ARTICLE Received 31 May 2011 | Accepted 10 Aug 2011 | Published 13 Sep 2011 DOI: 10.1038/ncomms1473 p53 and p16INK4A independent induction of senescence by chromatin-dependent alteration of S-phase progression Alexandre Prieur1,*, Emilie Besnard1,*, Amélie Babled1 & Jean-Marc Lemaitre1,2 Senescence is triggered by various cellular stresses that result in genomic lesions and DNA damage response activation. However, the role of chromatin and DNA replication in senescence induction remains elusive. Here we show that downregulation of p300 histone acetyltransferase activity induces senescence by a mechanism that is independent of the activation of p53, p21CIP1 and p16INK4A. This inhibition leads to a global H3, H4 hypoacetylation, initiating senescence-associated heterochromatic foci formation during S phase, together with a global decrease in replication fork velocity, and alteration of DNA replication timing. This replicative stress occurs without DNA damage and checkpoint activation, but results in a robust G2/M cell cycle arrest, within only one cell cycle. These results provide new insights into the control of S-phase progression by p300, and identify an unexpected chromatin- dependent alternative mechanism for senescence induction, which could possibly be exploited to treat cancer by senescence induction without generating further DNA damage. 1 INSERM, Avenir Team ‘Genome Plasticity and Aging’,–Functional Genomics Institute, 141 rue de la Cardonille, 34094 Montpellier, Cedex 05, France. 2 Centre Régional de Lutte contre le Cancer Val d’Aurelle-Paul Lamarque–208, rue des Apothicaires, Parc Euromédecine, 34298 Montpellier, Cedex 05, France. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.-M.L. (email: [email protected]). NatURE COMMUNicatiONS | 2:473 | DOI: 10.1038/ncomms1473 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1473 ellular senescence is characterized by a stable cell cycle arrest formation during S phase. This inhibition leads to a senescence-like that is triggered by various forms of stress stimuli including growth arrest in G2/M, within only one cell cycle. Interestingly, we Concogene activation (oncogene-induced senescence, OIS) or observed an alteration in the replication activity characterized by a telomere shortening (replicative senescence), and is considered as a global decrease in replication fork velocity, and a change in DNA barrier to tumourigenesis1. This arrest depends on the activation of replication timing, occurring in the absence of DNA damage and the cyclin-dependent kinase (CDK) inhibitors (p21CIP1 and p16INK4A), checkpoint activation. Finally, we show that the downregulation of components of the tumour-suppressor pathways that are governed p300 HAT activity leads to p53, p21CIP1 and p16INK4A-independent by the p53 and retinoblastoma (pRB) proteins, respectively2. Acti- senescence. These results reveal an unexpected and new mechanism vation of the DNA damage response (DDR), triggered by DNA for senescence induction involving inhibition of p300 activity. Our single-strand and/or double-strand breaks, has been described as a findings suggest that chromatin-dependent alteration can robustly common feature of telomere-initiated or OIS3,4. Moreover, dramatic induce senescence without activation of the commonly associated changes in chromatin structure seem to contribute to the irrevers- senescence pathways. ible nature of the senescent state, especially through the formation of senescence-associated heterochromatic foci (SAHF), which are Results characterized by hypoacetylation of histones, tri-methylation of Inhibition of p300 HAT activity induces senescence. We used two histone H3 on lysine 9 (H3K9me3) and the presence of facultative short hairpin RNAs (shp300-1 and shp300-2) to stably inhibit p300 heterochromatin proteins5–7. Chromatin organization is partly regu- expression in the hTERT-immortalized human diploid fibroblast lated by the balance of activity between histone acetyltransferases (HDF) cell line TIG3(et). In response to reduced p300 expression, (HATs) and histone deacetylases. The HAT p300, which is involved we observed the upregulation of the CDK inhibitors p16INK4A and in the regulation of cellular growth, differentiation and survival, can p21CIP1, an increase in senescence-associated β-galactosidase (SA- directly modify the chromatin state and interact with complexes that β-Gal) activity and SAHF formation (Fig. 1a–c). These changes are mediate chromatin metabolism8,9. We therefore decided to study the hallmarks of cellular senescence2. Assuming that cellular senescence role of p300 in senescence induction. can be triggered by different stresses, including oncogene activation Here demonstrate that repression of p300 HAT activity, which or telomere shortening2, we wondered whether inactivation of p300 induces global histone H3 and H4 hypoacetylation, initiates SAHF activity also occurs in these types of cellular senescence and may a shp300 b shp300 c shp300 kDa (–) #1 #2 Q (–) #1 #2 (–) #1 #2 2% 86% 68% t p300 230 23 p21CIP1 Hoechs 17 p16INK4A 3 46 β-Actin H3K9me Curcumin (µM) d TIG3(et) e f 45 Day 8 Day 12 0 µM 0 6 9 40 Curc. ( M) 6 µM kDa 0 6 9 0 6 9 µ 2% 58% 86% r 23 CIP1 35 9 µM p21 12 µM 30 15 µM 17 INK4A 25 30 µM p16 TIG3(et) 20 46 β-Actin 2% 62% 92% 15 10 Relative cell numbe 5 BJ(et) 0 1 3 5 7 9 11 13 Days g Hoechst H3K9me3 HP1γ Overlay Hoechst HMGA1 Overlay 16 0 µM BJ(et) 14 0 12 6 µM 10 9 µM 8 M) µ 12 µM ( 6 6 4 Relative cell number 2 0 Curcumin 1 3 5 7 9 11 13 Days 9 Figure 1 | Inhibition of p300 HAT activity induces senescence. (a–c) Stable downregulation of p300 by expression of two distinct shRNAs #1, #2 in TIG3(et) cells. Cells were collected and stained 12 days after selection. (a) Immunoblot analysis with β-actin as a loading control and Q for quiescent cells. (b) Images of SA-β-gal-stained (upper panel) and Hoechst-stained (lower panel) cells to detect SAHF. Scale bars are equal to 5 µm. (c) Images of a large field with H3K9me3 and Hoechst staining to detect SAHF. Scale bars are equal to 10 µm. (d–g) TIG3(et) and BJ(et) cells treated with different doses of curcumin or vehicle (0) as control. (d) Analysis by proliferation curve. Means of three experiments with standard deviation. (e) Immunoblot analysis with β-actin as a loading control for TIG3(et) cells. (f–g) Cells were collected and stained 12 days after selection. (f) SA-β-gal activity. Scale bars are equal to 40 µm. (g) Immunofluorescence analysis with specific antibodies for co-localization with SAHF for TIG3(et) cells. Scale bars are equal to 5 µm. (b,f) Percentage of SA-β-gal-positive cells is indicated in insert. NatUre cOMMUNicatiONS | 2:473 | DOI: 10.1038/ncomms1473 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. NatURE COMMUNicatiONS | DOI: 10.1038/ncomms1473 ARTICLE participate in cell cycle arrest. Interestingly, we observed a dramatic Because the G2/M senescence-like growth arrest occurs within decrease in p300 expression levels in HDF cells in which premature one cell cycle, we also hypothesized that S-phase progression might senescence was induced by overexpressing the oncogene RasV12 also be required for senescence induction by p300 HAT inhibi- (Supplementary Fig. S1a,b). Similarly, induction of replicative tion. To test this possibility, we co-treated curcumin-treated cells senescence on serial passaging in non-immortalized fibroblasts also with mimosine, an inhibitor of the G1/S transition, and found lead to a strong decrease in p300 expression (Supplementary Fig. that ongoing replication was necessary for SAHF formation in this S1c,d). Finally, we did not observe downregulation of p300 expression context (Fig. 2e). in quiescent cells (Fig. 1a), suggesting that downregulation of p300 Finally, we wondered whether p300 inhibition could promote a might be a common event specifically associated with the senescence SAHF-dependent induction of senescence. We inhibited the expres- induction process. sion of HMGA1, which is known to block SAHF formation and To obtain further insight into the mechanisms involved, we spe- senescence induction6, with a specific shRNA. We observed that cifically inhibited the HAT activity of p300, which is the major activ- SAHF formation was required for the G2/M senescence-like growth ity of this protein responsible for acetylation of histone H3 and H4. arrest induced by inhibition of p300 HAT activity (Fig. 2f-h), estab- We treated the HDF TIG3(et) cells for 12 days with increasing doses lishing a direct link between SAHF formation and cell cycle arrest. of curcumin, a potent inhibitor of p300 HAT activity10. We observed a proliferation arrest even at low doses ( < 12 µM) (Fig. 1d). This cell Alteration of S-phase progression by p300 HAT inhibition. cycle arrest was associated with the upregulation of CDK inhibitors, Because SAHF formation is described as a multistep process involv- p16INK4A, and p21CIP1, an increase in SA-β-Gal activity and formation ing acetylation changes of chromatin7, we investigated the effect of of SAHF, which co-localized with HP1γ, H3K9me3 and HMGA1, p300 inhibition on histone acetylation. Interestingly, we observed an as previously described6 (Fig. 1e,f,g). We confirmed these results immediate and global hypoacetylation of histones H3 and H4 in cur- in another hTERT-immortalized HDF cell line, BJ(et) (Fig. 1d and cumin-treated and p300kd HDF cells (Fig. 3a), which was also pre- f), demonstrating that inhibition of p300 HAT activity by curcu- vented by CTPB, the specific activator of p300 HAT activity Fig.( 3b). min induces premature senescence in HDF, thus recapitulating the Moreover, the chromatin loading of the minichromosome mainte- effects of p300 downregulation. To further confirm that inhibition nance protein 7 (MCM7), a member of the pre-replicative complex, of p300 HAT activity is involved in senescence induction, we used and the sliding clamp protein PCNA, were impaired, revealing that CTPB, a specific activator of p300 activity10.