Nuclear pore density controls heterochromatin reorganization during senescence

Here, Boumendil et al. show that an increased nuclear pore density during oncogene-induced senescence (OIS) is responsible for senescence-associated heterochromatin focus (SAHF) formation. They show that the nucleoporin TPR is necessary for both formation and maintenance of SAHF, identify a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei, and demonstrate the importance of heterochromatin organization for a specific gene activation program.

Three-dimensional (3D) genome organization is governed by a combination of polymer biophysics and biochemical interactions, including local chromatin compaction, longrange chromatin interactions, and interactions with nuclear structures. One such structure is the nuclear lamina (NL), which coats the inner nuclear membrane and is composed of lamins and membrane-associated proteins, such as Lamin B receptor (LBR). Electron microscopy (EM) reveals large blocks of heterochromatin associated with the nuclear periphery (Capelson and Hetzer 2009), and mapping genome interactions with laminB1 identifies >1000 lamina-associated domains (LADs). LADs are associated with heterochromatic histone marks (H3K27me3 or H3K9me3) (Guelen et al. 2008). Altered NL composition in the photoreceptors of nocturnal mammals leads to the loss of heterochromatin from the nuclear periphery and its accumulation at the center of the nucleus (Solovei et al. 2013).
Another situation in which there is a dramatic reorganization of heterochromatin is in oncogene-induced senescence (OIS)-a cell cycle arrest program triggered by oncogenic signaling. OIS cells undergo striking chromatin reorganization with loss of heterochromatin and constitutive LADs (Lenain et al. 2017) from the nuclear periphery and the appearance of internal senescence-associated heterochromatin foci (SAHFs). SAHFs appear consecutive to cell cycle arrest and are not observed in nontransformed replicating cells (Narita et al. 2003). SAHF formation results from a reorganization of pre-existing heterochromatin-regions decorated with H3K9me3, H3K27me3, macroH2a, and HP1α,β,γ-rather than de novo heterochromatin formation on new genomic regions (Narita et al. 2003;Zhang et al. 2005;Chandra et al. 2012;Sadaie et al. 2013). Known factors implicated in SAHF formation include activation of the pRB pathway (Narita et al. 2003), certain chromatin-associated nonhistone proteins (Narita et al. 2006), and the histone chaperones HIRA and Asf1a (Zhang et al. 2005(Zhang et al. , 2007. The NL has also been implicated in SAHF formation: LaminB1 and LBR expression are decreased in OIS, and their experimental depletion can facilitate, but is not sufficient for, SAHF formation (Sadaie et al. 2013;Lukášová et al. 2017).
The nuclear envelope is perforated by nuclear pores that control transport between the cytoplasm and nucleus. The nuclear pore complex (NPC) is a large transmembrane complex consisting of ∼30 proteins called nucleoporins ( Fig. 1A; Kim et al. 2018). In contrast to the adjacent NL, EM and superresolution light microscopy show that the nuclear area underneath nuclear pores is devoid of heterochromatin (Schermelleh et al. 2008;Capelson and Hetzer 2009), and nuclear pore density in different neurons and glial cell types from the rat cerebellar cortex anticorrelates with compact chromatin (Garcia-Segura et al. 1989). The nucleoporin TPR has been shown to be responsible for heterochromatin exclusion zones at the NPC (Krull et al. 2010).
The composition and density of the NPC change during differentiation and tumorigenesis Raices and D'Angelo 2012;Sellés et al. 2017;Rodriguez-Bravo et al. 2018). We therefore hypothesized that the NPC could contribute to global chromatin organization and that, specifically, heterochromatin organization could result from a balance of forces attracting heterochromatin to the NL and forces repelling it away from the NPC (Fig.  1B). In support of this hypothesis, we show here that nuclear pore density increases during OIS and that this increase is necessary for heterochromatin reorganization into SAHFs. We identified TPR as a key player in this reorganization. Furthermore, we demonstrated the functional consequences of heterochromatin reorganization in OIS for the programmed activation of inflammatory cytokine gene expression: the senescence-associated secretory phenotype (SASP).

