Didier Fesquet

David Llères

Charlotte Grimaud

Cristina Viganò

Francisca Méchali

Séverine Boulon

Robert Feil

Olivier Coux

Catherine Bonne-Andrea

Véronique Baldin
email: veronique.baldin@crbm.cnrs.fr

The 20S proteasome activator PA28γ controls the compaction of chromatin

Keywords: PA28γ, Proteasome, HP1, Heterochromatin, FLIM-FRET

PA28γ, a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell proliferation, but its precise cellular functions remain unclear. Here, we show that PA28γ is crucial for chromatin compaction. In human cells, we find that a small fraction of PA28γ co-localizes with HP1β, and PA28γ is present at HP1β-containing repetitive-DNA regions. PA28γ-depletion induces a decompaction of pericentromeric heterochromatin, as observed upon HP1β-knockdown. Using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living cells, we show that PA28γ controls chromatin compaction more broadly. Importantly, HP1β on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, PA28γ is necessary to maintain the level of H3K9 tri-methylation, as well as H4K20 mono-and tri-methylation, modifications required for heterochromatin establishment.

Overall, our findings demonstrate the implication of a proteasome regulator in chromatin organization.

Introduction

Proteasome-mediated protein degradation is a central pathway that controls the stability and the function of numerous proteins in most cellular processes [START_REF] Collins | The Logic of the 26S Proteasome[END_REF].

The various functions of the proteasome are actually performed by a family of proteasome complexes resulting from the association of different regulators/activators with the catalytic core-called the 20S proteasome [START_REF] Rechsteiner | Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors[END_REF][START_REF] Coux | The Proteasome System in Health and Disease[END_REF]. Among the proteasome family, the best-characterized member is the 26S proteasome, composed of the 20S core proteasome and the 19S regulator, which degrades poly-ubiquitylated proteins in an ATP-dependent manner [START_REF] Collins | The Logic of the 26S Proteasome[END_REF][START_REF] Bard | Structure and Function of the 26S Proteasome[END_REF]. In the nucleus, two other regulators, PA200 [START_REF] Ustrell | PA200, a nuclear proteasome activator involved in DNA repair[END_REF][START_REF] Rechsteiner | Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors[END_REF] and the homoheptamer PA28γ (also known as REGγ or Ki antigen) [START_REF] Ma | Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain)[END_REF][START_REF] Wilk | Properties of the nuclear proteasome activator PA28gamma (REGgamma)[END_REF][START_REF] Mao | REGgamma, a proteasome activator and beyond?[END_REF], are present and function in an ATP-and ubiquitin-independent manner. Among their many functions, it is now well established that proteasome complexes are associated with chromatin and enriched at specific sites in the genome [START_REF] Geng | Similar temporal and spatial recruitment of native 19S and 20S proteasome subunits to transcriptionally active chromatin[END_REF][START_REF] Kito | Cell cycle-dependent localization of the proteasome to chromatin[END_REF]. Notably, they are specifically recruited during transcription or in response to DNA damage, thereby suggesting a direct role for chromatin-associated proteasome complexes in genomic processes [START_REF] Mccann | Functions of the proteasome on chromatin[END_REF].

Chromatin exists mainly in two distinct compaction states: whereas euchromatin is a relaxed state that is generally transcriptionally active, heterochromatin corresponds to a highly compacted and often repressed state, rich in repetitive sequences such as satellite repeats, transposable elements and ribosomal DNA [START_REF] Lippman | Role of transposable elements in heterochromatin and epigenetic control[END_REF][START_REF] Nishibuchi | Biochemical and structural properties of heterochromatin protein 1: understanding its role in chromatin assembly[END_REF][START_REF] Saksouk | Constitutive heterochromatin formation and transcription in mammals[END_REF][START_REF] Janssen | Heterochromatin: Guardian of the Genome[END_REF]. Heterochromatin is paramount to the stability of eukaryotic genomes. Indeed, loss of control over these repetitive DNA regions can lead to transcriptional perturbation and DNA recombination, all of which events are at the root of oncogenic transformation [START_REF] Ayarpadikannan | The impact of transposable elements in genome evolution and genetic instability and their implications in various diseases[END_REF][START_REF] Klement | DNA double strand break responses and chromatin alterations within the aging cell[END_REF].

Multiple evidence from genetic and cell biology point to an important involvement of Heterochromatin Protein-1 (HP1) [START_REF] Maison | HP1 and the dynamics of heterochromatin maintenance[END_REF][START_REF] Verschure | In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation[END_REF] and trimethylation of histone H3 lysine 9 (H3K9me3) [START_REF] Martin | The diverse functions of histone lysine methylation[END_REF][START_REF] Saksouk | Constitutive heterochromatin formation and transcription in mammals[END_REF] and of histone H4 lysine 20 (H4K20me3) [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF][START_REF] Oda | Monomethylation of histone H4-lysine 20 is involved in chromosome structure and stability and is essential for mouse development[END_REF][START_REF] Beck | PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription[END_REF][START_REF] Bosch-Presegue | Mammalian HP1 Isoforms Have Specific Roles in Heterochromatin Structure and Organization[END_REF] in establishing and maintaining heterochromatic states. Two proteins of the HP1 family [START_REF] Nielsen | Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9[END_REF][START_REF] Thiru | Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin[END_REF], HP1α and β, are recruited through their binding to H3K9me3, and participate in the folding of chromatin into higher-order structures [START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF][START_REF] Maison | HP1 and the dynamics of heterochromatin maintenance[END_REF][START_REF] Machida | Structural Basis of Heterochromatin Formation by Human HP1[END_REF]. Recent reports identify a liquid phase-like HP1 population that generates a dynamic phase transition compartment surrounding the stable chromatin-bound HP1 fraction [START_REF] Larson | Liquid droplet formation by HP1alpha suggests a role for phase separation in heterochromatin[END_REF][START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF]. Such a compartmentalization mechanism may facilitate the access of proteins and their rapid exchange, necessary for the dynamic structural changes of heterochromatin during cell cycle progression and in the DNA damage response. Thus, it has been suggested that HP1 would act as a platform that selectively favors local concentration of different proteins to fulfill their chromatin-related functions [START_REF] Grewal | Heterochromatin revisited[END_REF]. However, the mechanism by which HP1 folds H3K9me3containing chromatin into higher-order structures, and the requirement of other factors have not been fully elucidated.

The roles of the different proteasome complexes in chromatin organization remain still elusive as well. Although PA28γ-20S proteasome complexes constitute only a minor fraction of the whole proteasome population (below 5%) [START_REF] Fabre | Label-free quantitative proteomics reveals the dynamics of proteasome complexes composition and stoichiometry in a wide range of human cell lines[END_REF], PA28γ is important for cell growth and proliferation. Indeed, among the limited number of identified proteins whose degradation is mediated by PA28γ-20S proteasome complexes, many of these such as CKIs (p21, p19, p16) and Myc [START_REF] Chen | Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome[END_REF][START_REF] Li | Ubiquitin-and ATPindependent proteolytic turnover of p21 by the REGgamma-proteasome pathway[END_REF][START_REF] Li | Regulation of c-Myc protein stability by proteasome activator REGgamma[END_REF] are involved in the control of cell proliferation. Consistent with this, PA28γ-knockout mice show a decrease in body size [START_REF] Murata | Growth retardation in mice lacking the proteasome activator PA28gamma[END_REF][START_REF] Barton | Immune defects in 28-kDa proteasome activator gamma-deficient mice[END_REF]. In addition, PA28γ plays a role in intranuclear organization, since it is involved in the dynamics of various nuclear bodies, including Cajal bodies [START_REF] Cioce | UV-induced fragmentation of Cajal bodies[END_REF][START_REF] Jonik-Nowak | PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28gamma[END_REF], nuclear speckles [START_REF] Baldin | A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking[END_REF], and promyelocytic leukemia protein bodies (PML) [START_REF] Zannini | REGgamma/PA28gamma proteasome activator interacts with PML and Chk2 and affects PML nuclear bodies number[END_REF]. A potential role of PA28γ in chromatin has also been proposed, since it has been linked to chromosome stability [START_REF] Zannini | REGgamma proteasome activator is involved in the maintenance of chromosomal stability[END_REF] and DNA repair [START_REF] Levy-Barda | Involvement of the nuclear proteasome activator PA28gamma in the cellular response to DNA double-strand breaks[END_REF].

