Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.


Introduction
Promyelocytic Leukemia Nuclear Bodies (PML NBs) are membrane-less organelles, also called biomolecular condensates (Banani et al. 2017), that concentrate proteins at discrete sites within the nucleoplasm thus participating in the spatio-temporal control of biochemical reactions (Lallemand-Breitenbach and de Thé 2018; Corpet et al. 2020;Li et al. 2020).PML NBs are 0.1-1µm diameter hollow sphere structures that vary in size and number depending on cell type, cell-cycle phase, or physiological state, highlighting their stress-responsive nature.The tumor-suppressor PML protein is the primary scaffold of PML NBs and forms an outer shell, together with the SP100 nuclear antigen, surrounding an inner core of dozens of proteins that localize constitutively or transiently in PML NBs.PML (also known as TRIM19) is a member of the tripartite motif (TRIM)-containing protein superfamily characterized by a conserved N-terminal RBCC motif essential for PML polymerization.Several isoforms of PML exist, all containing the RBCC motif and three well-characterized small-ubiquitin-related modifier (SUMO) modification sites at lysines K65, K160 and K490 and a SUMO interacting motif (SIM) enabling its interaction with SUMOylated proteins (Uggè et al. 2022;Corpet et al. 2020).SUMO E2 conjugating enzyme UBC9-mediated SUMOylation of PML enforces PML-PML interactions via intermolecular SUMO-SIM interactions.It also drives the multivalent recruitment of inner core protein clients through their SIM, possibly via liquid-liquid phase separation (LLPS) mechanisms (Corpet et al. 2020;Li et al. 2020;Sahin et al. 2014).
PML NBs have been involved in a wide variety of biological processes such as senescence, antiviral response, DNA damage response, or stemness suggesting that they are fully significant structures.The molecular mechanisms through which they exert their broad physiological impact are not fully elucidated yet.While PML NBs are in general devoid of DNA, except in specific cases (for review (Corpet et al. 2020)), they reside in the interchromatin nuclear space (Boisvert et al. 2000) and can associate with specific genomic loci (Shiels et al. 2001;Wang 2004;Kurihara et al. 2020;Ching et al. 2013;Kumar et al. 2007;Chang et al. 2013;Delbarre et al. 2017).PML NBs have been found associated with both transcriptionally-active domains (Boisvert et al. 2000;Wang 2004;Kurihara et al. 2020), as well as heterochromatin regions such as telomeres suggesting an important function in chromatin domain organization and regulation of their transcriptional state (for review (Delbarre and Janicki 2021)).
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The copyright holder for this preprint this version posted May 23, 2022.; https://doi.org/10.1101/2021.11.30.470516 doi: bioRxiv preprint 4 Targeted deposition of histones variants is crucial for chromatin homeostasis and the maintenance of cell identity (Allis and Jenuwein 2016).Among histone H3 variants, H3.3 is expressed throughout the cell cycle and is incorporated onto DNA in a DNA-synthesis independent manner by dedicated histone chaperone complexes (Martire and Banaszynski 2020).Histone cell cycle regulator A (HIRA) chaperone complex, composed of HIRA, ubinuclein 1 or ubinuclein 2 (UBN1 or UBN2) and calcineurin-binding protein CABIN1 is responsible for H3.3 deposition in transcriptionally active regions including enhancers, promoters and gene bodies, as well as in nucleosome-free regions and DNA damage sites (Ray-Gallet et al. 2002;2011;Goldberg et al. 2010;Zhang et al. 2017) (for review (Martire and Banaszynski 2020;Ricketts and Marmorstein 2016)).HIRA has also been recently shown to be involved in the transcription-mediated recycling of parental H3.3 (Torné et al. 2020).While HIRA complex is diffusively distributed in the nuclei of proliferating somatic cells, it relocalizes in PML NBs upon various stresses such as senescence entry (Rai et al. 2011;Zhang et al. 2005;Banumathy et al. 2009;Jiang et al. 2011), viral infection (Cohen et al. 2018;McFarlane et al. 2019;Rai et al. 2017), or interferon type I (IFN-I) treatment (Rai et al. 2017;McFarlane et al. 2019).These latter events underscore a role of HIRA in intrinsic anti-viral defense via chromatinization of incoming viral genomes (Rai et al. 2017;Cohen et al. 2018) as well as stimulation of innate immune defenses in the case of viral infection (McFarlane et al. 2019).
However, the exact significance of HIRA localization in PML NBs upon inflammatory stress response, as well as the role of the PML NBs themselves, remain to be defined.PML NBs may act as buffering/sequestration/degradation structures for various chromatinrelated proteins, and be a means to target them to specific chromatin regions juxtaposed to PML NBs.Here we investigated the molecular mechanisms of HIRA localization in PML NBs.We show that HIRA localizes in PML NBs in a SIM-SUMO-dependent manner upon IFN-I treatment.We provide evidence that PML is required for interferon-stimulated genes (ISGs) expression, and that ISGs loci juxtapose to PML NBs.ChIP-Seq analysis reveals a long-lasting H3.3 deposition on the 3' end of ISGs, which is partly dependent on HIRA and PML, but independent of HIRA localization in PML NBs.Instead, we uncover that PML NBs rather act as nuclear depot centers for HIRA, depending upon histone availability and dynamics.Together, our results put forward a dual role for PML/PML NBs during the inflammatory response both regulating ISGs transcription as well as H3.3 (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

