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1 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 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

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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 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 , 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 . 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 (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

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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 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

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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. 3a) and protein induction (Fig 3b, c, Extended

109 Data Fig. 4d). This result was confirmed with four individual TPR siRNAs (Extended Data Fig. 4e). To

110 rule out that SAHF and SASP loss upon TPR depletion was due to a general defect in nuclear transport,

111 we assessed NFkB import into the nucleus upon induction of paracrine senescence2,3,21,22 (Fig. 3d). We

112 observed no difference in NFkB import in cells depleted with TPR or scrambled siRNAs (Fig. 3e, f),

113 suggesting that TPR depletion does not lead to nuclear protein transport defects that could explain

114 SAHF and SASP loss.

115 Similarly to some other nucleoporins, TPR has been shown to be present in the nucleoplasm as well as

116 at nuclear pores23. To assess whether it is the increase in nuclear pore density – and consequent

117 increased TPR abundance at the nuclear periphery - in OIS that is necessary for SASP or whether TPR

118 has an independent role in the SASP, we assessed SASP upon POM121 depletion. POM121 is only

119 present within the NPC. We confirmed that decreased nuclear pore density upon POM121 depletion did

120 not affect cell-cycle arrest (Extended Data Fig. 5a), but the SASP was impaired (Extended Data Fig. 5b-

121 d).

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122 Our results suggest that heterochromatin reorganization into SAHF is necessary for SASP during OIS.

123 To further support this hypothesis, and to rule out the possibility that nuclear pores regulate SASP

124 through another independent mechanism, we used a different mean to deplete SAHF in ER Ras cells.

125 The histone chaperone ASF1a has been shown to be required for SAHF formation6,9 and indeed its

126 depletion (Fig. 4a) led to a loss of SAHF in ER-Ras cells, (Fig. 4b, c), similarly to what we had observed

127 in TPR or POM121 depleted cells6,9. ASF1a depletion does not affect nuclear pore density (Fig. 4d, e),

128 but as for TPR and POM121 depletion, there is a dramatic loss of SASP upon ASF1a depletion in ER

129 Ras cells (Fig. 4f), further confirming that SAHF are necessary for SASP.

130 Our data demonstrate that an increase in nuclear pore density is responsible for the eviction of

131 heterochromatin from the nuclear periphery by TPR and the consequent formation of SAHF in OIS.

132 Similar mechanisms could be conserved in other type of senescence as nuclear pore density is also

133 increased in replicative senescence24. The organisation of chromatin relative to the nuclear periphery

134 has generally been considered from the point of view of interactions between (hetero)chromatin and

135 components of the nuclear lamina. Here we demonstrate that the repulsion of heterochromatin by

136 nuclear pores is another important principle of nuclear organisation and it will be interesting to establish

137 whether the modulation of nuclear pore density also influences the 3D organisation of the genome during

138 development.

139

140

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141 Methods

142 Cell culture

143 Cells culture were cultured in standard conditions, 37°C, 5% CO2, in Dulbecco’s Modified Eagle Medium

144 (DMEM) supplemented with 10% fetal bovine serum (FBS). IMR90 cells were obtained from ATCC.

145 IMR90 ER: RAS and ER:Stop cells were produced by retroviral infection of IMR90 human diploid

146 fibroblast cells with pLNC-ER:RAS and pLXS-ER:Stop retroviral vectors respectively as described3. To

147 induce Ras translocation in the nucleus 4-hydroxy-tamoxifen (Sigma) diluted in DMSO was added to

148 cells to a concentration of 100 nM. During senescence induction 4HT containing-medium was changed

149 every 3 days.