Results and Discussion
Nuclear pore density increases during OIS To assess the role of the NPC in SAHF formation during OIS, we induced the activity of oncogenic Ras (RAS G12V ) by addition of 4-hydroxy-tamoxifen (4HT) in human IMR90 cells, leading to OIS, activation of p53 and p16, and expression of SASP proteins ( Fig. 1C; Supplemental Fig. S1A; Acosta et al. 2013). Nuclear pores disassemble upon entry into mitosis but are very stable during interphase (Daigle et al. 2001;Dultz and Ellenberg 2010). In quiescent cells, nuclear pore density is stabilized by down-regulation of nucleoporin mRNAs (D'Angelo et al. 2009). However, expression profiling in OIS cells (ER: Ras) showed that, compared with control ER:Stop (Stop codon) cells, nucleoporin mRNA levels are unchanged during senescence (Supplemental Fig. S1B). Nucleoporin protein accumulation in senescent cells was confirmed by immunoblotting for POM121 (an integral membrane protein of the NPC central ring) (Funakoshi et al. 2011) and TPR (a large coiled-coil protein of the nuclear basket) (Fig. 1A,D; Cordes et al. 1998). Immunofluorescence and structured illuminated microscopy (SIM) (Schermelleh et al. 2008) showed that increased nucleoporin levels during OIS results in an increased nuclear pore density ( Fig. 1E-G).

Decreasing nuclear pore density leads to loss of SAHF formation
To assess whether the increased nuclear pore density is responsible for heterochromatin reorganization into SAHFs, we used siRNAs to deplete POM121 (Supplemental Fig.  S2A) during the entire course of OIS induction ( Fig. 2A). As expected, since POM121 is required for NPC assembly during interphase (Dultz and Ellenberg 2010;Funakoshi et al. 2011), this led to a decrease in nuclear pore density (

The nucleoporin TPR is necessary for SAHF formation and maintenance
TPR is the last nucleoporin to be incorporated in new NPCs (Bodoor et al. 1999) through its interaction with  Hase and Cordes 2003). TPR has been shown to establish heterochromatin exclusion zones at nuclear pores (Krull et al. 2010) and influence HIV integration sites by maintaining an open chromatin architecture near the NPC (Lelek et al. 2015).
To determine whether it is the increased abundance of TPR at the nuclear periphery of OIS cells-as a result of elevated nuclear pore density-that is responsible for SAHF formation, we depleted TPR during OIS induction (Supplemental Fig. S3A,B). Contrary to a recent report, TPR depletion did not affect nuclear pore density (Supplemental Fig. S3C; McCloskey et al. 2018). However, similar to POM121 depletion, TPR depletion led to the loss of SAHFs (Fig. 3A,B). We confirmed these results with four independent siRNAs targeting TPR (Supplemental Fig.  S3D-F). We conclude that TPR is necessary for the formation of SAHFs during OIS.
The effect of TPR knockdown on heterochromatin relocalization during OIS does not appear to be due to obvious changes in the amount of laminB1 at the NL (Supplemental Fig. S4A).
To assess whether TPR is necessary for maintenance as well as the formation of SAHFs, we used a time course to determine when SAHFs are formed. The percentage of cells containing SAHFs increased gradually after 4HT treatment of ER:Ras cells, reaching a maximum at 6 d (Supplemental Fig. S4B). We therefore depleted TPR 6 d after 4HT addition, when SAHFs have already formed (Fig.  3C). We observed a dramatic reduction of cells containing SAHFs 2 d later (day 8) (Fig. 3D,E). siRNA depletion under these conditions was only partial, and we observed loss of SAHFs in cells specifically depleted for TPR, whereas SAHFs were maintained in cells where knockdown was incomplete (Supplemental Fig. S4C). In some cells with partial TPR depletion, there was a relocalization of heterochromatin to the nuclear periphery in patches that corresponded to sites of TPR depletion (Fig. 3F) but that still contained nuclear pores as detected by MAB414 staining (Fig. 3G). We conclude that exclusion of heterochromatin from the nuclear periphery by TPR is necessary for both the formation and maintenance of SAHFs during OIS.