HIRA accumulation in PML NBs correlates with an increase in PML valency
Several stimuli, such as IFN-I treatment (Rai et al. 2017;McFarlane et al. 2019), can trigger HIRA accumulation in PML NBs.Previous studies suggest that a valency-dependent switch-like partitioning of a given client protein in the condensed PML NB phase is controlled by the multivalent interactions between its SIM motifs and SUMOylated lysines on the PML protein, as shown for the H3.3 histone chaperone DAXX (Banani et al. 2016;Sahin et al. 2014), which localizes constitutively in PML NBs (Ishov et al. 1999).As a first step towards deciphering the mechanism of HIRA localization in PML NBs, we investigated the valency-dependent recruitment of HIRA in PML NBs.Treatment of human primary foreskin diploid fibroblast BJ cells with the TLR3 ligand poly(I:C), a strong stimulant of the IFN-I pathway, or with the Tumor necrosis factor a (TNFa) cytokine, triggered a strong accumulation of HIRA in PML NBs (Figures 1A-B), similarly to a control IFN-I treatment (Sup. Figure 1A).The accumulation was abrogated by addition of ruxolitinib, an inhibitor of the JAK-STAT pathway downstream of the IFN-I receptor, underscoring the involvement of the IFN-I signaling pathway in primary cells (Figures 1A-B and Sup. Figure 1A).IFNb, poly(I:C) and TNFa induced an IFN-I dependent increase of PML and its SUMOylated forms (Figure 1C), confirming previous data (Stadler et al. 1995;Gao et al. 2008).Treatment with other pro-inflammatory cytokines such as IL-6 or the IL-8 chemokine, did not increase PML protein levels or SUMOylation (Sup. Figure 1B), nor affected HIRA localization that remained pan-nuclear (Sup.Figure 1C).These results suggest that an IFN-I-dependent increase of PML valency (increase in protein levels and SUMOylation), but not of HIRA (Sup. Figure 1D) is part of the mechanism for HIRA accumulation in PML NBs.

Accumulation of HIRA in PML NBs depends on SUMO-SIM interactions
We hypothesized that HIRA's partitioning in PML NBs could be regulated by SUMO-SIM interactions.We first investigated whether HIRA and PML/SUMO could interact together in cellulo.Proximity Labelling Assay (PLA) allows the detection of closely interacting protein partners in situ at distances below 40nm (Sahin et al. 2016).Using PLA, we

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detected interaction foci between PML and SUMO2/3 as expected (Sahin et al. 2016), with the number of interaction foci increasing significantly upon IFNb treatment (Figures 2A-B), which is known to stimulate PML SUMOylation (Stadler et al. 1995).We then assessed the interactions between HIRA and PML or HIRA and SUMO2/3.We could detect a significant interaction between these proteins in presence of IFNb (Figures 2A-B), accordingly to the accumulation of HIRA in PML NBs.Positive PLA signal between HIRA and SUMO2/3 could either mean that HIRA is SUMOylated or that HIRA interacts with other SUMOylated proteins.The molecular mass of HIRA remained unchanged upon IFNb treatment of primary cells (Sup. Figure 1D), as previously described (McFarlane et al. 2019), which is not in favor of post-translational modification of HIRA with SUMO groups.
In addition, ectopic HIRA mutated on K809, identified as a possible SUMOylated lysine in a SUMO screen (Schimmel et al. 2014;Hendriks et al. 2014), was still recruited in PML NBs similar to the wild-type protein (Sup.Figure 1E), suggesting that at least K809 SUMOylation is dispensable for HIRA recruitment in PML NBs.We thus conclude that HIRA can interact with SUMOylated proteins in situ.
To confirm that SUMOylation of cellular proteins, including PML, is required for HIRA partitioning in PML NBs, we depleted the pool of SUMO1/2/3 by siRNA treatment (Figure 2C).Depletion of SUMOs led to a significant decrease of HIRA accumulation in PML NBs upon IFNb treatment (Figure 2D).Of note, in absence of SUMOs, PML NBs appear as large aggregates devoid of DAXX (Sup. Figure 2A), reminiscent of the alternative PML NBs structures observed during mitosis, in human embryonic stem cells or in sensory neurons (Corpet et al. 2020).Thus, presence of SUMO proteins, that can undergo LLPS in vitro (Banani et al. 2016), seems key to promote partitioning of HIRA.Increasing the pool of free SUMOs by ectopic expression did not trigger HIRA accumulation in PML NBs (Sup. Figure 2B) suggesting that SUMOs need to be conjugated to specific proteins to trigger HIRA partitioning in PML NBs.
To further substantiate the requirements for non-covalent SUMO-SIM interactions in mediating HIRA accumulation in PML NBs, we used the Affimer technology, previously known as Adhiron.Affimers are artificial protein aptamers consisting of a scaffold with two variable peptide presentation loops that can specifically bind with high affinity and high specificity to their binding partners.A recent screen identified several Affimers that inhibit SUMO-dependent protein-protein interactions mediated by SIM motifs (Hughes et al. 2017).We selected the S1S2D5 Affimer that specifically targets both SUMO1 and (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint this version posted May 23, 2022.; https://doi.org/10.1101/2021.11.30.470516 doi: bioRxiv preprint SUMO2/3-mediated interactions and which possesses a consensus SIM motif (Hughes et al. 2017).Inducibly-expressed S1S2D5-His Affimer showed a nuclear staining with accumulation of the Affimer in PML NBs (Figures 3A-B), as expected for a synthetic peptide that exhibits a SIM domain (Hughes et al. 2017;Banani et al. 2016).S1S2D5-His Affimer expression prevented the accumulation of HIRA in PML NBs upon IFNb treatment (Figure 3C), without affecting HIRA nor PML protein levels (Sup.Figure 2C).Collectively, our results demonstrate that SUMO-SIM interactions play an important role in the targeting of HIRA in PML NBs in response to IFNb.
PML is known to be mainly SUMOylated on lysines K65, K160 and K490 (Kamitani et al. 1998).Immortalized Pml-/-mouse embryonic fibroblasts (MEFs) reconstituted with a doxycyclin-inducible wild-type Myc-tagged version of human PML (Myc-PML WT) or a PML mutated on its three main SUMOylation sites (Myc-PML 3K) were used to investigate the specific requirements for PML SUMOylation in HIRA partitioning (Figure 3D).We first verified that HIRA accumulation in PML NBs was conserved in wild-type but not Pml-/-MEFs upon activation of the IFN-I pathway (Sup.Figure 2D).Upon doxycyclin induction, Myc-PML WT or its mutated form were expressed at high levels in Pml-/-MEFs (Figure 3D).Despite a diminution in the amount of the ectopic PML proteins following addition of mouse IFNa (Figure 3D), the wild type PML rescued HIRA accumulation in ectopically formed PML NBs unlike the PML 3K (Figure 3E).Of note, super resolution microscopy analyses of PML 3K-expressing MEFs reveal that PML 3K form spherical structures exactly like WT PML (Sahin et al. 2014).These data demonstrate that PML SUMOylation on K65, K160 and K490 is required for HIRA recruitment in PML NBs.
Multivalent interactions between client SIM motifs and SUMOylated lysines on the PML protein are implicated in client recruitment in PML NBs, as shown for DAXX (Banani et al. 2016;Sahin et al. 2014).Using JASSA (Beauclair et al. 2015) and GPS-SUMO (Zhao et al. 2014), we selected a set of 5 putative SIM motifs in HIRA protein sequence and tested whether they were involved in HIRA recruitment in PML NBs by mutating them individually (Figure 3F).Cells expressing the wild-type (WT) tagged version of HIRA (HIRA-HA WT) displayed ectopic HIRA accumulation in PML NBs upon IFNb treatment (Figure 3F).HIRA-HA mSIM1 and mSIM3 mutants did not show sufficient expression in individual cells to analyze their localization.HIRA-HA mSIM4 and mSIM5 mutants showed a normal accumulation in PML NBs (Sup. Figure 2E).Interestingly, the HIRA-HA mSIM2 showed a significant decrease in its accumulation in PML NBs upon IFNb treatment (Figure 3F).This data confirms the importance of the SUMO-SIM interaction pathway in general, and particularly at least, the putative SIM2 motif in HIRA for its recruitment in PML NBs.Unfortunately, the levels of HIRA-HA mSIM2 expression remained very low at the cell population level compared to HIRA-HA WT (Sup. Figure 2F Figures 3A-C).Overall, our data argue for a multistep molecular mechanism involving PML SUMOylation, a putative SIM motif on HIRA and SP100 in the accumulation of HIRA in PML NBs following activation of the IFN-I signaling pathway.