150

151 SiRNA transfection

152 2x105 IMR90, ER-STOP and ER-Ras cells were transfected using Dharmafect transfection reagent

153 (Dharmacon) with a 30 nM final concentration of siRNAs. Predesigned siRNAs (Dharmacon) were used

154 to knock down expression as follows:

155 Scr: on target plus non targeting control pool D-001810-10-59

156 TPR: on target plus smart pool L-010548-00

157 TPR-6: on target plus J-010548-06

158 TPR-7: on target plus J-010548-07

159 TPR-8: on target plus J-010548-08

160 TPR-9: on target plus J-010548-09

161 POM121: on target plus smart pool L-017575-04

162 ASF1a: on target plus smart pool L-020222-02-0020

163

164 mRNA expression analysis

165 mRNA expression was determined by IonTorrent mRNA sequencing using the Ion AmpliSeq™

166 Transcriptome Human Kit. 6 independent biological replicates were analysed and

167 adjusted p-value were calculated by Benjamini and Hochberg (BH) and FDR multiple test correction.

168 Data analysis was performed using Babelomics-5 (http://babelomics.bioinfo.cipf.es).

169

170 Immunoblotting

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171 1x106 cells were lysed in RIPA buffer and protein concentration was determined using the Pierce BCA

172 protein analysis kit. 15 µg of proteins were loaded and ran into NuPage 3-8% Tris acetate gels

173 (Invitrogen). Transfer of proteins into a nitrocellulose membrane was performed using the iBlot 2 gel

174 transfer device (Thermofisher). Immunoblotting was done using the following antibodies at the indicated

175 dilutions:

176 TPR: Abcam, ab84516 (1:200)

177 Pom121: Millipore AB6041 (1:500)

178 Actin: Santa Cruz Biotechnology, sc-1616 (1:2000)

179

180 Immunofluorescence

181 2x105 cells were seeded on coverslips and grown during the course of senescence induction. Cells were

182 fixed in 4% paraformaldehyde (pFa) for 10 min at room temperature, permeabilized in 0.1% Triton X100

183 for 10 min, blocked in 1% BSA for 30 min, incubated with primary antibodies diluted in 1% BSA for 1h

184 and with fluorescently labelled secondary antibodies (Life Technologies) for 45 min. Coverslips were

185 counterstained with DAPI and mounted in Vectashield (Vectorlabs).

186

187 Imaging by Structured Illumination Microscopy (SIM) and measurement of nuclear pores density

188 The bottom plane of cells was imaged by 3D SIM (Nikon N-SIM) and reconstructed using NIS element

189 software after immunofluorescence with following antibodies at the indicated diutions:

190 Pom121 (Millipore AB6041) (1:250)

191 TPR (Abcam, ab84516) (1:500)

192 MAB414 (Abcam, ab24609) (1:50)

193 15 nuclei were imaged for each condition and 5 ROI of 100x100 pixels were analyzed/nucleus. Individual

194 nuclear pore complexes in each ROI were counted manually.

195

196 Measurement of SAHF positive cells

197 Cells were stained with DAPI and observed using epifluorescence microscopy. 100-200 cells were

198 counted per condition and the percentage of SAHF positive cells was calculated.

199

200 BrdU incorporation

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201 Cells in culture were incubated with 10 μM 5-Bromo-2’ – deoxyuridine BrdU (Sigma) for 16 hours prior

202 to fixation. Immunofluorescence staining was conducted as described in the immunofluorescence

203 section and detected using the BrdU Antibody (BD Pharmingene 555627) in the presence of 1 mM

204 MgCl2 and 0.5 U/ul DNaseI (Sigma D4527).

205

206 b-Galactosidase staining

207 SA-β-Gal staining solution was prepared using 20x KC(100 mM K3FE (CN)6 and 100 mM K4Fe (CN)

208 6*3H2O in PBS), 20x X-Gal solution (ThermoFisher Scientific) diluted to 1x in PBS/1 mM MgCl2 at pH

209 5.5-6. Staining was conducted overnight on cells fixed with glutaraldehyde.