In this study, we highlight a still unsuspected function of PA28γ in the control of chromatin compaction. By studying the localization of the 20S proteasome in human cells, we found that a fraction of the 20S proteasome localizes in nuclear foci, together with its regulator PA28γ and HP1β. Subsequent investigations revealed that PA28γ is associated with repetitive DNA sequences abundant in heterochromatin, and is necessary to sustain the compaction of chromatin. This function of PA28γ, possibly independent of the 20S proteasome, depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are both involved in heterochromatin formation.

Results

A fraction of PA28γ-20S proteasome complexes co-localizes with HP1 proteins.

To investigate the subcellular location of the 20S proteasome in human cells, we established a stable U2OS cell line expressing an inducible alpha 4 (α4) protein, one of the constitutive subunits of the 20S proteasome, fused with GFP (α4-GFP). Biochemical characterization of this cell line demonstrated that the α4-GFP subunit was correctly incorporated into proteolytically active proteasomes (Fig S1A). As reported for the endogenous α4 subunit, ectopic α4-GFP was detected throughout the cell, with an accumulation in the nucleoplasm [START_REF] Britton | A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair[END_REF]. These observations suggest that α4-GFP foci are closely linked to chromatin. Combined, these findings reveal a particular nuclear localization of PA28γ-20S proteasome complexes upon ectopic expression of α4 subunit.

Visualization of α4-GFP in living U2OS cells by time-lapse video microscopy revealed that these α4-GFP nuclear foci are dynamic, as their presence fluctuates throughout the cell cycle (Movie-1). α4-GFP displayed a diffused localization during mitosis, after which it started to assemble into foci, whose number and size gradually increased during interphase.

Detection of nuclear α4-GFP foci in synchronized cells indicated that the percentage of cells with α4-GFP foci reached a peak in late-S/early-G2 phases (Fig S1C).

Next, we investigated the nature of the α4-GFP nuclear foci. Among different proteins forming nuclear foci, we focused our attention on a member of the HP1 family, HP1β. Newly synthetized HP1β was reported to also accumulate into nuclear foci with a characteristic peak in late-S/early-G2 phases when GFP-HP1β was overexpressed [START_REF] Dialynas | Methylation-independent binding to histone H3 and cell cycledependent incorporation of HP1beta into heterochromatin[END_REF]. By performing immunostaining against HP1β protein, we revealed the presence of endogenous HP1β in α4-GFP foci (Fig 1D). Immunoblot analyses of PA28γ and GFP immunoprecipitation experiments performed in U2OS-α4-GFP cell extracts showed that HP1β is present in . We then wondered whether HP1β plays a role in the formation of PA28γ-20S proteasome foci. After depletion of HP1β by siRNAs in U2OS-α4-GFP cells (Fig 1F ), we observed a significant decrease of the percentage of cells containing α4-GFP foci (∼14%) compared to the control cells (∼36%) (Fig 1F,bar graph). We also noted that PA28γ was no longer detected at identifiable nuclear foci in the absence of HP1β. Importantly, PA28γ depletion significantly decreased the percentage of cells containing α4-GFP foci (∼9.7%) (Fig 1F), suggesting that PA28γ is required for their formation. First, these results indicate that HP1β plays a role in the recruitment and/or retention of PA28γ-20S proteasome to these foci when α4 is overexpressed. Second, these data reveal that PA28γ is required to recruit the 20S proteasome to nuclear foci. Altogether, the above findings demonstrate that ectopic α4 overexpression promotes the enrichment of PA28γ-20S proteasome complexes in HP1β-containing nuclear foci.

Without α4 overexpression, a fraction of PA28γ co-localizes also with HP1β.

Immunostaining analyses of PA28γ in U2OS cells revealed only a diffuse distribution of PA28γ within the nucleus. We hypothesized that the presence of foci was due to the amplification of a natural process that most likely occurs in cells, but was only easily detectable when exacerbated by α4 overexpression. To test this hypothesis, we asked whether PA28γ co-localizes with HP1 proteins in U2OS cells without α4 ectopic expression.

Since both PA28γ and HP1β proteins are abundant in the nucleus, we pre-extracted the soluble nuclear proteins by treating the U2OS cells with 0.5%Triton X-100/PBS [START_REF] Guillot | Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells[END_REF] To strengthen this result, we used the in situ Proximity-Ligation Assay (is-PLA), which allows the detection of the close proximity between two proteins within cells (less than 40 nm, i.e. likely to be an interaction) [START_REF] Soderberg | Direct observation of individual endogenous protein complexes in situ by proximity ligation[END_REF]. Using both PA28γ and HP1β antibodies, is-PLA revealed a characteristic dotted pattern throughout the nuclei of U2OS Recently, we identified an important regulator of PA28γ, the protein PIP30 (PSME3IP1), which affects PA28γ interactions and localization in Cajal bodies [START_REF] Jonik-Nowak | PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28gamma[END_REF].

Since PIP30 was detected in α4-GFP foci, we tested whether PIP30 modulates the colocalization of PA28γ and HP1β. We found that PIP30 had no effect on this process since no difference in the number of PLA-dots between PA28γ and HP1β was observed in wild-type and PIP30-knockout (KO-PIP30) U2OS cells (Fig S2D).

Taken together, these results demonstrate that a fraction of PA28γ and the 20S proteasome is in close physical proximity (and thus is likely to interact either directly or indirectly) to a fraction of the heterochromatin-binding protein HP1β.

PA28γ controls pericentromeric heterochromatin compaction.

The co-localization of PA28γ and HP1β prompted us to investigate whether PA28γ was associated with chromatin comprising repetitive sequences characteristic of heterochromatin, such as interspersed (HERV-K), pericentromeric (Satellite II and α Satellite) and Major Satellite (L1 LINE) DNA repetitive sequences [START_REF] Padeken | Repeat DNA in genome organization and stability[END_REF]. Quantitative chromatin immunoprecipitation (ChIP-qPCR) experiments performed on parental (WT) versus KO-PA28γ U2OS cells revealed that PA28γ was associated with all four heterochromatin sequences tested (Fig 3A), as was HP1β (Fig S3). Note that PA28γ is also associated with a DNA sequence corresponding to Cyclin E2 promoter, located in euchromatin (Fig 3A).