PML depletion but not HIRA impairs ISGs expression
IFN-I is responsible for the upregulation of hundreds of ISGs as part of the innate immune response participating in the inhibition of virus replication (Shaw et al. 2017).Since HIRA and PML are involved in transcriptional regulation, we investigated whether the partitioning of HIRA in PML NBs upon IFN-I stimulation could play a role in the transcription of ISGs.
We first analyzed the expression of a selected set of ISGs, MX1, OAS1, ISG15 or ISG54 (IFIT2), in cells treated with IFNb for 6 or 24 h.mRNA levels of these ISGs increased strongly after IFNb treatment, peaking at 6 h of treatment.Transcripts levels then gradually decreased over time, but remained highly expressed at 24 h post addition of IFNb (Sup. Figure 4A).We then analyzed ISGs mRNA levels in cells depleted of HIRA or PML (Sup. Figure 4B).Depletion of HIRA had no significant impact on ISGs expression at 6 and 24 h of IFNb stimulation (Figure 4A), consistent with previous reports (Rai et al. 2017;McFarlane et al. 2019).Compared to ISGs transcription climax at 6h post IFNb, the peak of accumulation of HIRA in PML NBs at 24h post addition of IFNb (Sup. Figure 3C) suggests that the latter might not be directly involved in ISGs transcriptional upregulation.In contrast, PML depletion led to a significant reduction in ISGs expression both at 6 and 24 h after IFNb stimulation (Figure 4A).MX1, OAS1, ISG15 or ISG54 mRNA levels were only 27%, 16%, 28% or 35 % of the levels observed in 6h IFNb-treated cells, respectively.Thus, the results suggest an essential role of PML in the IFN-I-dependent transcriptional upregulation of ISGs, which is independent of HIRA.
Interferon-stimulated gene loci are juxtaposed to PML NBs after IFN-I stimulation 9 PML NBs make direct physical contacts with surrounding chromatin regions and these associations may serve to modulate genome functions and gene expression (Corpet et al. 2020).In the context of the IFNg inflammatory response, genes within the MHCII locus are located in proximity of PML NBs (Gialitakis et al. 2010).Given the above results, we thought to examine the spatial connection between PML NBs and specific ISG loci.We performed immunostaining of the PML protein, together with fluorescence in situ hybridization (immuno-FISH) to detect the PML, MX1, OAS1 gene loci in cells treated or not with IFNb.PML was used as a positive ISG control since previous immuno-trap analyses found a specific interaction between PML NBs and the PML gene locus upon IFNa treatment (Ching et al. 2013).To evaluate the specificity of potential spatial changes, we also scored localization of the Grehlin and Obestatin Prepropeptide (GHRL) locus, which is not an ISG (Eggenberger et al. 2019) and is localized in heterochromatin regions (Becker et al. 2017).Visual inspection showed an overall closer locus-to-PML NB proximity of PML, MX1 and OAS1, but not GHRL, in IFNb-treated cells relative to untreated cells (Figure 4B).To quantify the association of PML NB with ISG loci, we calculated the mean minimal distance (mmd) between each locus and the center of the closest PML NB per nucleus in untreated and treated cells.A decreased distance could be a consequence of the increased number and size of PML NBs upon IFNb treatment (Sup. Figure 4D).We thus normalized the mmd for the ISGs to the one calculated for the GHRL locus.A marked decrease in the mmd of PML NBs with the three loci was scored at 48h, which was significant for PML and MXI loci, reaching a calculated mmd of 0.67µm and 0.72µm, respectively, as compared to 1.06µm for the GHRL locus (Figure 4C).We also confirm the presence of HIRA in PML NBs juxtaposed to ISGs by triple labelling (Figure 4D).The requirement of PML for the acute peak of transcription of ISGs at 6h of IFNb (Figures 4A),