210

211 Detecting SASP, tumour suppressors and BrdU by high content microscopy

212 Detection of SASP proteins, tumour suppressors and BrdU positive cells by high content microscopy is

213 described at : https://doi-org.ezproxy.is.ed.ac.uk/10.1007/978-1-4939-6670-7_9

214

215 Quantitative reverse transcriptase PCR

216 Total RNA was extracted using the RNeasy minikit (QIAGEN). Complementary DNAs were generated

217 using Superscript II (Life technologies). PCR reactions were performed on a Lightcycler 480 (Roche)

218 using SYBR Green PCR Master Mix (Roche). Expression was normalized to β-actin. Sequences for the

219 primers used are as follows:

220 Pom121-Fw TTCAACGTGAGCAGCACAAC

221 Pom121-Rev CAAAAGTGTTGCCGAAAGGTG

222 TPR-Fw CTGAAGCAATTCATTCGCCG

223 TPR-Rev GGCATATCTTCAGGTGGCCC

224 ASF1a-Fw CAGATGCAGATGCAGTAGGC

225 ASF1a-Rev CCTGGGATTAGATGCCAAAA

226 Actin-Fw CATGTACGTTGCTATCCAGGC

227 Actin-Rev CTCCTTAATGTCACGCACGAT

228 IL1a-Fw AGTGCTGCTGAAGGAGATGCCTGA

229 IL1a-Rev CCCCTGCCAAGCACACCCAGTA

230 IL1b-Fw TGCACGCTCCGGGACTCACA

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231 IL1b-Rev CATGGAGAACACCACTTGTTGCTCC

232 IL6-Fw CCAGGAGCCCAGCTATGAAC

233 IL6-Rev CCCAGGGAGAAGGCAACTG

234 IL8-Fw GAGTGGACCACACTGCGCCA

235 IL8-Rev TCCACAACCCTCTGCACCCAGT

236

237 Statistics

238 All experiments were performed in a minimum of 3 biological replicates. Error bars are standard error

239 of the mean. P-values were obtained by two sample equal variance, 2 tails t-test.

240

241 References

242 1. Ito, Y., Hoare, M. & Narita, M. Spatial and Temporal Control of Senescence. Trends Cell Biol. 243 27, 820–832 (2017). 244 2. Acosta, J. C. et al. Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence. 245 Cell 133, 1006–1018 246 3. Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls 247 paracrine senescence. Nat Cell Biol 15, 978–990 (2013). 248 4. Coppé, J.-P., Desprez, P.-Y., Krtolica, A. & Campisi, J. The Senescence-Associated Secretory 249 Phenotype: The Dark Side of Tumor Suppression. Annu. Rev. Pathol. Mech. Dis. 5, 99–118 (2010). 250 5. Narita, M. et al. Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes 251 during Cellular Senescence. Cell 113, 703–716 (2003). 252 6. Zhang, R. et al. Formation of MacroH2A-Containing Senescence-Associated Heterochromatin 253 Foci and Senescence Driven by ASF1a and HIRA. Dev. Cell 8, 19–30 (2005). 254 7. Chandra, T. et al. Independence of Repressive Histone Marks and Chromatin Compaction 255 during Senescent Heterochromatic Layer Formation. Mol. Cell 47, 203–214 (2012). 256 8. Narita, M. et al. A Novel Role for High-Mobility Group A Proteins in Cellular Senescence and 257 Heterochromatin Formation. Cell 126, 503–514 (2006). 258 9. Zhang, R., Chen, W. & Adams, P. D. Molecular Dissection of Formation of Senescence- 259 Associated Heterochromatin Foci. Mol. Cell. Biol. 27, 2343–2358 (2007). 260 10. Sadaie, M. et al. Redistribution of the Lamin B1 genomic binding profile affects rearrangement 261 of heterochromatic domains and SAHF formation during senescence. Genes Dev. 27, 1800–1808 262 (2013). 263 11. Lenain, C. et al. Massive reshaping of genome–nuclear lamina interactions during oncogene- 264 induced senescence. Genome Res. 27, 1634–1644 (2017). 265 12. Capelson, M. & Hetzer, M. W. The role of nuclear pores in gene regulation, development and 266 disease. EMBO Rep. 10, 697 (2009). 267 13. Dultz, E. & Ellenberg, J. Live imaging of single nuclear pores reveals unique assembly kinetics 268 and mechanism in interphase. J. Cell Biol. 191, 15 (2010). 269 14. Daigle, N. et al. Nuclear pore complexes form immobile networks and have a very low 270 turnover in live mammalian cells. J. Cell Biol. 154, 71 (2001). 271 15. Rabut, G., Doye, V. & Ellenberg, J. Mapping the dynamic organization of the nuclear pore 272 complex inside single living cells. Nat. Cell Biol. 6, 1114 (2004). 273 16. D’Angelo, M. A., Raices, M., Panowski, S. H. & Hetzer, M. W. Age-Dependent Deterioration of 274 Nuclear Pore Complexes Causes a Loss of Nuclear Integrity in Postmitotic Cells. Cell 136, 284–295 275 (2009). 276 17. Doucet, C. M., Talamas, J. A. & Hetzer, M. W. Cell cycle-dependent differences in nuclear 277 pore complex assembly in metazoa. Cell 141, 1030–1041 (2010). 278 18. Funakoshi, T., Clever, M., Watanabe, A. & Imamoto, N. Localization of Pom121 to the inner