Based on these results, we then investigated whether PA28γ could regulate heterochromatin compaction. For this purpose, we used a previously described U2OS cell clone (F42B8) carrying lacO DNA repeats stably integrated within constitutive heterochromatin, at a pericentromeric region [START_REF] Jegou | Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line[END_REF]. This lacO array forms a single heterochromatic locus that can be visualized in cells following the transient expression of the GFP-LacI construct. The GFP signal allows us to measure the area occupied by the lacO locus and thus to quantify the variations of its compaction state. We first examined the effect of HP1β depletion on lacO array compaction by transfecting these cells with siRNAs directed against HP1β (si-HP1β) or luciferase (si-Luc), and with a GFP-LacI-expressing construct. The efficiency of si-HP1β was verified by immunoblot (Fig 3B,upper panel), and changes in heterochromatin compaction state were monitored 48 hours post-transfection by fluorescence microscopy (Fig 3B,lower panel). LacO locus appeared as a small dot with a surface area that was not significantly affected by the transfection of si-Luc (0.390 ± 0.045 µm 2 vs 0.370 ± 0.052 µm 2 in control cells). Upon HP1β knockdown, we observed a significant increase of the GFP-LacI dot surface area (0.730 ± 0.069 µm 2 ). This corresponds to an expansion of the surface area occupied by the lacO DNA repeats due to heterochromatin decompaction (Fig 3C). We then examined the effect of PA28γ knockdown (Fig 3D -E). Upon PA28γ-depletion we observed a significant increase in the GFP-LacI dot surface area (0.636 ± 0.014 µm 2 vs 0.370 ± 0.052 µm 2 and 0.443 ± 0.011 µm 2 in control cells and si-Luc treated cells, respectively) (Fig 3E). Thus, as observed in absence of HP1β, the pericentromeric heterochromatin is significantly decompacted in the absence of PA28γ.

PA28γ controls chromatin compaction in living cells, independently from its interaction

with the 20S proteasome.

To complete and expand the results obtained on fixed cells and at a specific genome locus, we performed quantitative FLIM-FRET (Fluorescence Lifetime Imaging Microscopy-Förster Resonance Energy Transfer) measurements of chromatin compaction at the nanometer-scale in a single living cell. For this study, we used HeLa cells that stably co-expressed histone H2B-GFP and mCherry-H2B (HeLa H2B-2FPs ). FRET was measured between the fluorophoretagged histones incorporated into the chromatin and in this assay an increase in FRET efficiency corresponds to an increase in the occurrence of close proximity (<10 nm) between nucleosomes [START_REF] Lleres | Quantitative analysis of chromatin compaction in living cells using FLIM-FRET[END_REF].

We first confirmed the co-localization between PA28γ and HP1β proteins at some nuclear sites in HeLa cells as seen in U2OS cells, by using is-PLA (Fig S4). Then, we established a stable CRISPR/Cas9 PA28γ knockout HeLa cell line (Fig 4A), expressing either H2B-GFP alone (HeLa H2B-GFP -KO-PA28γ) or both H2B-GFP and mCherry-H2B (HeLa H2B-2FPs -KO-PA28γ). We verified that PA28γ depletion affected neither H2B-GFP nor mCherry-H2B expression levels by immunoblot and microscopy analysis (Fig S5A -B).

Next, we analyzed by FLIM-FRET microscopy the levels of nanoscale chromatin compaction in asynchronous interphase parental and KO-PA28γ cells. In wild-type (WT) HeLa H2B-2FPs cells, a heterogeneous FRET efficiency map was apparent throughout nuclei on representative images using continuous pseudocolors (Fig 4B,left panel). We found that the areas with highest FRET signal (red-orange population) decreased in left panel). This effect was confirmed by the determination of the mean FRET efficiency percentage, which shows a major reduction in the level of chromatin compaction compared to the WT cells (Fig 4B,right panel). As a positive control for chromatin decompaction, we treated HeLa H2B-2FPs cells with Trichostatin A (TSA), an inhibitor of histone deacetylases used to provoke large-scale chromatin decompaction [START_REF] Lleres | Quantitative analysis of chromatin compaction in living cells using FLIM-FRET[END_REF][START_REF] Otterstrom | Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo[END_REF].

As expected, after 24 hours of TSA treatment, the mean FRET efficiency percentage dropped drastically, consistent with a massively decompacted interphase chromatin ( show that PA28γ plays an important role in regulating the compaction of chromatin in cells, with a particular impact on the highly structured chromatin.

An important question was to determine whether the 20S proteasome is required for this function of PA28γ. For this, we used a mutant of PA28γ deleted of its last C-terminal 14 amino acids (called ΔC), which is unable to bind and therefore to activate the 20S proteasome [START_REF] Ma | PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus[END_REF][START_REF] Zannini | REGgamma proteasome activator is involved in the maintenance of chromosomal stability[END_REF]. We stably expressed this mutant in HeLa H2B-2FPs -KO-PA28γ cells (named KO/KI-ΔC) at an expression level comparable to that observed in WT cells (Fig S5E). The inability of this PA28γ mutant to bind the 20S proteasome was confirmed by co-immunoprecipitation experiments from cell extracts treated (Fig 4E) or not (Fig S5F) with the proteasome inhibitor MG132, known to increase the association between PA28γ and the 20S proteasome [START_REF] Welk | Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes[END_REF]. As shown in Fig 4E andS5F, the 20S proteasome was detected by the presence of its α4 subunit in PA28γ immunoprecipitations from HeLa H2B- 2FP WT and KO/KI-WT cell extracts, but not in KO/KI-ΔC and KO-PA28γ cells. Chromatin compaction was then analyzed by FLIM-FRET in living asynchronous cells. Surprisingly, we found that the expression of PA28γ-ΔC mutant restored the level of chromatin compaction in PA28γ-KO cells to a FRET efficiency value (24.7%) similar to the one observed in WT cells (23.03%) (Fig 4F). These results demonstrate that the compaction of chromatin requires PA28γ, but not its interaction with the 20S proteasome.

PA28γ is required for HP1 to mediate chromatin compaction.

To further characterize the PA28γ/HP1β connection, we performed siRNA-mediated Intriguingly, we noted that PA28γ-knockdown by siRNA had a stronger effect than the PA28γ-knockout. This difference can be explained by potential compensatory mechanisms developed by the PA28γ-KO cell line to maintain homeostasis. Finally, depletion of both PA28γ and HP1β had no additional effect compared to PA28γ alone. Altogether, these results demonstrate that PA28γ and HP1β control the compaction of the chromatin, and that PA28γ is required for this process to occur properly.

In order to determine the impact of chromatin decompaction induced by PA28γ depletion on physiological events, we next investigated the consequences of PA28γ depletion on the cell cycle. Analysis of cell-cycle profiles of KO-PA28γ cells by fluorescence-activated cell sorting (FACS) revealed a minor but significant increase of 10% in the number of G1phase cells, and a decrease of 7% in the number of S-phase cells relative to WT cells (Fig S6A), as previously described in PA28γ -/-MEFs [START_REF] Murata | Growth retardation in mice lacking the proteasome activator PA28gamma[END_REF]. To obtain more insight into these perturbations, we analyzed the progression of both cell lines during the cell cycle after their release from a G1/S boundary block induced by double thymidine treatment. As

PA28γ contributes to the maintenance of heterochromatin marks.

Besides the recruitment of HP1 proteins, methylations of histone H3 on lysine 9 (H3K9me) [START_REF] Maison | HP1 and the dynamics of heterochromatin maintenance[END_REF][START_REF] Do | Dynamics of the HP1beta-PCNAcontaining complexes in DNA replication and repair[END_REF][START_REF] Saksouk | Constitutive heterochromatin formation and transcription in mammals[END_REF] and histone H4 on lysine 20 (H4K20me) [START_REF] Shoaib | Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing[END_REF] have been shown to be important for chromatin compaction. Since tri-methylation of histone H4 (H4K20me3) is also an evolutionarily-conserved mark of heterochromatin [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF][START_REF] Balakrishnan | Decoding the histone H4 lysine 20 methylation mark[END_REF][START_REF] Beck | PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription[END_REF], we tested whether PA28γ regulates these heterochromatinenriched modifications. By analyzing the levels of H3K9me3 in total cell extracts by immunoblotting, no change in the steady-state level of H3K9me3 was observed in KO-PA28γ cells compared to WT cells (Fig 6A). By contrast, PA28γ depletion led to a decrease (∼20%) in the steady-state level of H4K20me3 (Fig 6B). This result was particularly interesting since it has recently been suggested that HP1β mediates a direct functional link with H4K20me3 [START_REF] Bosch-Presegue | Mammalian HP1 Isoforms Have Specific Roles in Heterochromatin Structure and Organization[END_REF]. As H4K20me3 depends on the mono-methylation of histone H4 lysine K20 (H4K20me1) [START_REF] Tardat | PR-Set7-dependent lysine methylation ensures genome replication and stability through S phase[END_REF], we also analyzed the steady-state level of H4K20me1. We observed a strong reduction of H4K20me1 (∼40%) (

Discussion

The key finding of this study is that PA28γ, known as a nuclear activator of the 20S proteasome, is an essential factor in the process of chromatin compaction.