TPR is necessary for the SASP
SAHFs are proposed to be involved in silencing promitotic genes, contributing to stable cell cycle arrest (Narita et al. 2003(Narita et al. , 2006Zhang et al. 2007). However, TPR-depleted OIS cells did not show defective cell cycle arrest as assayed by 5-bromo-2 ′ -deoxyuridine (BrdU) incorporation and activation of p16, p21, and p53 (Supplemental Fig.  S5A-C). This suggests that SAHFs are dispensable for cell cycle arrest, in agreement with the fact that not all senescent cells form SAHFs (Kosar et al. 2011). Furthermore, SAHFs have been shown to be insufficient to maintain cell cycle arrest, as inactivation of p53 or ATM in OIS cells leads to senescence escape without SAHF alteration (Di Micco et al. 2011). An important characteristic of OIS is activation of the SASP, which is responsible for the non-cell-autonomous effects of senescence. The SASP consists of the expression and secretion of cytokines, chemokines, extracellular matrix proteases, growth factors, and other signaling molecules. The SASP is a tumor-suppressive mechanism that reinforces cell cycle arrest and leads to paracrine senescence but can also promote tumor progression in premalignant lesions (Coppé et al. 2010;Acosta et al. 2013). Strikingly, in the absence of SAHFs after TPR depletion, we observed a complete loss of the SASP, as exemplified by a lack of IL1α, IL1β, IL6, and IL8 mRNA and protein ( Fig. 4A-C; Supplemental Fig. S5D,E). SAHF and SASP loss upon TPR depletion does not seem to be due to a general defect in nuclear transport, as we detected NFκB nuclear import upon induction of paracrine senescence (Supplemental Fig. S6A-C; Acosta et al. 2008Acosta et al. , 2013Chien et al. 2011).
Similarly to some other nucleoporins, a fraction of TPR is present in the nucleoplasm as well as at nuclear pores (Frosst et al. 2002). To assess whether it is the increase in nuclear pore density in OIS (and consequent increased TPR abundance at the nuclear periphery) that is necessary for the SASP or whether TPR has an independent role, we assessed the SASP upon depletion of POM121, which is present only within the NPC. Decreased nuclear pore density upon POM121 depletion did not affect cell cycle arrest (Supplemental Fig. S7A), but the SASP was impaired (Supplemental Fig. S7B-D).
The nuclear pore basket nucleoporin NUP153 (Fig. 1A) is necessary for the association of TPR with the NPC (Hase and Cordes 2003). To further confirm that the role of TPR in SAHF formation and the SASP depends on its presence at the NPC rather than in the nucleoplasm, we depleted NUP153 (Supplemental Fig. S8A). NPC density was unchanged (Supplemental Fig. S8B), but, consistent with the role of NUP153 in TPR-nuclear basket association, TPR-containing NPC density decreased upon NUP153 depletion (Supplemental Fig. S8C). Concomitantly, the percentage of SAHF-containing cells decreased (Supplemental Fig. S8D,E), and the SASP was lost (Supplemental Fig. S8F). We conclude that it is TPR association with the NPC that is necessary for SAHF formation and SASP activation in OIS.