IFN-I stimulation triggers accumulation of endogenous H3.3 in the 3' end region of transcribed ISGs
Using a tagged version of H3.3 in MEF cells, previous studies showed an increased and prolonged deposition of ectopic H3.3 in the transcription end sites (TES) region of ISGs 10 upon IFN-I stimulation (Tamura et al. 2009;Sarai et al. 2013).We thus wondered whether PML and HIRA could functionally impact endogenous H3.3 deposition on ISGs using an H3.3-specific antibody previously validated in ChIP (Lee et al. 2019).The amount of H3.3 remained unaffected by 24 h of IFNb stimulation excluding a putative ISG-like behavior (Sup.Figure 5A).We first investigated H3.3 incorporation on MX1, OAS1 and ISG54 (IFIT2).Three distinct regions of the selected ISGs were analyzed: the promoter region, located just upstream (-120pb) of the transcriptional start site (TSS), the middle of the coding region (mid), and a distal site in the coding region near the TES (see map in Figure 5A).A slight decrease of H3.3 occupancy at promoter regions was measured (Figure 5A).This reduction following IFN stimulation likely reflects transcription-induced nucleosome depletion known to happen for many genes upon stimulation (Workman 2006).Remarkably, IFNb stimulation induced H3.3 incorporation most noticeably over the distal sites of the coding regions (Figure 5A).This was concomitant with an increase in H3K36me3, a histone mark added by the methyltransferase SETD2, which moves with RNA pol II during transcription (Sup.Figure 5B).Use of a control IgG antibody did not lead to any significant amount of immunoprecipitated DNA (% input) in any of the conditions highlighting the specificity of our ChIP experiment (Sup.Figure 5C).In addition, no change in H3.3 occupancy was observed at an enhancer region known to be enriched with H3.3 (Pchelintsev et al. 2013), underscoring the specificity of H3.3 accumulation in ISGs (Figure 5A).Normalization of H3.3 signal over the total H3 histones signal, which showed no major changes in histone density, confirmed the increased amount of H3.3 at ISGs with a preference for the TES regions (Sup.Figure 5D).This fits with the known replicationindependent replacement of canonical H3 histones with H3.3 during transcription (Ahmad and Henikoff 2002;Mito et al. 2005;Workman 2006).No noticeable H3.3 increase was observed at representative mid or TES regions at 6 or 12 h of IFNb treatment (Sup. Figure 5E).Therefore, H3.3 increased deposition most likely takes place after the peak of ISGs transcription at 6 h of IFNb (Sup. Figure 4A).Importantly, H3.3 deposition continued to increase for an extended period of time and was even higher at 48 h of IFNb, suggesting that it could leave a long-lasting chromatin mark on ISGs (Sup. Figure 5E).
We then performed ChIP-Seq analysis for endogenous H3.3 on cells treated or not with IFNb for 24 h.We first examined H3.3 enrichment over the gene bodies of a published panel of equivalent sized ISGs or non-ISGs (McFarlane et al. 2019).The levels of H3.3 significantly increased on ISGs in IFNb-treated cells with a clear bias towards the TES (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint this version posted May 23, 2022.; https://doi.org/10.1101/2021.11.30.470516 doi: bioRxiv preprint 11 regions of the genes (Figure 5B).In contrast, no significant difference of H3.3 enrichment could be observed across the non-ISGs (Figure 5C).We selected genes with the highest difference in H3.3 enrichment at the TES region between IFNb treated and non-treated cells, and performed Gene Ontology (GO) analysis.GO analysis showed a clear enrichment in genes involved in IFNa and IFNg response comforting the specific enrichment of H3.3 on the TES region of ISGs as a prolonged response to an IFN-I stimulus (Figure 5D).To evaluate the identity of H3.3-enriched genes in an unbiased manner, we performed an independent GO analysis on all genes found in the ChIP peak calls.This yielded similar results with IFNa and IFNg responses being the most highly significant GO terms (Sup. Figure 5F).Thus, these findings establish that IFN-I triggers a specific long-lasting H3.3 deposition on ISGs following IFN-I stimulus.

H3.3 deposition on ISGs is impaired upon HIRA or PML depletion but is independent of the localization of HIRA in PML NBs
We next wondered whether HIRA and/or PML was essential for H3.3 deposition at ISGs.Cells were depleted of HIRA or PML (Figure 5E, left) and treated with IFNb for 24 h, before performing ChIP on H3.3.Absence of HIRA or PML led to a modest but consistent decrease in H3.3 at mid or TES regions of selected ISGs, suggesting the implication of these two proteins for the long-lasting H3.3 deposition on ISGs (Figure 5E, right).ChIP-Seq analysis confirmed a modest, but still significant, decrease in the loading of H3.3 at the TES on the panel of ISGs (p-value = 4,76e-03 for HIRA knock-down (KD) or 1.262e-03 for PML KD, as assessed by a paired Student's t-test) (Figure 5F, Sup. Figure 5G).Representative STAT1 and GCH1 genes, confirmed the deficit in H3.3 loading at the TES region of ISGs in the absence of HIRA or PML (Sup. Figure 5H).We thus conclude that HIRA and PML both contribute to the increased long-lasting H3.3 deposition at the TES region of ISGs following the transcriptional peak associated to IFN stimulus.
We then investigated if the accumulation of HIRA in PML NBs was a prerequisite for the increased deposition of H3.3 on ISGs.The low level of expression of the HIRA-HA mSIM2 mutant, that does not accumulate in PML NBs following IFN-I stimulus (Sup. Figure 2F), unfortunately precluded its use for biochemical analyses.We therefore chose to deplete SP100 that also strongly impairs HIRA recruitment in PML NBs (Sup. Figure 3  PML NBs act as storage sites to buffer HIRA according to histones availability and dynamics Quantification of nucleoplasmic levels of HIRA outside PML NBs showed a significant decrease after IFNb treatment (Figure 6A).Similarly, ectopic expression of HIRA led to its accumulation in PML NBs without the need of IFN-I stimulation (Figure 6B), suggesting that PML NBs could help buffering an excess of HIRA proteins.H3.3-H4 deposition/recycling on chromatin is tightly regulated by the HIRA complex (Martire and Banaszynski 2020).We thus reasoned that an increase of the nucleoplasmic pool of H3.3-H4 could modulate HIRA accumulation in PML NBs upon IFN-I treatment.We generated human primary BJ cells expressing an inducible HA-tagged form of H3.3 (BJ eH3.3i).
Treatment with doxycyclin triggered a strong, yet highly variable, expression of eH3.3 (Figure 6C, Sup.Figures 6A-B), consistent with the polyclonal nature of the BJ eH3.3i.We verified that doxycyclin did not impact HIRA localization in absence of IFNb and that IFNb alone triggered a normal accumulation of HIRA in PML NBs in these cells (Sup. Figure 6A).Addition of doxycyclin that triggers H3.3 overexpression did not significantly alter HIRA accumulation in PML NBs upon IFNb on a population level (Sup.Figure 6A).However, close examination of individual cells showed an impaired accumulation of HIRA in PML NBs in cells with a strong overexpression of eH3.3 (Figure 6C).This was not observed in low eH3.3 expressing cells, in which HIRA was detected in PML NBs together with eH3.3 (Figure 6C).Quantification of the mean eH3.3 nuclear fluorescence intensity showed that it was low in average in nuclei showing accumulation of HIRA in PML NBs upon IFNb, while a significant shift to higher eH3.3 nuclear intensities was observed in nuclei showing absence of HIRA in PML NBs, underscoring a strong antagonism between HIRA detection in PML NBs and high expression of eH3.3 (Figure 6D).Similar results were obtained in human primary lung fibroblasts (Sup.Figure 6C).Therefore, presence of an excessive H3.3 pool in the nucleoplasm prevents HIRA accumulation in PML NBs upon IFN-I stimulation.
Finally, we wondered if increasing histone dynamics could also prevent HIRA accumulation in PML NBs by forcing its requirement on chromatin.We therefore treated 13 BJ cells with trichostatin A (TSA), a drug that selectively inhibits class I and II mammalian histone deacetylases, inducing chromatin decompaction and therefore increasing histone exchange on chromatin (Nozaki et al. 2017).Interestingly, TSA treatment significantly impaired the accumulation of HIRA in PML NBs seen under IFNb stimulation (Figure 6E), without noticeable change in HIRA levels (Sup.Figure 6D).Thus, TSA-induced hyperacetylation of chromatin seems to redirect HIRA from PML-NBs, possibly to ensure H3.3 deposition/recycling on the hyperactive decondensed chromatin.