We demonstrate that PA28γ plays a key role in this process by showing that: i) the lack of PA28γ causes the decompaction of lacO DNA repeats that are stably integrated into pericentromeric heterochromatin, as does the loss of HP1β, ii) the depletion of PA28γ induces a decompaction of the highly structured fraction of the chromatin, even in the presence of HP1 proteins, as visualized in living cells with the quantitative FLIM-FRET measurements of chromatin compaction. In line with these observations, we also found that PA28γ knockout decreases significantly the level of H3K9 and H4K20 tri-methylation, as well as monomethylation of H4K20.

Importantly, our data show that in interphase PA28γ and HP1β are both required to control the most condensed states of chromatin as defined by our experimental approaches. Whether PA28γ and HP1β act independently or in concert will need further investigation.

Nevertheless, based on our data obtained in α4-GFP expressing cells, HP1β seems to be required for the recruitment of PA28γ-20S proteasome complexes in nuclear foci. We show that a fraction of PA28γ and HP1β co-localize in normal cells, although this co-localization is difficult to detect, probably because they are located in much more dynamic and/or transient structures than when α4 is overexpressed. It is thus tempting to speculate that the formation of these foci is an early step in the chromatin condensation process.

Using cells overexpressing α4-GFP, we found that PA28γ-20S proteasome complexes gradually concentrate into foci through mid-to-late S-phase to G2-phase, which is reminiscent of the accumulation of newly-synthetized GFP-HP1β at heterochromatin foci, whose formation has been shown to require passage through S-phase [START_REF] Dialynas | Methylation-independent binding to histone H3 and cell cycledependent incorporation of HP1beta into heterochromatin[END_REF].

Interestingly, we found that the loss of PA28γ decreases the S phase duration by one hour.

Considering this point, it is tempting to hypothesize that, following the progression of replication forks, PA28γ might regulate the re-establishment of modifications on the newly incorporated histones on each of the daughter DNA strands. To support this hypothesis, in the absence of PA28γ, a significant decrease of H3K9me3 and H4K20me3 marks was observed by ChIP-qPCR experiments at specific heterochromatin regions. Furthermore, an important decrease in the steady-state level of H4K20me1 was found. In addition, the longer G1-phase observed upon the loss of PA28γ is a cell cycle effect comparable to the G1-to-S phase transition delay reported upon a drop in H4K20me levels [START_REF] Evertts | H4K20 methylation regulates quiescence and chromatin compaction[END_REF] or upon the depletion of the methyltransferase PR-Set7 [START_REF] Liao | Histone methyltransferase KMT5A gene modulates oncogenesis and lipid metabolism of papillary thyroid cancer in vitro[END_REF]. Altogether these data argue for a possible role of PA28γ in the regulation of these histone marks. At this stage it is important to note that the 20S proteasome, which bears the proteolytic activity, is not necessary for PA28γ-mediated chromatin compaction; this precludes any direct regulation of this process by a PA28γ-dependent proteolysis event. PA28γ could potentiate the function of the lysine methyltransferases Suv39h, PR-Set7 or Suv4-20h, which are responsible for H3K9 trimethylation, H4K20 mono-methylation and H4K20 tri-methylation, respectively. It cannot be excluded that PA28γ could facilitate the action of specific histone deacetylases (HDACs) [START_REF] Almouzni | Maintenance of Epigenetic Information[END_REF] and/or the recruitment of chromatin remodeling factors.

Interestingly, recent studies show that HP1 proteins have the capacity to form liquid-like droplets resulting from a liquid-liquid phase separation (LLPS) mechanism [START_REF] Larson | Liquid droplet formation by HP1alpha suggests a role for phase separation in heterochromatin[END_REF][START_REF] Strom | Phase separation drives heterochromatin domain formation[END_REF]. This property could facilitate the enrichment of transient supramolecular complexes/machineries that could rapidly assemble and disassemble, and exchange their components. This plasticity in their composition allows them to efficiently respond to the structural changes of heterochromatin, and might thus stimulate PA28γ's interactions with other proteins required for controlling chromatin compaction. In this context, it is important to underline that PA28γ is involved in the dynamics of various membraneless nuclear bodies [START_REF] Cioce | UV-induced fragmentation of Cajal bodies[END_REF][START_REF] Baldin | A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking[END_REF][START_REF] Zannini | REGgamma/PA28gamma proteasome activator interacts with PML and Chk2 and affects PML nuclear bodies number[END_REF][START_REF] Jonik-Nowak | PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28gamma[END_REF], also considered to be liquid-like protein droplet organelles [START_REF] Erdel | Formation of Chromatin Subcompartments by Phase Separation[END_REF][START_REF] Sawyer | Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing[END_REF].

The reason behind the recruitment of 20S proteasome into foci is still an intriguing open question, since its interaction with PA28γ is not required for PA28γ-mediated chromatin compaction. To explain the 20S proteasome recruitment, one possibility is that the catalytic activity of PA28γ-20S proteasome complexes is involved in specific steps of chromatin compaction, such as the control of heterochromatin spreading, as it was shown for the 26S proteasome in yeast [START_REF] Seo | The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast[END_REF]. Another possibility, in line with a possible link with LLPS droplet organelle formation [START_REF] Sawyer | Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing[END_REF], is that the recruitment of the 20S proteasome by PA28γ could constitute a reservoir of active proteasome that can rapidly be mobilized upon cellular stress. In the cellular response to DNA double-strand breaks (DSBs), PA28γ is required for the rapid mobilisation of the 20S proteasome at DNA damage sites and contributes to the repair-pathway choice (Levy- [START_REF] Levy-Barda | Involvement of the nuclear proteasome activator PA28gamma in the cellular response to DNA double-strand breaks[END_REF]. In this regard, HP1β was also shown to be recruited at DSB sites and to favor the initiation of DNA damage response (DDR) by promoting chromatin changes [START_REF] Ayoub | HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response[END_REF][START_REF] Luijsterburg | Heterochromatin protein 1 is recruited to various types of DNA damage[END_REF][START_REF] Bartova | Function of heterochromatin protein 1 during DNA repair[END_REF], it would therefore be interesting to investigate whether the mobilization of PA28γ to DNA damage sites is also dependent on the presence of HP1β.

It is noteworthy that, over the years, PA28γ has been implicated in several chromatinrelated processes, such as maintenance of chromosomal stability [START_REF] Zannini | REGgamma proteasome activator is involved in the maintenance of chromosomal stability[END_REF], DNA repair (Levy- [START_REF] Levy-Barda | Involvement of the nuclear proteasome activator PA28gamma in the cellular response to DNA double-strand breaks[END_REF] and control of rDNA transcription [START_REF] Sun | Regulation of energy homeostasis by the ubiquitinindependent REGgamma proteasome[END_REF]. Our present observations suggest that the role of PA28γ in the regulation of chromatin compaction could be the common mechanism that links these processes to PA28γ. Although much remains to be understood regarding the biological functions of PA28γ in this process, our data reveal that PA28γ is a crucial factor in the regulation of chromatin compaction and this discovery undoubtedly opens new perspectives for a deeper understanding of the proteasome functions and the complex mechanisms that control chromatin organization, particularly during cell cycle.