Chromatin reorganization controls the SASP
Our results suggest that heterochromatin reorganization is necessary for the SASP during OIS. To exclude that nuclear pores regulate the SASP through another independent mechanism, we used a different means to deplete SAHFs. The histone chaperone ASF1a is required for SAHF formation (Zhang et al. 2005(Zhang et al. , 2007, and, indeed, its depletion led to a loss of SAHFs in ER:Ras cells ( Pores regulate heterochromatin foci density (Fig. 5D), but, as for TPR and POM121 depletion, there is a dramatic loss of the SASP upon ASF1a depletion in ER:Ras cells (Fig. 5E). While we cannot completely rule out that intact nuclear pores are needed for SASP activation independent of chromatin reorganization, this result supports the hypothesis that heterochromatin reorganization is necessary for the SASP. Our data suggest that an increase in nuclear pore density is responsible for the eviction of heterochromatin from the nuclear periphery by TPR and the consequent formation of SAHFs in OIS. Similar mechanisms could be conserved in other types of senescence, as nuclear pore density is also increased in replicative senescence (Maeshima et al. 2006). Chromatin organization relative to the nuclear periphery has generally been considered from the point of view of interactions between (hetero) chromatin and components of the NL. Here we demonstrate that the repulsion of heterochromatin by nuclear pores is another important principle of nuclear organization, and it will be interesting to establish whether the modulation of nuclear pore density also influences the 3D organization of the genome during development.

Materials and methods
Cell culture IMR90 cells were infected with pLNC-ER:RAS and pLXS-ER:Stop retroviral vectors to produce ER:Ras and ER:Stop cells, respectively (Acosta et al. 2013). Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM. 4HT-containing medium was changed every 3 d.

RNA expression analysis
mRNA expression profiling was by IonTorrent mRNA sequencing using the Ion AmpliSeq transcriptome human gene expression kit. Six biological replicates were analyzed, and adjusted P-values were calculated by Benjamini and Hochberg (BH) and false discovery rate multiple test correction. Data analysis was performed using Babelomics-5 (http://babelomics. bioinfo.cipf.es).
For individual mRNAs, total RNA was extracted using the RNeasy minikit (Qiagen), and cDNAs were generated using SuperScript II (Life technologies). Real-time PCR was performed on a LightCycler 480 (Roche) using SYBR Green PCR master mix (Roche) using the primers listed in Supplemental Table S3. Expression was normalized to β-actin.

Immunoblotting
Cells (1 × 10 6 ) were lysed in RIPA buffer, and protein concentration was determined using a Pierce BCA protein analysis kit. Fifteen micrograms of proteins was run into NuPage 3%-8% Tris acetate gels (Invitrogen). After transfer onto nitrocellulose with a iBlot 2 gel transfer device (Thermo Fisher), immunoblotting was done using antibodies listed in Supplemental  Table S2.

Immunofluorescence and SAHF measurement
Cells (2 × 10 5 ) were seeded and grown on coverslips during senescence induction. Cells were fixed in 4% paraformaldehyde (pFa) for 10 min at room temperature, permeabilized in 0.1% Triton X-100 for 10 min, blocked in 1% BSA for 30 min, and incubated with primary antibodies di-luted in 1% BSA for 1 h and with fluorescently labeled secondary antibodies (Life Technologies) for 45 min. Coverslips were counterstained with DAPI and mounted in VectaShield (Vector Laboratories).
To detect replicating cells, cells were incubated with 10 μM BrdU (Sigma) for 16 h prior to fixation and immunodetection using a BrdU antibody (BD Pharmingene, 555627) in the presence of 1 mM MgCl 2 and 0.5 U/µL DNaseI (Sigma, D4527).
Detection of SASP proteins, tumor suppressors, and BrdU-positive cells by high-content microscopy is described in Hari and Acosta (2017). The percentage of SAHF-positive cells was determined by manual examination of 100-200 DAPI-stained cells.

SIM and measurement of nuclear pore density
The bottom plane of cells was imaged by 3D SIM (Nikon N-SIM) and reconstructed using NIS element software after immunofluorescence with antibodies as indicated in Supplemental Table S2. Fifteen nuclei were imaged for each condition, and five regions of interest (ROIs) of 100 × 100 pixels were analyzed per nucleus. Individual NPCs in each ROI were counted manually.

Statistics
All experiments were performed in a minimum of three biological replicates. Error bars are standard error of the mean. P-values were obtained by two-sample equal variance two-tailed t-test.