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279 nuclear membrane is required for an early step of interphase nuclear pore complex assembly. Mol. 280 Biol. Cell 22, 1058–1069 (2011). 281 19. Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D 282 structured illumination microscopy. Science 320, 1332–1336 (2008). 283 20. Krull, S. et al. Protein Tpr is required for establishing nuclear pore-associated zones of 284 heterochromatin exclusion. EMBO J. 29, 1659–1673 (2010). 285 21. Chien, Y. et al. Control of the senescence-associated secretory phenotype by NF-κB 286 promotes senescence and enhances chemosensitivity. Genes Dev. 25, 2125–2136 (2011). 287 22. Kuilman, T. et al. Oncogene-Induced Senescence Relayed by an Interleukin-Dependent 288 Inflammatory Network. Cell 133, 1019–1031 (2008). 289 23. Frosst, P., Guan, T., Subauste, C., Hahn, K. & Gerace, L. Tpr is localized within the nuclear 290 basket of the pore complex and has a role in nuclear protein export. J. Cell Biol. 156, 617–630 (2002). 291 24. Maeshima, K. et al. Cell-cycle-dependent dynamics of nuclear pores: pore-free islands and 292 lamins. J. Cell Sci. 119, 4442 (2006). 293 25. Hoelz, A., Debler, E. W. & Blobel, G. The Structure of the Nuclear Pore Complex. Annu. Rev. 294 Biochem. 80, 613–643 (2011). 295 296

297 Acknowledgments

298 C.B. was supported by a H2020 Marie-Curie IF (655350 – NPCChr) and by a prize for young researchers

299 from the Bettencourt-Schueller foundation. JCA is supported by a Career Development Fellowship from

300 Cancer Research UK. W.A.B is supported by a Medical Research Council (MRC) University Unit grant

301 MC_PC_U127527202. We thank Robert Illingworth (MRC HGU, Edinburgh) for the Volcano plot of

302 mRNA expression.

303

304 Authors contribution

305 CB and WAB conceived the experiments and designed the experiments together with JCA. P.H

306 performed qRT-PCRs and immunostaining for cytokines of the SASP and siRNA transfections for many

307 of the experiments. CB conducted most of the other experiments including; super-resolution microscopy,

308 counting of nuclear pore densities and identification of cells containing SAHFs. KCFO assisted with

309 immunoblotting. CB and WAB wrote the manuscript with input from all authors.