Materials and methods

Plasmids

A human cDNA encoding the full length of 20S proteasome subunit α4 (PSMA7), with or without a final stop codon, was PCR amplified from a human fibroblast cDNA library and inserted into pcDNA 3 or pML1-EGFP [START_REF] Baldin | A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking[END_REF][START_REF] Farras | JunB breakdown in mid-/late G2 is required for down-regulation of cyclin A2 levels and proper mitosis[END_REF][START_REF] Feuvre | High yield bacterial expression and purification of active recombinant PA28alphabeta complex[END_REF]. The cDNA encoding α4-EGFP was then inserted into the pTRE2 vector [START_REF] Kowarz | Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines[END_REF]. The resulting vector was co-transfected with pCMV(CAT)T7-SB100 (gift from Zsuzsanna Izsvak, Addgene plasmid #34879) into recipients cells, and puromycin-resistant single colonies were selected for re-expression of PA28γ WT or ΔC proteins. pEGF-LacI [START_REF] Jegou | Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line[END_REF] was a generous gift from Prof. K. Rippe (DKFZ, Heidelberg, Germany).

Antibodies

The following antibodies were used at 1:1000 dilution for immunoblotting and 1-3 µg/ml for immunoprecipitation: anti-PA28γ (rabbit polyclonal BML-PW8190), anti-α4 (1:2000), anti- Signaling Technology); anti-α -tubulin (mouse monoclonal, T9026, Sigma-Aldrich, 1:6,000).

Fluorescent secondary antibodies conjugated either to Alexa Fluor 488, 594 and 680

(1:1,000), or to DyLight 680 and 800 (1:10,000) were purchased from Thermo Fisher Scientific. Secondary antibodies conjugated to HRP were purchased from Bio-Rad SA

(1:10,000). GFP-TRAP-A® beads were from ChromoTek.

Cell culture, transfections, cell synchronization and FACS analysis

HeLa (CCL-2) and U2OS (HTB-96) cells, obtained from ATCC, were grown in DMEM (Lonza) containing 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (Biowest), 2 mM glutamine, 100 U/ml penicillin and 10 µg/ml streptomycin (Lonza). A U2OS Tet-Off stable inducible cell line expressing α4-GFP was established as previously described for α7-GFP [START_REF] Baldin | A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking[END_REF], and cultured in medium supplemented with 250 µg/ml G418 (Sigma), 200 µg/ml hygromycin B (Calbiochem) and 2 µg/ml tetracycline (Sigma-Aldrich) to inhibit the expression of the exogenous protein. Single clones were then expanded and analyzed by western blotting using GFP or α4 antibodies. After cell washing with PBS (4 times), α4-GFP expression was induced for 24 to 36 hours in fresh medium, in the absence of tetracycline. For positive clones, the functionality of α4-GFP was controlled by testing its incorporation into the 20S, and the activity of these chimeric 20S proteasomes in a proteasome activity assay (see Appendix Fig S1 B-D). U2OS-LacO (F42B8) cells (a generous gift of Prof. K. Rippe, DKFZ, Heidelberg, Germany) were grown in the same media as U2OS but containing G418 (500 µg/ml) [START_REF] Jegou | Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line[END_REF]. Establishment and characterization of parental HeLa H2B-GFP and HeLa H2B-2FPs (H2B-GFP and mCherry-H2B) cell lines were previously described [START_REF] Lleres | Quantitative analysis of chromatin compaction in living cells using FLIM-FRET[END_REF]. Of note: after thawing, cells were cultured for one week before seeding, for all experiments.

For transient PA28γ and HP1β knockdown experiments, U2OS and/or HeLa (H2B-GFP or 2FPs) cells were transfected with 20 nM of Luciferase targeting siRNA (si-Luc, 5'-CGTACGCGGAATACTTCGA-3') used as negative control, or -PA28γ (PSME3), and -HP1β (CBX1) targeting siRNA (si-PA28γ: 5'-GAAUCAAUAUGUCACUCUA-3'; si-HP1β:

5'-AGGAAUAUGUGGUGGAAAA-3') purchased from Eurofins Genomics, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and examined after 2 days. When indicated, cells were transiently transfected with 0.5 µg/ml DNA using JetPEI TM (Ozyme), according to the manufacturer's instructions and analyzed after one day. Stable U2OS (Jonik-permeabilized prior the fixation with 0.5% Triton X-100 in PBS for 5 min at 4°C for the colocalization between endogenous PA28γ and HP1β in U2OS cells.

Images and Z-stack images were acquired with 63X/1.32 NA or 100X/1.4 NA oil immersion objective lenses using a DM 6000 microscope (Leica). Microphotographs were taken with a 12-bit CoolSnap HQ2 camera. Images were acquired as TIFF files using MetaMorph imaging software (Molecular Devices). For quantitative analyses of PLA dots, Z-stacks were acquired every 0.3 µm (Z step) with a range of 6-7.5 µm. For endogenous detection, images (as a Z stack, slices every 200 nm) were also acquired on a Zeiss LSM 880 point scanning confocal microscope equipped with a 63x Plan-Apochromat 1.4NA oil immersion objective (Zeiss) and using the 488 nm and 561 nm laser lines with the Airyscan detector. The Zeiss Zen black software was used to process the Airyscan raw Images. Co-localization in 3D, between PA28γ and HP1β, was analyzed using the Imaris (Bitplane) co-localization module.

The number of PLA-dots and the size of GFP-LacI dots were detected with ImageJ (1.49v).

FLIM-FRET analysis

FLIM-FRET experiments were performed in HeLa cells stably expressing H2B-GFP alone (HeLa H2B-GFP ) or with mCherry-tagged histone H2B (HeLa H2B-2FPs ). 5 x 10 4 cells were seeded in a Fluorodish 35 (FD35-100, World Precision Instruments). For siRNA experiments, 24 hours after seeding, cells were transfected with 20 nM of siRNA (against Luciferase, PA28γ or

Time-lapse video microscopy

U2OS-α4-GFP cells seeded on a 6-well plate were induced to expressed α4-GFP 24 hours previously, and imaged on an Olympus IX83 microscope with a 40x objective and equipped with an Andor Zyla 4.2 sCMOS camera [START_REF] Van Dijk | Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation[END_REF]. Both systems were driven by MetaMorph software. Image were then processed with the imageJ package and saved in Avi Format Schneider, C.A., [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Immunoprecipitation and immunoblotting

For immunoprecipitation of GFP-fusion or endogenous proteins, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1% IGEPAL CA630, 0.5% DOC, 0.1% SDS, 1 mM DTT, 5 mM EDTA, 50 mM NaF and 1 mM Na 3 VO 4 ) in the presence of complete EDTA-free protease inhibitor cocktail (Roche Life Science) for 20 min at 4°C.

Lysates were clarified by centrifugation for 10 min at 10,000 x g and the protein concentration of the supernatant was determined using BSA as a standard (CooAssay protein dosage reagent, Interchim). Total lysate (200 µg) was pre-cleared for 30 min, and immunoprecipitations were performed using either GFP-TRAP beads or the antibodies indicated and protein A or G magnetic beads (Dynal, Lake Success, NY) for 2 hours at 4°C

with constant gentle stirring. After several washes, bead pellets were boiled in 2x Laemmli buffer, separated by SDS-PAGE, and subjected to immunoblotting. Note that proteasome activity assay, cells were homogenized in a modified lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM ATP, 1 mM DTT, 10% Glycerol, 0.5% IGEPAL CA630, 0.3% Triton X-100) in the same.