Discussion
There have been considerable efforts in defining the multiple roles of PML and PML NBs in the recent years including in chromatin dynamics.After having dissected the molecular mechanisms responsible for HIRA accumulation in PML NBs upon IFN-I treatment, we investigated the functional role of the PML NBs-HIRA-H3.3axis in inflammatory response.
Our work underscores two independent roles for PML/PML NBs in (1) regulating the transcriptional status of ISGs and the incorporation of H3.3 at these loci and in (2) acting as storage centers to modulate HIRA complex nucleoplasmic availability upon inflammatory stress.

HIRA accumulation in PML NBs upon inflammatory stresses is dependent on functional SUMO-SIM interactions
While senescence was the first stress shown to induce accumulation of HIRA complex in PML NBs (Zhang et al. 2005;Banumathy et al. 2009;Rai et al. 2011;Jiang et al. 2011), IFN-I signaling pathway was recently shown to be responsible for similar behavior of HIRA upon a viral infection (Rai et al. 2017;Cohen et al. 2018;McFarlane et al. 2019).Here, we first extend and corroborate these findings by showing that various inflammatory stresses, including TNFa, or a synthetic dsRNA (PolyI:C), can also mediate HIRA recruitment in PML NBs.We show that HIRA partitioning in PML NBs is mediated by SUMO-SIM interactions, that can be inhibited with specific Affimers.Our data using Pml -/- MEFs reconstituted with PML WT or PML 3K underline the importance of PML main SUMOylation sites in recruiting HIRA complex.Remarkably, SIM-containing clients, as exemplified with DAXX (Sahin et al. 2014), are favored for partitioning in endogenous PML NBs containing a higher valency of SUMO sites compared to SIM sites (Banani et al. 2016).Here, we identified a putative SIM motif on HIRA sequence that participate in its recruitment in PML NBs.Interestingly, the VLRL SIM motif identified is followed by a Serine in the position 229 (S229).Phosphorylation adjacent to SIM motifs can lead to an increased affinity towards SUMO1 lysine residues (Cappadocia et al. 2015).Other posttranslational modifications such as phosphorylation could thus be important in regulating HIRA partitioning by changing the affinity between HIRA and SUMOylated PML proteins/partners.Of note, glycogen synthase kinase 3 b (GSK-3b) mediatedphosphorylation of HIRA on S697 was suggested to drive HIRA accumulation in PML NBs upon senescence entry (Ye et al. 2007).Thus, HIRA accumulation in PML NBs after IFN-I treatment mechanistically relies on SUMO-SIM interactions.Whether this is mediated by direct/indirect interactions with SUMOylated PML or SP100 ((McFarlane et al. 2019) and this study), remains to be investigated.Nevertheless, the use of SUMO-specific Affimers opens interesting avenues to interfere with client recruitments in PML NBs including HIRA.

PML regulates ISGs transcription and PML NBs associate with ISGs loci
We next investigated the functional role of the accumulation of HIRA in PML NBs by first focusing on the impact of HIRA and PML depletion in ISGs transcription and H3.3 deposition.While HIRA binding to ISGs is increased after IFN-I (McFarlane et al. 2019;Rai et al. 2017), its depletion did not affect ISGs expression, consistent with previous reports (McFarlane et al. 2019;Rai et al. 2017).In contrast, our data underscore the importance of the PML protein for the initial burst of transcription of ISGs at 6 h of IFNb stimulation.This is consistent with both the association of PML NBs with transcriptional sites after IFNb stimulation, as shown by visualization of nascent transcripts (Fuchsová et al., 2002) and consistent with the role of PML in ISGs induction following viral infection (Alandijany et al., 2018).PML proteins could be recruited to transcriptionally active ISGs by a specific, yet to be defined, protein-protein interaction.Previous studies showed that the nuclear DNA helicase II (NDH II), which is essential for gene activation, relocalizes in PML NBs in a transcription-dependent manner (Fuchsová et al. 2002).The authors suggested PML NBs could play a role in the transcriptional regulation of ISGs attached to PML NBs, although this was not investigated (Fuchsová et al. 2002).Here, by using immuno-FISH, we demonstrate for the first time a juxtaposition of a subset of ISG loci with PML NBs at late time-points of IFNb stimulation.This data underscores the likely importance of a PML NBs-gene locus association as a marker of the physiological state of the cell.Since, PML targeting at specific gene loci is sufficient to induce de novo formation of PML NBs (Brouwer et al., 2009;Chung et al., 2011;Erdel et al., 2020;Kaiser et al., 2008;Wang et al., 2018), we hypothesize that chromatin-bound PML proteins involved in ISGs transcription could then act as seeds to mediate neonucleation of new PML NBs at ISG loci (see model in Figure 6F).Alternatively, movement of ISGs to preexisting PML NBs could still be at play to explain this closer association upon IFNb stimulation.The use of ALaP-Seq method to map chromatin regions proximal to PML NBs (Kurihara et al., 2020) could nicely complement the immuno-FISH approach to identify genome-wide changes in PML-NBschromatin associations upon IFN-I stimulation.

