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bioRxiv preprint doi: https://doi.org/10.1101/374033; this version posted July 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Nuclear pore density controls heterochromatin reorganization during 2 senescence 3 4 Charlene Boumendil1, Priya Hari2, Karl C. F. Olsen1, Juan Carlos Acosta2, Wendy A. 5 Bickmore1* 6 7 1MRC Human Genetics Unit, Institute oF Genetics and Molecular Medicine, University 8 of Edinburgh, Crewe Road South, Edinburgh EH4 2XU 9 10 2Cancer Research UK Edinburgh Centre, Institute oF Genetics and Molecular Medicine, 11 University oF Edinburgh, Crewe Road South, Edinburgh EH4 2XR 12 13 *correspondence to WAB, MRC Human Genetics Unit, Institute oF Genetics and Molecular 14 Medicine, University oF Edinburgh, Crewe Road South, Edinburgh EH4 2XU. 15 [email protected] 16 17 18 19 Abstract 20 Oncogene induced senescence (OIS) is a cell cycle arrest program triggered by oncogenic signalling. 21 An important characteristic oF OIS is activation oF the senescence associated secretory phenotype 22 (SASP)1 which can reinForce cell cycle arrest, lead to paracrine senescence but also promote tumour 23 progression2–4. Concomitant with cell cycle arrest and the SASP activation, OIS cells undergo a striking 24 nuclear chromatin reorganization, with loss oF heterochromatin From the nuclear periphery and the 25 appearance oF internal senescence-associated heterochromatin Foci (SAHF)5. The mechanisms by 26 which SAHF are Formed, and their role in cell cycle arrest and expression oF the SASP, remain poorly 27 understood. Here we show that nuclear pore density increases during OIS and is responsible For SAHF 28 Formation. In particular, we show that the nucleoporin TPR is required For both SAHF Formation and 29 maintenance. The TPR-induced loss oF SAHF does not aFFect cell cycle arrest but completely abrogates 30 the SASP. Our results uncover a previously unknown role oF nuclear pores in heterochromatin re- 31 organization in mammalian nuclei and in senescence, which uncouples the cell cycle arrest From the 32 SASP. 33 34 1 bioRxiv preprint doi: https://doi.org/10.1101/374033; this version posted July 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 Main 36 SAHF Formation results From a reorganization oF pre-existing heterochromatin – genomic regions 37 decorated with repressive histone marks such as H3K9me3, H3K27me3, MacroH2a and 38 heterochromatin proteins HP1a,b,g - rather than de novo Formation oF heterochromatin on new genomic 39 regions5–7. SAHF appear consecutive to cell cycle arrest and are never observed in replicating cells5. 40 Factors implicated in the Formation oF SAHF include; activation oF the pRB pathway,5 certain chromatin- 41 associated non-histone proteins8, and the histone chaperones HIRA and AsF1a6,9. SAHF are proposed 42 to participate in the silencing oF pro-mitotic genes, contributing to stable cell cycle arrest in senescence8. 43 However, whether SAHF Formation is necessary For cell cycle arrest, or For expression oF the SASP 44 phenotype, has not been investigated. 45 In non-senescent cells, a major proportion oF heterochromatin is associated with the nuclear lamina 46 (lamina associated domains - LADs), that underlines the nuclear envelope. We hypothesized that a 47 modiFication oF the nuclear envelope could lead to the Formation oF SAHF, by decreasing the Forces 48 localising heterochromatin to the nuclear lamina and/or by increasing Factors that repel heterochromatin 49 (Fig. 1a). In agreement with this hypothesis, there is a loss oF LADs during OIS10,11, with the released 50 heterochromatin then presumably Forming SAHF. LaminB1 expression is decreased in OIS and its 51 experimental depletion can Facilitate (but is not suFFicient For) SAHF Formation10. 52 Contrasting with studies on the nuclear lamina, the role oF nuclear pores in the Formation oF SAHF has 53 not been studied. Nuclear pores perForate the nuclear membrane, allowing selective import and export 54 oF macromolecules into and out oF the nucleus. The 120 MDa nuclear pore complex (NPC) is composed 55 oF a highly ordered arrangement oF nucleoporins (Fig 1b). Whereas most oF the nuclear envelope is 56 associated with heterochromatin, the area underneath the nuclear pores is completely devoid oF it and 57 NPCs have been proposed to actively exclude heterochromatin12. We therefore considered that the 58 relative density oF nuclear pores could be important in the balance oF Forces attracting or repelling 59 heterochromatin at the nuclear periphery and decided to assess the role oF NPCs in the Formation oF 60 SAHF in OIS. 61 We used a system in which the activity oF oncogenic Ras (RASG12D) is induced by addition oF 4-hydroxy- 62 tamoxiFen (4HT) in human IMR90 cells, leading to