ChIP-qPCR

ChIP experiments with U2OS were performed as described previously [START_REF] Brustel | Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication[END_REF].

Briefly, cells were fixed with 1% formaldehyde (10 min) and quenching was performed with 125 mM Glycine. After a PBS wash, cells were resuspended in buffer A (10 mM Tris-HCl pH 8, 10 mM KCl, 0.25% Triton X-100, 1 mM EDTA, 0.5 mM EGTA) for 5 min on ice.

After centrifugation, nuclei were extracted with buffer B (10 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA for 10 minutes on ice. To extract chromatin, nuclei were resuspended in Lysis buffer (10 mM Tris-HCl pH 8, 140 mM NaCl, 0.1% SDS, 0.5% Triton X-100, 0.05% Na-Deoxycholate, 1 mM EDTA, 0.5 mM EGTA). After sonication with EpiShear probe sonicator (Active Motif) to obtain chromatin fragments less than 800 bp, ChIP was performed with 15-30 µg of sheared chromatin incubated with protein A magnetic beads (Invitrogen, Thermo Fisher) coupled with the appropriate antibody, as follows: anti- A. Stable asynchronously growing U2OS (Tet-Off) α4-GFP cells were induced for the expression of GFP-tagged α4 subunit of the 20S proteasome (α4-GFP, green) for 24 hours in the absence of tetracycline, then fixed and stained with DAPI (blue). Scale bar, 10 µm.

B. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were immunostained with antibody raised against alpha 6 subunit of the 20S proteasome (α6, left panel), the regulatory complex PA28γ (PA28γ, middle panel) and a subunit of the 19S regulatory complex (Rpt6, right panel), all in grey. Scale bar, 10 µm.

C. α4-GFP foci are associated with chromatin. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were not treated (-CSK buffer) or treated (+ CSK buffer) with an extraction CSK buffer, then fixed and immunostained with anti-PA28γ antibodies (red). Scale bar, 5 µm.

D.

Induced U2OS-α4-GFP cells (α4-GFP, green) were immunostained with anti-HP1β antibodies (grey). A merge image of GFP and HP1β signal is shown. Scale bar, 10 µm.

Arrows indicate sites of co-localization between HP1β and α4-GFP.

E.

Co-immunoprecipitation of PA28γ and HP1β in asynchronous induced U2OS-α4-GFP cells. Induced U2OS-α4-GFP cells, as in A, were lysed and subjected to pull-down with either an antibody raised against PA28γ or GFP-TRAP, or the appropriate isotype control (CTL). An immunoblot of the pull-down (IP) and supernatant (SN) from whole-cell extracts (WCE) was probed with the antibodies indicated.

F. U2OS-α4-GFP cells were transfected with si-Luc, si-PA28γ or si-HP1β. One day later, the expression of α4-GFP was induced, and cells were recovered 48 hours after siRNA treatment.

Immunostaining was performed to detect PA28γ (red) and HP1β (magenta) in cells treated 3 0.9 0.8 0.9 0.9 0.7 0.8 1 0.2 0.2 0.2 0.2 1.1 1.7 1.2 1 0.2 0.2 0.2 0.4 1.3 2 1 1 2.9 2.1 2 1.3 0.6 0.3 0.6 1 2.5 2.1 1.9 0.9 0.8 0.

(

  Fig 1A). However, it was also present in nuclear foci of a fraction of asynchronously growing cells (∼ 25-30%) (Fig 1A-B). A similar location to nuclear foci was observed in U2OS cells expressing an ectopic untagged α4 subunit, thus excluding a potential mislocalization due to the fusion with the GFP moiety (Fig S1B). Co-immunostaining experiments showed that other subunits of the 20S proteasome, such as α6, were detected in α4-GFP foci as well (Fig 1B), reflecting the presence of the entire 20S proteasome. In addition to the 20S proteasome, we detected its PA28γ regulator (Fig 1B), but not the 19S complex, as shown by the absence of accumulation of the 19S subunit Rpt6 within α4-GFP foci (Fig 1B). Interestingly, PA28γ-containing α4-GFP foci were resistant to an extractionfixation buffer treatment (Fig 1C), used to reveal chromatin-associated proteins by immunofluorescence

  , and performed immunostaining of HP1β and PA28γ proteins. Analysis of widefield microscope images (Figs 2A and S2A) showed a potential co-localization between HP1β and PA28γ in some discrete areas of the nucleus (Fig 2A, merge image and higher magnifications). Z-stack acquisition on an Airyscan confocal microscope (Fig 2B, left panel), followed by image analysis with Imaris software module, confirmed the co-localization between both proteins in U2OS cells (∼32 co-localization sites per nucleus) (Fig 2B, right panel).

  cells (Fig 2C, upper middle panel), strongly supporting the close proximity of the two proteins. Silencing of PA28γ expression with siRNAs (Fig 2C, left panel), used as a negative control, abolished the PLA dots, demonstrating the specificity of the PLA signal (Fig 2C, lower middle panel). Quantification of the number of PLA dots per nucleus (see Materials and methods) indicated on average 37 and 1.4 dots per nucleus, in the presence and absence of PA28γ, respectively (Fig 2C, bar graph). We also evidenced that PA28γ co-localizes in part with HP1α (Fig S2B). Finally, we also found a close proximity between the 20S proteasome and HP1β by is-PLA, using antibodies against the α4 subunit of the 20S proteasome (Fig 2D).

  Fig S5C). By extracting the FRET efficiency distribution curves related to the FRET efficiency map of individual nuclei in both WT and KO-PA28γ cell lines, we observed upon loss of PA28γ a marked reduction of the high FRET population corresponding to high levels of chromatin compaction (Fig 4C, black curve vs blue curve). In contrast, the low-FRET population corresponding to chromatin regions with the lowest degree of chromatin compaction remained poorly affected. Therefore, PA28γ seems to impact preferentially, if not exclusively, the most condensed chromatin domains during interphase. To ascertain that the relaxation of the chromatin was due to the absence of PA28γ in the above experiments, we re-expressed PA28γ in PA28γ-KO cells (two different clones named KO/KI-WT#6 and # 8 were selected) at a level comparable to that of the endogenous protein (Fig S5D). Remarkably, for KO/KI-WT#6 and # 8 clones, the FRET efficiency was restored to values similar to WT cells (Fig 4D) indicating the re-establishment of normal chromatin compaction. Thus, these results

  depletion of both PA28γ and HP1β proteins (Fig 5A), and analyzed the effects on chromatin compaction levels by FLIM-FRET. FRET measurements revealed a marked decompaction of chromatin upon PA28γ-knockdown that was even stronger than upon HP1β depletion (Fig 5B). This decompaction was correlated with the clear disappearance of the most compacted states of the chromatin within nuclei (Fig 5C, left panel). To complete these data, the quantitative analysis of the FRET distribution profiles revealed a less pronounced decompaction of the chromatin with still some remaining high-FRET values upon si-HP1β depletion than after si-PA28γ depletion (Fig 5C right panel, compare red and black curves). Most likely the lower effect of HP1β knockdown can be explained by the presence of HP1α and/or by the redundancy of the HP1 isoforms in regulating chromatin compaction (Bosch-Presegue et al., 2017). Importantly, these results indicate that even in the presence of HP1 proteins the lack of PA28γ results into a strong decompaction of chromatin. We noted that the transfection of the siRNA-Luc only (blue curve) caused an increase in the high-FRET population (Fig 5C right panel) as compared to parental cells (Fig 4C right panel).

  shown in Fig S6B, the depletion of PA28γ in U2OS cells caused an early exit from S phase (left panel) correlated to an early entrance in G2 phase as compared to the parental U2OS cells (right panel). This significant shortening of S phase (≈ 1 hour) in KO-PA28γ cells was also confirmed by an immunoblot using cell cycle markers including Cyclin E and the phosphorylation of histone H3 on serine 10 (Fig S6C). Together, these results indicate that the duration of S phase is reduced in the absence of PA28γ, suggesting that the chromatin decompaction induced by the lack of PA28γ could facilitate the accessibility to DNA for the replication machinery.