310

311 Competing interests: The authors declare that they have no competing interests.

312

313 Materials and correspondence:Correspondence and material requests should be addressed to WAB

10 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. ER:STOP ER:Ras a b d kDa

121 POM121

Cytoplasmic filaments 42 Actin cytoplasm Cytoplasmic ring c IMR90-ER:STOP Transmembrane nucleoporins ER:STOP ER:Ras nuclear envelope POM121 Inner ring Nucleoplasmic ring 270 TPR 4HT nucleus

proliferation Nuclear basket IMR90-ER:Ras TPR 42 Actin

4HT senescence

e TPR staining f TPR g POM121

*** 10 10 2 2 8 8 H.S

6 6 ER STOP

4 4

Nuclear pores/µm 2 Nuclear pores/µm 2

0 0 ER:STOP ER:Ras ER:STOP ER:Ras ER STOP ER Ras

MAB414 staining

h siRNA siRNA siRNA i j transfection transfection transfection fixation TPR

+ 4HT Scr 8

day 0 3 5 8 2 6

* siRNA: 4

Nuclear pores/µm 2 POM121

0 siRNA: Scr POM121 ER:STOP ER:STOP ER:Ras ksiRNA: Scr Scr POM121 l

70 60

50 * 40

30

20 % SAHF +ve cells

10 HS 0 siRNA: Scr Scr POM121

ER:STOP ER Ras

Figure 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.

Figure 1. Increase of nuclear pores density in OIS is necessary for SAHF formation

a) Schematic showing the balance of forces attracting heterochromatin to the nuclear lamina and

repelling heterochromatin from nuclear pores.

b) Model of the nuclear pore complex indicating the position of the nucleoporins TPR and POM121,

adapted from 25.

c) Schematic of OIS induction in IMR90 cells by 4HT and continued proliferation in control ER-Stop

cells.

d) Western blot showing POM121 (left panel) and TPR (right panel) protein levels in 4HT treated ER-

Stop and ER-Ras cells.

e) TPR Immunostaining in ER-STOP and ER-Ras cells treated with 4HT. Left: bottom plane of nucleus

imaged by SIM. Right: enlargement of the insets. Scale bars 2 µm.

f) Mean (+/- SEM) nuclear pore density (pores/µm2) in 4HT treated ER-Stop and ER-Ras cells as

counted by TPR staining in 3 independent biological replicates, ***p=0.0001.

g) As for f) but for Pom121 staining. H.S = highly significant p=1.3E-06.

h) Schematic showing depletion experiment as performed for panels J) and K)

i) MAB414 (antibody recognizing several nucleoporins) immunostaining in ER STOP cells treated

with 4HT after 2 days-knockdown with scramble (Scr) or POM121 siRNAs. Left: bottom plane of

nucleus imaged by SIM. Right: enlargement of the insets. Scale bars 2 µm.

j) Mean (+/- SEM) nuclear pore density (pores/µm2) in 4HT treated ER-Stop cells after knock down

with scramble (Scr) or POM121 siRNAs as assayed by TPR staining in 3 independent biological

replicates, *= p<0.05.

k) DAPI staining of 4HT-treated ER-Stop and ER-Ras cells in controls (Scr) and upon POM121

depletion (siPOM121). Scale bars 10 µm. Bottom: enlargement of the insets.

l) Mean (+/- SEM) % of cells containing SAHF in 4HT-treated ER-Stop and ER-Ras cells after

knockdown with scramble (Scr) siRNAs and in 4HT-treated ER Ras cells with POM121 SiRNAs.

Data from 3 independent experiments. *=p<0.05 HS=highly significant.

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. a ER:STOP ER:Ras b siRNA: Scr Scr TPR 90 80 70 60 50 ** 40 30

% SAHF +ve cells 20 10 HS 0 siRNA: Scr Scr TPR ER:STOP ER:Ras

c 90 80 70 60 50 40 30

% SAHF +ve cells 20 10 0 day 0 1 2 34 5 9876 10 0 1 2 34 5 9876 10 ER:STOP ER:Ras

ER:STOP ER:Ras d siRNA e f transfection fixation + 4HT 90 80 day 0 3 6 8 70 Si:Scr 60 ** 50 40

30 % SAHF +ve cells 20 HS 10

Si:TPR 0 siRNA: Scr Scr TPR ER:STOP ER:Ras

g DAPI TPR merge ER:STOP siTPR ER:Ras

siSCR

Figure 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.