H3.3-induced deposition in the TES region of transcribed ISGs is partially mediated by HIRA and PML
We then investigated the role of PML and HIRA in the H3.3-mediated deposition on ISGs.Endogenous H3.3 deposition shows a strong preference for the ISGs TES regions, consistent with previous reports obtained in mouse cells overexpressing exogenous H3.3 (Tamura et al. 2009;Sarai et al. 2013).Also, our data highlight a long-lasting deposition of H3.3 up to 48h after IFN-I stimulation, well beyond the peak of transcription of the ISGs.
Deposition of endogenous H3.3 was reduced in the absence of PML consistent with the role of PML NBs in targeting H3.3 to chromatin (Delbarre et al. 2013) and in line with the role of PML in chromatinization of latent viral genomes (Cohen et al. 2018).Since PML depletion impairs transcription of ISGs (Figure 4A) and (Alandijany et al. 2018), it could as well indirectly affect H3.3 deposition at TES regions, which has been shown to be linked to the transcriptional activity of ISGs per se (Sarai et al. 2013).PML has been shown to be implicated in the loading of HIRA on ISGs (McFarlane et al. 2019), anticipating a role of a PML-HIRA axis in H3.3 deposition on these loci.HIRA depletion indeed mildly impaired H3.3 deposition at ISGs TES regions, which is consistent with its known function in H3.3nucleosome assembly.HIRA interacts with RNA pol II and also with H3K36me3 (Torné et al. 2020;Ray-Gallet et al. 2011), a histone mark added by the methyltransferase SETD2, which moves with RNA pol II during transcription.In MEFs, HIRA has been shown to interact with WHSC1, another H3K36me3 methyltransferase that recruits HIRA for prolonged H3.3 deposition on ISGs (Sarai et al. 2013).Since H3K36me3 was also found to be enriched at the TES part of ISGs in IFNb treated cells (Sup. Figure 5B), one could anticipate a similar recruitment of HIRA via an H3K36methyltransferase/H3K36me3 axis.Of note, HIRA impact on H3.3 levels could also be explained by its role in H3.3 recycling during ISGs transcription (Torné et al. 2020).Since H3.3 deposition is only moderatly affecting following HIRA depletion, it is not unlikely that other H3.3 chaperones could compensate for the absence of HIRA as already observed for viral genomes chromatinization (Cohen et al. 2018).Indeed, the DAXX/ATRX complex, which localizes constitutively in PML NBs, could participate in H3.3 deposition on ISGs, although its activity is mainly associated to chromatin silencing.Alternatively, the remodeling protein CHD2 has been shown to be recruited to the promoters of myogenesis genes to incorporate H3.3 (Harada et al. 2012, Siggens et al., 2015).It could be interesting to see if CHD2 is recruited to PML NBs, and if it participates to H3.3 deposition at ISGs loci after IFN-I induction.Nonetheless, these data indicate the importance of a PML-HIRA axis to regulate H3.3 dynamics on ISGs loci.
The role of the prolonged H3.3 deposition on ISGs can be multiple.First, this long-lasting mark could contribute to the acquisition of a functional IFN response memory.Indeed, H3.3 was shown to mediate memory of an active state upon nuclear transfer in Xenopus laevis (Ng and Gurdon 2007).In addition, in MEFs, IFNb stimulation creates a transcriptional memory of a subset of ISGs, which coincides with acquisition of H3.3 and H3K36me3 on chromatin (Kamada et al. 2018).A second stimulation with IFNb allows a faster and greater transcription of so called "memory ISGs", which is dependent on H3.3 deposition during the first stimulation phase (Kamada et al. 2018).In HeLa cells, PML was shown to be required for the stronger re-expression of HLA-DRA after IFNg restimulation, a locus that remained juxtaposed to PML NBs after transcription shut-off (Gialitakis et al. 2010).Second, H3.3 deposition may also serve to directly regulate ISGs expression.In mouse cells, H3.3 was found to be phosphorylated on Serine 31 on macrophages-induced genes following bacterial lipopolysaccharide stimulation, a post-translational mark serving as an ejection switch for the ZMYND11 transcriptional repressor, and allowing the transcriptional amplification of the target genes (Armache et al. 2020).Whether H3.3S31P is increased on ISGs upon IFN-I remains to be investigated.
Given the accumulation of HIRA in PML NBs upon IFNb stimulation, an important question was to address whether this accumulation was required for HIRA function in H3.3 deposition/recycling at ISGs.Our ChIP analysis after SP100 depletion, which prevents HIRA localization in PML NBs under IFN-I stimulation is impaired, did not show any decrease of H3.3 enrichment at ISGs loci but rather an increase.This result suggests that HIRA accumulation in PML NBs is not a prerequisite for H3.3 deposition on ISGs, at least following a first wave of IFN-I stimulation, and might rather serve as a nuclear depot for subsequent activities.