  Fig 6B). To examine the variation of H3K9me3 and H4K20me3 at specific heterochromatin sequences, we carried out ChIP assays on parental (WT) and KO-PA28γ U2OS cells using antibodies against H3K9me3 and H4K20me3, and performed qPCR using the same primers as in Fig 3A. We observed a significant decrease in H3K9me3 precipitation levels at these specific sequences (Fig 6C) that was not detected by immunoblot analyses on total cell extract (Fig 6A). Note that the difference observed between immunoblot and ChIP assay could result from the use of different antibodies in the two experiments and/or be due to the highly enrichment of H3K9me3 in pericentric heterochromatin. We confirmed the decrease of H4K20me3 (≥ 60%) in KO-PA28γ versus WT U2OS cells (Fig 6D). As expected, this effect was stronger than the one found in total extract. As summarized in Fig 6E, our data suggest that PA28γ participates to the regulation of histones H3K9 and H4K20 methylation states that are required for heterochromatin establishment.

α6 ( 1

 1 :2000), and anti-Rpt6/Sug1 (mouse monoclonal, BML-PW8120, BML-PW8100 and BML-PW9265, ENZO Life Sciences, respectively); anti-PA28γ (mouse monoclonal, 611180, BD Transduction); anti-HP1α (rabbit polyclonal, 2616S, Cell Signaling); anti-HP1β (rabbit monoclonal (D2F2), 8676S, Cell Signaling, mouse monoclonal (1MOD-1A9) 39979, Active Motif); anti-MCM7 (monoclonal, sc-9966, Santa-Cruz); anti-PIP30 (PSME3IP1) (Jonik-Nowak et al., 2018); anti-GFP (mouse monoclonal (Clone 7.1), 11814460001, ROCHE, Sigma); anti-RFP (rat monoclonal, 5F8, Chromotek); anti-β-actin (rabbit monoclonal, 13E5, Cell Signaling); anti-H3K9me3 (mouse monoclonal (clone 2AG-6F12-H4) 39285, Active Motif); anti-H3 (rabbit polyclonal, ab1791, Abcam); anti-H4K20me1 (rabbit polyclonal, #9724, Cell Signaling Technology); anti-H4K20me3 (rabbit monoclonal, #5737, Cell

  Specific macros were created to automatically quantify these different parameters. The script allows the creation of a mask of DAPI image to isolate the nucleus of each cell and create a maximum intensity projection (MIP) of the Z-stacks or the image. The mask is used in the MIP to count the number of PLA-dots of each nucleus via an appropriate threshold. The "Analyze Particles" tool of ImageJ was used to calculate the size of each GFP-LacI dots. FLIM-FRET Microscopy. FLIM-FRET data were acquired with a Zeiss LSM 780 laser scanning microscope coupled to a 2-photon Ti:Saphire laser (Chameleon Ultra II tunable 680-1080 nm, Coherent) producing 150-femtosecond pulses at 80 MHz repetition rate and a Time Correlated Single Photon Counting (TCSPC) electronics (SPC-830; Becker & Hickl GmbH) for time-resolved detection. Enhanced green fluorescent protein (EGFP) and mCherry fluorophores were used as a FRET pair. The two-photon excitation laser was tuned to 890 nm for selective excitation of the donor fluorophore. The LSM780 microscope is equipped with temperature-and CO 2controlled environmental black wall chamber. Measurements were acquired in live cells at 37°C, 5% CO 2 with 63x/1.4 oil Plan-Apochromat objective lens. A short-pass 760-nm dichroic mirror was used to separate the fluorescence signal from the laser light. Enhanced detection of the emitted photons was afforded by the use of the HPM-100 module (Hamamatsu R10467-40 GaAsP hybrid PMT tube). The FLIM data were processed using SPCimage software (Becker & Hickl GmbH).

  HP1β) and FLIM-FRET experiments were performed 48 hours later. 30 min prior to imaging, the culture medium was changed to complete DMEM medium without phenol red. An acquisition time of 90 s was set up for each FLIM experiment. The analysis of the FLIM measurements was performed by using SPCImage software(Becker & Hickl, GmbH).Because FRET interactions cause a decrease in the fluorescence lifetime of the donor molecules (EGFP), the FRET efficiency was calculated by comparing the FLIM values obtained for the EGFP donor fluorophores in the presence (HeLaH2B-2FPs ) and absence (HeLa H2B-GFP ) of the mCherry acceptor fluorophores. FRET efficiency (E FRET) was derived by applying the following equation: E FRET = 1-(τDA / τD) at each pixel in a selected region of interest (nucleus) using SPCImage software. τDA is the mean fluorescence lifetime of the donor (H2B-EGFP) in the presence of the acceptor mCherry-H2B in HeLa H2B-2FPs cells and τD is the mean fluorescence lifetime of H2B-EGFP (in the absence of acceptor) in HeLa H2B-GFP cells. The FRET distribution curves from nuclei were displayed from the extracted associated matrix using SPCImage and then normalized and graphically represented using Microsoft Excel and GraphPad Prism software. For each experiment, FLIM was performed on multiple cells from several independent experiments (see figure legends).

H3pan ( 1

 1 µl/ChIP, C15310135 Diagenode), anti-H3K9me3 (2 µl/ChIP, C15410056 Diagenode), anti-H4K20me3 (2 µl/ChIP, C15410207, Diagenode), anti-PA28γ (0.5 µl /ChIP, ENZO Life Sciences). ChIP experiments were performed at least three times from independent chromatin preparations and quantitative PCR analyses of ChIP DNAs were performed using a SYBR green quantitative PCR kit (Invitrogen, Thermo Fisher) and a LightCycler 480 II instrument (Roche) under conditions standardized for each primer set. The amount of DNA in ChIP samples was extrapolated from standard curve analysis of chromatin DNA before immunoprecipitation (input), and values were represented as the ratio between the percentage of input obtained for each antibody to the ones obtained for H3. Primer sets used for qPCR: HERV-K For 5'-TGCCAAACCTGAGGA AGAAGGGAT-3' and HERV-K sur le Cancer (SFI20111203984, to SB/ PJA20181207962, to DL), Institut National du Cancer (INCa) (PLBIO18-KiMec, to RF).

Figure legends Figure 1 .

 legends1 Figure legendsFigure 1. PA28γ and HP1β co-localize in α4-GFP-20S proteasome foci.

  with the siRNA indicated. Representative images are shown (left). Arrows indicate cells with α4-GFP and PA28γ foci. Scale bar, 10 µm. The percentage of cells with α4-GFP foci is shown in the bar graph (right). Error bars derived from 3 independent experiments represent the mean ± SD, n ≥ 47 cells per condition. One-way ANOVA analysis, p = 0.0001 (****) for siRNA-PA28γ and -HP1β versus WT.

Figure 2 .