OIS - activation oF p53 and p16 and expression oF 63 SASP proteins3 (Fig. 1c; Extended Data Fig. 1a). Nuclear pores disassemble upon entry into mitosis but 2 bioRxiv preprint doi: https://doi.org/10.1101/374033; this version posted July 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 are very stable during interphase13–15. To stabilise nuclear pore density during diFFerentiation, quiescent 65 cells down-regulate the expression oF mRNA encoding nucleoporins16. However, RNA expression 66 profiling in OIS cells (ER-Ras) showed that, compared with control ER-STOP (STOP codon) cells, 67 nucleoporin mRNA levels remain unchanged during senescence (Extended Data Fig. 1b). This 68 suggests a potential accumulation oF nucleoporins in senescent cells and we conFirmed this by 69 immunoblotting for two nucleoporins; POM121 – an integral membrane protein oF the NPC central 70 ring17,18 and TPR – a large coiled-coil protein oF the nuclear basket (Fig. 1b, d). ImmunoFluorescence 71 and structured illuminated microscopy (SIM)19 showed that the increase in nucleoporin levels during OIS 72 results in an increased nuclear pore density (Fig. 1e -g). 73 As components oF the NPC have been shown to interact preFerentially with euchromatin12, we 74 hypothesized that NPCs may actively exclude heterochromatin and that the density oF nuclear pores – 75 i.e. the relative area oF the nuclear periphery that repels heterochromatin, may be a critical Factor in 76 determining whether heterochromatin is retained at the nuclear periphery or released From it to selF- 77 associate in SAHF in the nucleoplasm (Fig. 1a). 78 To assess whether the increased nuclear pore density we observe in OIS cells is responsible For 79 heterochromatin reorganization into SAHF, we used siRNAs to deplete POM121 (Extended Data Fig. 80 2a) during the entire course oF OIS induction (Fig. 1h). As expected, since POM121 is required For NPC 81 assembly during interphase13,18, this led to a decrease in nuclear pore density (Fig. 1i, j and Extended 82 Data Fig. 2b). Consistent with our hypothesis, POM121 depletion resulted in a dramatic reduction oF 83 OIS cells containing SAHF (Fig. 1k, l). 84 TPR has been shown to establish heterochromatin exclusion zones at nuclear pores20 and so the 85 increased abundance oF TPR at the nuclear periphery oF OIS cells, as a result oF the elevated nuclear 86 pore density, might be responsible For SAHF Formation. We thereFore depleted TPR during OIS induction 87 (Extended Data Fig. 3a, b). This did not aFFect nuclear pore density (Extended Data Fig. 3c). However 88 similarly to POM121 depletion, TPR depletion led to the loss of SAHF (Fig. 2a, b). To rule out oFF target 89 eFFects, we conFirmed these results with Four independent siRNAs targeting TPR (Extended Data Fig. 90 3d-F). We conclude that TPR is necessary For the formation of SAHF. 91 To assess whether TPR was necessary for the maintenance as well as the Formation oF SAHF, we 92 perFormed a time course experiment to determine when SAHF are Formed aFter 4HT addition. The 3 bioRxiv preprint doi: https://doi.org/10.1101/374033; this version posted July 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 percentage oF cells containing SAHF increased From 1 day aFter 4HT treatment of ER-Ras cells, reaching 94 a maximum at 6 days (Fig. 2c). We thereFore depleted TPR with siRNAs 6 days upon 4HT addition, 95 when SAHF have already Formed (Fig. 2d). We observed a dramatic reduction oF cells containing SAHF 96 two days later (day 8) (Fig. 2e, F). TPR staining revealed that our siRNA depletion under these conditions 97 was only partial and we could observe loss oF SAHF in cells speciFically depleted For TPR, whereas 98 SAHF were maintained in cells where knockdown was incomplete (Extended Data Fig. 3g). Closer 99 observation revealed that in cells with partial depletion oF TPR there was a relocalization oF 100 heterochromatin towards the nuclear periphery in patches that correspond to sites oF TPR-depletion 101 (Fig. 2g). We conclude that exclusion oF heterochromatin From the nuclear periphery by TPR is 102 necessary For both the Formation and maintenance of SAHF during OIS. 103 We next wanted to investigate the phenotypic consequences oF SAHF loss in OIS ER-Ras cells depleted 104 For TPR. OIS cells depleted For TPR did not show any deFect in cell-cycle arrest as assayed by BrdU 105 incorporation (Extended Data Fig. 4a, b) and there was proper activation oF p16, p21 and p53 cell cycle 106 regulators (Extended Data Fig. 4c). This suggests that SAHF are dispensable For cell-cycle arrest. 107 However, in the absence oF SAHF aFter TPR depletion, we observed a complete loss oF SASP as 108 exempliFied by lack oF IL1a, IL1b, IL6 and IL8 mRNA (Fig.

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