HIRA levels
Importantly, our study unveils a second aspect of the PML NBs-HIRA interplay, with PML NBs seemingly acting as depot centers to regulate the pool of nucleoplasmic/chromatinbound HIRA, independently of their roles in ISGs transcription/H3.3deposition.We first show that HIRA intensity level in the nucleus, outside PML NBs, decreases upon IFN-I treatment, while steady state HIRA protein amount remains unchanged (our study and (Rai et al. 2017)).Acute stress could thus induce changes in HIRA requirements in the nucleus, to retarget it to specific chromatin loci, leaving a pool of "unemployed" HIRA, which then accumulates in PML NBs and could be released later for further tasks.Hence, it is interesting to mention that overexpression of an ectopic HIRA is also sufficient to induce its accumulation in PML NBs in untreated cells ( (Ye et al. 2007) and this study), underscoring the fact that an excess of HIRA protein is indeed buffered in PML NBs.
Although SUMO-SIM interactions play an essential role in mediating HIRA accumulation in PML NBs, our study unveils the availability of free H3.3 as a novel important parameter controlling this accumulation.Indeed, overexpression of a pool of free soluble H3.3 in the nucleoplasm impairs HIRA accumulation in PML NBs upon IFN-I treatment, possibly by driving HIRA outside PML NBs to handle this pool of H3.3-H4 histones.Similarly, TSAinduced hyperacetylation, which results in global decompaction of chromatin and increased histone exchange/dynamics (Nozaki et al. 2017), also strongly impairs HIRA localization in PML NBs upon IFN-I treatment.
Accordingly, we propose a role for PML/PML NBs in regulating ISGs transcription and H3.3 deposition as follows (Figure 6F): PML is required for ISGs transcription promoting H3.3 loading on these genes.In addition, PML could serve as a plateform to load HIRA on ISGs (McFarlane et al. 2019).While HIRA depletion does not affect ISGs transcription per se, it could participate in H3.3 deposition/recycling at ISGs, a function which does not seem to require its accumulation in PML NBs.In addition, PML NBs plays a role of 'nuclear storage center' for HIRA complex, to buffer its availability in the nucleoplasm, and thus possibly regulating its activity (Figure 6F).As mentioned above, H3.3 deposition at ISGs after a first stimulus allows faster and greater transcription of ISGs upon restimulation (Kamada et al. 2018).Juxtaposition of PML NBs with ISGs at late times after IFN-I treatment could help to keep a memory of the physiological state of the cell.PML would remain in close proximity to ISGs to regulate them upon a second wave of IFN-I stimulation and HIRA accumulation in PML NBs could also be a mean for the cell to make the chaperone complex available much faster in case of a second inflammatory wave.
In conclusion, our study highlights two important functional and independent roles for PML NBs in the inflammatory response, which add to their pivotal involvment in various stress responses.

Antibodies
All the primary antibodies used in this study, together with the species, the reference and the dilutions for immunofluorescence and western blotting, are summarized in Sup.Table 3.

Proximity Ligation Assay (PLA)
Proximity Ligation Assays were performed with the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, DUO92101).Cells on coverslips were fixed in 2% PFA for (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint this version posted May 23, 2022.; https://doi.org/10.1101/2021.11.30.470516 doi: bioRxiv preprint 20 12 minutes at RT°C and then permeabilized in PBS 0,2 % Triton X-100 for 5 minutes at RT°C.Cells were then treated according to the manufacturer's instructions (see Sup. Table 3 for dilutions of primary antibodies).Coverslips were mounted in Duolink In Situ Mounting Medium with DAPI and stored at 4°C before observation.
After performing classic immunofluorescence as described above (without the DAPI staining step), cells were post-fixed in 2% PFA for 12 minutes at RT°C and then permeabilized and deproteinized in PBS 0,5% Triton X-100 0,1M HCl for 10 minutes at RT°C.Samples were dehydrated in successive EtOH baths (2x 70% EtOH, 2x 85% EtOH and 2x 100% EtOH).After co-denaturation at 80°C for 5 minutes, cells' DNA was hybridized with FISH probes diluted at 1/5 O/N at 37°C in dark and humid chamber.Cells were then washed 5 minutes in Saline-Sodium Citrate (SSC) 0,5X at 68°C, 2 minutes in SSC 1X at RT°C and incubated in DAPI diluted in SSC 2X for 5 minutes at RT°C.Coverslips were mounted in Fluoromount-G and stored at 4°C before observation.

Microscopy, imaging, and quantification
Images were acquired with the Axio Observer Z1 inverted wide-field epifluorescence microscope (100X or 63X objectives/N.A. 1.46 or 1.4) (Zeiss) and a CoolSnap HQ2 camera from Photometrics.Identical settings and contrast were applied for all images of the same experiment to allow data comparison.Raw images were treated with Fiji software or with Photoshop (Adobe).HIRA complex accumulation in PML NBs was attested by manual counting of a minimum of 100 cells for each condition and per replicate.PML-NBs and genes loci proximity was measured using the Fiji RenyiEntropy mask on PML and FISH staining.X and Y coordinates for the center of the spots were recovered and all distances between each PML NBs and gene loci were calculated using the formula != #(%1 − %2) * + (,1 − ,2) * to find the minimal distance in each nucleus.Quantification of nuclear intensities was performed with Fiji.Briefly, DAPI and PML stainings were used to define masks of nuclei and of PML NBs.We quantified mean HA fluorescence intensity within each nucleus with the measure function applied on the red (HA) channel.To quantify HIRA intensity outside PML NBs, we first created a mask of nuclei devoid of PML NBs (Image calculator function of Fiji) and then applied the measure function on HIRA channel.
Western Blot was performed as in (Corpet et al. 2014) (see Sup. Table 3 for antibodies dilution).Signal was revealed on ChemiDoc Imaging System (Bio-Rad) by using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, RPN2236) or Clarity Max Western ECL Blotting Substrate (Bio-Rad, 1705062).