 2 Figure 2. Endogenous HP1β, PA28 and the 20S proteasome co-localize in U2OS cells. A. Nuclear localization of endogenous HP1β (left panel) and PA28γ (middle panel) by immunofluorescence in asynchronous U2OS cells after pre-permeabilization with 0.5% Triton

Figure 4 .

 4 Figure 4. PA28γ controls chromatin compaction, independently of its interaction with the 20S proteasome. A. Immunoblot analysis of PA28γ expression level in total extracts from parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B-2FPs cells. Tubulin was used as a loading control. B. FRET analysis in asynchronous parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B- FPs cells. FLIM-FRET measurements were performed and the spatial distribution of the FRET efficiency is represented in a continuous pseudo-color scale ranging from 0 to 30 % (left panel). Scale bars, 10 µm. Right panel, statistical analysis of the mean FRET efficiency percentage in WT and KO-PA28γ HeLa H2B-2FPs nuclei, presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. Data represent the means ± SD from 4-6 independent experiments, the total number of analyzed cells is n = 154 nuclei (WT) and n = 132 nuclei (KO-PA28γ), **** p < 0.0001 (Student's T-test). C. Spatial distribution of the FRET efficiency (percentage) in representative WT and KO-PA28γ HeLa H2B-2FPs nuclei. The FRET percentage distribution is depicted in a continuous pseudo-color scale ranging from 0 to 30% (left panel). Scale bars, 10 µm. FRET distribution graph shows distinct populations of FRET efficiency in WT and KO-PA28γ cells (blue and black curves, respectively) (right panel). D. Spatial distribution of the FRET efficiency (percentage) in representative WT, KO-PA28γ and KO/KI-WT #6, KO/KI-WT #8 HeLa H2B-2FPs nuclei. The FRET percentage distribution is depicted as in C. Scale bars, 10 µm. Quantification of the mean FRET efficiency was represented as box-and-whisker plots. Data represent the means ± SD from 3 independent experiments, the total number of analyzed cells is n = 102 nuclei (WT), n = 90 (KO-PA28γ), n = 53 (KO/KI-WT #6), n = 54 (KO/KI-WT #8). n.s not significant, **** p <0.0001 (Student's T-test).E.Cell extracts from parental HeLa H2B-FPs (WT), PA28γ-knockout (KO-PA28γ) cells and KO cells re-expressing the wild-type form (KO/KI-WT) or the ΔC-mutant (KO/KI-ΔC) of PA28γ, treated for 2 hours with 25 µM of MG132, were subjected to immunoprecipitation using anti-

Figure S6 .

 S6 Figure S6. PA28γ depletion decreases the S phase duration. A. Asynchronous parental (WT) and KO-PA28γ U2OS cells were fixed and stained with propidium iodide and then subjected to flow cytometry analysis. The histogram presents the repartition of the cells in the G1, S, and G2/M phases of the cell cycle. Data represent the means ± SD from four independent biological repeats. p-values were determined by 2-way ANOVA test, ns = not significant (p = 0.2407), *p < 0.05 (p = 0.0312) and ***p < 0.001 (p = 0.0006). B. Asynchronous parental U2OS and U2OS-KO-PA28γ cells, synchronized at the G1/S phase transition by a double thymidine block, then were released for the times indicated and subjected to FACS analysis. Histograms representing the percentage of the cells in S (left panel) and G2/M (right panel) phases of the cell cycle are shown. Data represent the means ± SD from three biological repeats. p values were determined with a 2-way ANOVA for each time point. p-values are presented in Table4.C.Total cell extracts of WT-or -KO-PA28γ U2OS cells, asynchronous (AS) and synchronized at the G1/S phase transition by a double thymidine block and then release procedure were analyzed by immunoblot using the antibodies indicated. β-actin was used as a loading control.

Table 4 :

 4 p values of the figure S6 (determined with the 2-way ANOVA) !

			!
		T-test (H3K9me3/H3)!	T-test (H4K20me3/H3)!
		U2OS vs KO PA28γ	U2OS vs KO PA28γ
	HERV-K!	6,47871E-06!	0,009044059!
	α sat!	0,00014585!	0,001004765!
	Sat II!	8,6538E-06	1,37015E-05
	L1 LINE!	2,80855E-05!	0,002330735!

Table 1 :

 1 p values of the figure 6C-D (ChIP)!

		T-test (si-Luc vs si-HP1β)!
	HERV-K!	0,057078569!
	Sat II!	0,066693611!
	α-sat	0,005250998!
	L1 LINE!	0,000382203!

Table 3 :

 3 p values of the figure S3 (ChIP)!Table 2: p values of the figure S1C !

	AS	HU	R4h	R6h	R8h	R10h	R11h
	AS		0,0011	0,0099	0,0723	0,0264	0,0047	0,0127
	HU			0,1957	0,0017	0,0013	0,7166	>0.9999
	R4h				0,0938	0,0124	0,827	0,6832
	R6h					0,0286	0,021	0,021
	R8h						0,0011	0,0041
	R10h							0,4511
	R11h

Acknowledgements

We thank Prof. Karsten Rippe (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for providing U2OS-LacO (F42B8) cell and the pEGFP-LacI vector, Dr. Philippe Fort for help with statistical analysis, Dr. Nathalie Morin for Airyscan acquisition and helpful discussions and Dr. Eric Julien for helpful discussions; and the Montpellier Ressources Imagerie (MRI) platform, a member of the National Infrastructure France-BioImaging supported by the French National Agency (ANR-10-INSB-04, Investments for the Future).

We thank Dr. James Hutchins for checking the scientific English.

Funding

Institutional support was provided by the Centre National de la Recherche Scientifique (CNRS) and the University of Montpellier. This work was also supported by grants from the People Programme (Marie Curie Actions) of the EU Seventh Framework Programme (FP7 REA agreement 290257, UPStream, to OC), Comité de l'Aude et Comité du Gard de la Ligue Nationale Contre le Cancer (2014 and 37-2015, to VB), Fondation ARC pour la Recherche

Author contribution

VB conceived the project and supervised the study. DF, DL, CG, CV, SB, CBA, and VB performed experiments and analyzed data. FM generated reagents. OC and RF provided conceptual advice on study and interpretation of the data. CBA and VB wrote the article with input from all of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

SUPPLEMENTARY DATA

Supplementary Methods

Proteasome Activity Assay

Proteasome peptidase activity was measured using black flat-bottom 96-well plates (Nunc) by incubating beads of immunopurified proteasome in 50 µl of activity buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM ATP, 1 mM DTT, 10% Glycerol) containing 100 µM suc-LLVY-AMC, in the presence or absence of 25 µM MG132 (Enzo Life Science), for 20 min at 37°C. Proteasome activity was determined by the detection of the free AMC fluorescence using a FLx800 microplate fluorescence reader (excitation 380 nm, emission 440 nm, Bio-Tek Instruments). p-values are presented in Table 3. D. Immunoblot analysis of PA28γ expression level in total extracts from parental (WT), PA28γ-knockout (KO-PA28γ) HeLa H2B-2FPs cells and two independent clones of HeLa H2B-2FPs cells knocked out for PA28γ in which wild-type PA28γ was stably re-expressed (KO/KI-WT #6, KO/KI-WT #8). Tubulin was used as a loading control.

Figure Supplementary

E. Whole-cell extracts from parental HeLa H2B-2FPs (WT), PA28γ-knockout (KO-PA28γ) cells and KO cells re-expressing the wild-type (KO/KI-WT#8) form or the ΔC-mutant (KO/KI-ΔC) of PA28γ. Cells were treated or not for 2 hours with MG132 (25 µM), and whole cell extracts analyzed by immunoblot using the antibodies indicated.