Post FIRST Lipofilling (immediate)	3-months post lipofilling	cc		% compared to volume injected
	1	592	534	58		22,3
	2	621	572	49		19,6
	3	765	725	40		19
	4	1 738	72 708	30	80	25
	5	2 541	55 507	34	135	17,9
	6 7 8 9 10	3 529 4 773 5 708 6 708 706	80 509 -733 -690 -651 675	20 40 18 57 31	72 120 72 64	11,7 20 16,3 21 18,8
	11	Mean 716	69 672	44	90,5	18,3
	12	763	703	60		20,7
	13	795	780	15		16,6
	14	567	508	59		23
	15	770	752	18		18,9
	Mean	686	648	38,2	19,3
	P value (P<0,005) = 0,7

Tab. 6 Comparative volumes in second fat grafting at 3 months post-op

		LGD + subfascial fat volume (cc)	Volume resorption
		Post SECOND	3-months		%
	PPN	Lipofilling (immediate)	post lipofilling	cc	compared to volume injected
	1	464	418	16	22,2%
	2	582	567	15	27,2%
	3	568	551	17	21,2%
	Mean	538	512	16	23,6
		Simple LGD volume (cc)	Volume resorption
		Post SECOND	3-months		%
	PPN	Lipofilling (immediate)	post lipofilling	cc	compared to volume injected
	1	614	595	19	24%
	2	707	679	28	21%
	3	797	783	14	19%
	4	828	807	21	18%
	5	579	556	23	32%
	6	573	562	11	17%
	7	693	674	19	45%
	8	697	679	18	72%
	9	659	639	20	13%
	Mean	686	667	19	29
	P

value (P<0,005) = 0,3

Table 7 . BMI analysis LGD + subfascial fat

 7

		Time zero	6-months postoperative	3-months post first lipofilling	3-months post second	P<0,05	Kruskal-Wallis
					lipofilling
	Mean BMI	23,3	23,4	23,2	23,4	0,9	0,7
				Simple LGD
	Mean BMI	22,8	22,2	22,3	22,5	0,9	0,4

Acknlowledgements

We would like to show our gratitude to Stephanie Weber who provided technical equipment and support to our research.

Funding: None

Conflicts of interest: None declared

Ethical approval: Not required