Figure 2. TPR is necessary for SAHF formation and maintenance

a) DAPI staining of non-senescent 4HT treated ER-Stop and OIS (ER-Ras) cells after control

scrambled (Scr) SiRNA and upon TPR depletion (siTPR). Scale bars 10 µm.

b) Mean (+/- SEM) % of cells containing SAHF in 4HT-treated ER-Stop and ER-Ras cells after

knockdown with scramble (Scr) siRNAs and in 4HT-treated ER Ras cells with TPR SiRNAs. Data

from 3 independent experiments. **=p<0.01 HS=highly significant.

c) Time course of mean (+/- SEM) % cells with SAHF after 4HT-treatment of control (ER-Stop) and

OIS cells (ER-Ras). Data from 3 independent experiments.

d) Time course for TPR depletion by siRNA late in the OIS programme as performed for panels e-g.

e) DAPI staining of 4HT-treated ER STOP and OIS cells ER:Ras in controls (Scr) and upon TPR

depletion (siTPR). Scale bars 2 µm.

f) Mean (+/- SEM) % of cells containing SAHF in 4HT-treated ER-Stop and ER-Ras cells after

knockdown with scramble (Scr) siRNAs and in ER-Ras cells with TPR SiRNAs. Data from 3

independent experiments. **=p<0.01 HS=highly significant.

g) DAPI (blue) and TPR (red) staining of 4HT-treated ER-STOP and ER-Ras upon TPR depletion

(siTPR) imaged by SIM. Right: enlargement of the insets. Scale bars 2 µm.

siRNA: d c a e

+ 4HT FCS: day siRNA: CM: siRNA: IL1ß IL1α Relative mRNA expression 0.2 0.4 0.6 0.8 1.0 0 ER STOP/Ras ER:Ras ER:STOP 0

ER:STOP 10% p65 DAPI bioRxiv preprint c c TPR Scr Scr ER:STOP Scr 3 Scr not certifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. doi:

https://doi.org/10.1101/374033 1% ER:Ras (CM) collect medium Scr HS IL8 IL6 IL1ß IL1α 9 TPR ER:Ras ; this versionpostedJuly21,2018. day siRNA: b % +ve cells 0 10 20 30 40 50 60 IMR90 0 TPR ER:STOP Scr siRNA: f

% +ve nuclear p65 cells HS 70 10 20 30 40 50 60 80 0 RSO MER:Ras CM ER:STOP CM c P c TPR Scr TPR Scr The copyrightholderforthispreprint(whichwas Scr 5 siRNA transfection ER:Ras **

8 7 serum starve for 12h TPR ** ** IL8 IL6 IL1ß IL1α Add CM for 30 min ***

n.s fixation Figure 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.

Figure 3. TPR is necessary for the SASP

a) Mean (± SEM) mRNA level, measured by qRT-PCR for SASP genes (IL1a, IL1b, IL6,IL8), in 4HT-

treated ER-Stop and ER-Ras cells after knockdown with scramble (Scr) siRNAs and in 4HT-treated

ER-Ras cells with TPR SiRNAs. Expression is shown relative to ER-Ras cells transfected with Scr

siRNAs. Data from 3 independent experiments.

b) Mean (± SEM) % of cells positive for SASP cytokines (IL1a, IL1b, IL6,IL8) in 4HT-treated ER-Stop

and ER-Ras cells after knockdown with scramble (Scr) siRNAs and in 4HT-treated ER-Ras cells

with TPR SiRNAs, measured by immunostaining. Data from 3 independent experiments. **=p<0.01