Chromatin immunoprecipitation (ChIP)
Cells were crosslinked directly in the culture dishes according to (Becker et al. 2017).
After the PBS washes, cell pellets were snap-frozen in liquid nitrogen and stored at -80°C before immunoprecipitation.Cells were de-frozen on ice and chromatin was prepared following the TruChIP protocol from Covaris, as described in (Cohen et al. 2018).We used the Covaris M220 Focused-ultrasonicator to shear through chromatin (7 minutes at (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

Reverse Transcription (RT)
TRIzol reagent protocol (Invitrogen, 15596026) was used to isolate total RNAs, resuspended in ddH2O according to the manufacturer instructions.Contaminant DNA was removed with the DNA-free DNA Removal Kit (Invitrogen, AM1906).We used 1μg of RNA for reverse transcription (RT).RNAs were annealed with Random Primers (Promega, C118A) and RT was performed with the RevertAid H Minus Reverse Transcriptase (Thermo Scientific, EP0452) according to the manufacturer instructions.
cDNAs were stored at -20°C before qPCR analysis.

Quantitative PCR (qPCR)
qPCRs were performed using the KAPA SYBR qPCR Master Mix (SYBR Green I dye chemistry) (KAPA BIOSYSTEMS, KK4618).Primers used for qPCR are described in Supplementary Table 4.

ChIP-Seq analysis
After ChIP, libraries were made in BGI and sequenced on a BGISEQ-500 sequencing platform (https://www.bgi.com).An average of 34 Million single-end 50bp reads was obtained for each library.Reads were trimmed using Trimmomatic and quality assessed with FastQC.Reads were aligned to the human genome hg38 using the BWA alignment software.Duplicate reads were identified using the picard tools script and only nonduplicate reads were retained.Broad peaks calling was performed with MACS2 (Zhang et al. 2008) ("--extsize 250 -q 0.01 --broad --broad-cutoff 0.05"), using input DNA as control.We defined all possible locations of H3.3 by merging broad peaks identified in our four conditions (n=190295), and annotated them with Homer (http://homer.ucsd.edu/homer/download.html ).We counted reads extended to 250bp falling into these possible locations, in the four ChIP and their corresponding inputs, using (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint this version posted May 23, 2022.; https://doi.org/10.1101/2021.11.30.470516 doi: bioRxiv preprint 23 bedtools-intersect.CPMs were obtained by dividing raw counts by the total number of mapped reads normalized to 1e6, and RPKMs by dividing CPMs by the peak length normalized to 1e3.Input RPKMs, used as background, was substracted from the respective ChIP RPKMs.We focused on 0,5% of the peaks with highest RPKM difference (n=951) between IFNb treated and not treated conditions, of which 711 were intragenic.These peaks allowed us to defined a set of 654 genes, on which we performed GO analysis, with MsigDB, using enrichR plaform (Kuleshov et al. 2016).
As a complementary approach, we measured the ChIP enrichment within the 1000bp regions spanning the TESs (-500 +500), extending all unique reads into 250bp fragments, and counting those falling within TES using bedtools-intersect.CPMs were obtained similarly, and input DNA CPMs, used as background, was substracted from ChIP CPMs.Genes with the log2 of differential TES enrichment between IFNb treated and not treated conditions being higher than 5 (log2(Fold Change)>5) were retained for GO analysis, as described above.
PlotProfile were generated using the DeepTools suite, starting from the MACS2 fold enrichment bigwig files, which take into account the read extension, the input DNA background and the library size normalization.The list of 48 core ISGs and 48 non-ISGs equal in size to the core ISGs was taken from (McFarlane et al. 2019).In order to reduce the noise on the profiles, we selected for each gene the transcript with the highest H3.3 enrichment at the TES in the IFNb treated condition.Genome browser snapshots of H3.3 enrichment were generated using Integrative Genomics viewer (IGV : https://software.broadinstitute.org/software/igv/).

Statistical analyses
Statistical analyses were performed using GraphPad Prism 6.To perform Student t test, we verified normal distribution of samples using Shapiro test and variance equality with

Materials availability
All plasmids and cell lines generated in this study can be accessed upon request to the corresponding authors.
), preventing any biochemical analyses.McFarlane et al. previously published that SP100 was required for HIRA localization in PML NBs (McFarlane et al. 2019), and we confirmed those data (Sup.
in comparison to the occurrence of the juxtaposition of PML NBs with the ISGs loci at 48h post addition of IFNb suggests that existing PML NBs are not directly involved in the transcriptional control of ISGs, but rather nucleate at ISGs loci from the PML proteins initially involved in ISGs transcription.
and(McFarlane et al. 2019)).SP100 depletion did not prevent H3.3 loading at the ISGs TES upon IFN-b stimulation, but on the contrary increased it (Figure5G).These data suggest 12 that although both HIRA and PML are essential for the long-lasting deposition of H3.3 on the TES of ISGs following an acute IFN-I stimulus, the accumulation per se of HIRA in the PML NBs serves a different purpose.

Figure 2 .Figure 3 .
Figure 2. HIRA recruitment to PML NBs is dependent on SUMO proteins.A. Fluorescence microscopy visualization of Proximity Ligation Assays (PLA) signals (red) obtained after incubation of anti-PML+anti-SUMO, anti-HIRA+anti-PML or anti-HIRA+anti-SUMO antibodies on BJ cells treated or not with IFNβ at 1000U/mL for 24h.Cell nuclei are visualized by DAPI staining (grey or blue on the merge).Scale bar represents 10μm.B. Box-and-whisker plot shows the number of PLA spots detected in cells described in A. In average, 200 nuclei/condition were analyzed from 3 independent experiments.The line inside the box represents the median of all observations.p-values (Mann-Whitney u-test): ****<0,0001.C. Western-blot visualization of SUMO-1 and SUMO-2/3 from total cellular extracts of BJ cells treated with 60nM of siRNAs against luciferase or SUMO-1+SUMO-2/3 for 48h and with IFNβ at 1000U/mL during the last 24h.Actin is a loading control.D. (left) Fluorescence microscopy visualization of HIRA (green) and PML (red) in BJ cells treated with siRNAs as described in C. Cell nuclei are visualized by DAPI staining (grey).Scale bar represents 10μm.(right) Histograms show quantitative analysis of cells with HIRA localization at PML NBs.Numbers represent the mean of 3 independent experiments (±SD).p-values (Student t-test): *<0,05.

FigureFigure 6 .
Figure 5 -continued (which was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.