***= p<0.001, HS=highly significant.

c) Immunostaining (green) for SASP cytokines IL1a and IL1b in DAPI (blue) stained nuclei of 4HT-

treated ER-Stop and ER-Ras cells subjected to RNAi with scrambled (Scr) siRNAs or siRNAs

targeting TPR. Scale bars 100 µm.

d) Schematic of NFkb nuclear import assessment as performed for panels e and f. Medium (CM) of

ER STOP and Ras cells was collected after 9 days of culture in presence of 4HT. CM was then

added to IMR90 cells upon scramble (Scr) or TPR depletion by SiRNA. NFkb-p65 import in the

nucleus of IMR90 cells was assessed 30 min after addition of CM.

e) Immunostaining (red) for p65 in DAPI (blue) stained cells for scrambled (Scr) or TPR (siTPR)

depleted IMR90 cells upon addition of collected medium (CM) from 4HT-treated ER-Stop and ER-

Ras cells. Scale bars 100µm.

f) Mean (+/- SEM) % of cells containing nuclear p65 staining in scrambled (Scr) or TPR (siTPR)

depleted IMR90 cells upon addition of collected medium (CM) from 4HT-treated ER-Stop and ER-

Ras cells. Data from 3 independent experiments. n.s=non significant.

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. a 1.0

0.8

0.6

0.4

0.2 * ASF1a relative mRNA expression ASF1a relative mRNA 0 siRNA: Scr Scr ASF1a ER:STOP ER:Ras

b ER:STOP ER:Ras c 80 siRNA: Scr Scr ASF1a 70 60 50 40 30 ***

% SAHF +ve cells 20 10 0 siRNA: Scr ASF1a Scr ASF1a ER:STOP ER:Ras

d MAB414 staining e TPR staining f n.s 1.0 n.s IL1α 4 4 IL1ß 0.8

2 2 IL6 3 3 IL8 0.6

2 2 0.4 *** Nuclear pores/µm Nuclear pores/µm 1 1

Relative mRNA expression 0.2 HS HS HS

0 0 0 siRNA: Scr ASF1a siRNA: Scr ASF1a siRNA: Scr Scr ASF1a ER:STOP ER:Ras

Figure 4 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.

Figure 4. SAHF formation is necessary for the SASP

a) Mean (± SEM) ASF1a mRNA level, established by qRT-PCR, in 4HT-treated ER-STOP and ER-

Ras cells after knockdown with scramble (Scr) or ASF1a siRNAs. Expression is shown relative to

ER-STOP cells transfected with Scr siRNAs. Data from 3 independent experiments. *= p<0.05.

b) DAPI staining of 4HT-treated ER-STOP and ER-Ras cells in controls (Scr) and upon ASF1a

depletion (siASF1a). Scale bars 2 µm

c) Mean (+/- SEM) % of cells containing SAHF in 4HT-treated ER-Stop and ER-Ras cells after

knockdown with scramble (Scr) siRNAs and in ER-Ras cells with ASF1a SiRNAs. Data from 3

independent experiments. ***=p<0.001.

d) Mean (+/- SEM) nuclear pore density (pores/µm2) in 4HT treated ER-Stop cells after knock down

with scramble (Scr) or ASF1a (siASF1a) siRNAs as counted by MAB414 staining in 3 independent

biological replicates, n.s= non significant.

e) As in d) but for TPR staining. n.s= non significant.

f) Mean (± SEM) mRNA levels, measured by qRT-PCR, for SASP genes (IL1a, IL1b, IL6, IL8), in

4HT-treated ER-Stop and ER-Ras cells after knockdown with scramble (Scr) siRNAs and in 4HT-

treated ER-Ras cells with ASF1a SiRNAs. Expression is shown relative to ER-Ras cells transfected

with Scr siRNAs. Data from 3 independent experiments. ***=p<0.001, HS=highly significant.