Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage-and Histone Methylation-Dependent Pathways

Downregulation of EZH2 Results in DNA Damage and Induces p21

Because global levels of H3K27me3 were not affected at the onset of replicative senescence or senescence induced by knockdown of EZH2, we examined the presence of H3K27me3 marks at the CDKN2A locus by performing chromatin immunoprecipitation (ChIP) coupled with qPCR (ChIP-qPCR). No significant changes in H3K27me3 enrichment were observed 4 days after infection with shEZH2, with a decrease becoming apparent at 8 days (Figures 2A and 2B). These data reflect closely the global levels of H3K27me3 and suggest that loss of EZH2 can induce senescence independent of epigenetic changes at the CDKN2A locus.

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Figure 2

Downregulation of EZH2 Induces DNA Damage and Senescence in a DNA Replication-Dependent Manner

(A) H3K27me3 enrichment at the CDKN2A locus was determined 4 and 8 days after shEZH2 infection using ChIP. For locations of primer pairs, see (B). Normal rabbit immunoglobulin G (IgG) was used as the immunoprecipitation (IP) control (**p < 0.01, n = 3).

(B) Schematic of the CDKN2A locus.

(C) The frequency of p21-expressing cells was scored by IF after knockdown of EZH2 (percent of total cells, random fields, more than 400 cells per condition, **p < 0.01).

(D) The frequency of p16-expressing cells was determined as in (C) (*p < 0.05).

(E) Early passage LF1 cells were grown in the presence of DZNep (5 μM), and the levels of p16, p21, and EZH2 proteins and H3K27me3 marks were examined by immunoblotting after 9 and 18 days of treatment.

(F) p21 shRNAs or the empty vector were combined with an EZH2 shRNA (+) or a shGFP control (−), and SA-β-Gal-positive cells were scored 4 days after infection (*p < 0.05, **p < 0.01, n = 3).

(G) 53BP1 foci were visualized by IF 4 and 8 days after EZH2 knockdown. The stacked bars depict the fraction of cells with 1 focus (blue) or 2 (red), 3 (green), and more than 4 (black) foci per nucleus (**p < 0.01, n = 4).

(H) shATM and shEZH2 knockdowns were combined, and SA-β-Gal-positive cells were scored as in (F) (**p < 0.01, n = 3).

(I) At the time of shEZH2 infection (time [t] = 0; Figure S1G), cells were reseeded into either normal medium (Pro, 15% FBS), quiescence-inducing medium (Qui, 0.25% FBS), or medium supplemented with thymidine (Thy). 53BP1 foci were scored after 4 days (**p < 0.01, n = 3).

(J) EZH2 was knocked down as in (A), and cells were pulse-labeled with EdU for 1 hr 2 days after infection. A representative image showing colocalization of EdU (red) and 53BP1 (green) signals is shown. Right: magnified views of the indicated areas. Scale bar, 3 μm.

Error bars represent SD. See also Figures S3 and S4 and Tables S1 and S2.

Four days after knockdown of EZH2, RNA-seq analysis of differential gene expression implicated p53 and p21 activation as the top upstream regulators (Figure S2C). To examine the activation of senescence pathways elicited by the downregulation of EZH2 in more detail, we monitored the time course of p21 and p16 expression changes. p21 mRNA was induced 4 days after EZH2 knockdown (Figure S3A), whereas p16 mRNA was unchanged at this time, becoming upregulated only later (8 days; Figure S3B). The earliest time after infection we could assess was 2 days, at which point SA-β-Gal staining showed that the majority of cells (93%) were already senescent (Figure 1G). Likewise, examining single cells by immunofluorescence, we found that, at the 2-day time point, 83% of cells were positive for p21 protein (Figure 2C). The frequency of p21-positive cells declined over time, with only 10% of cells being positive at 8 days.

A similar analysis of p16 showed no expression at early time points, with the appearance of p16-positive cells becoming significant at 6 and 8 days (Figure 2D). In agreement, treatment with the drug 3-deazaneplanocin A (DZNep), which is known to deplete cellular EZH2 protein levels (Tan et al., 2007), induced senescence characterized by early activation of p21 followed by upregulation of SASP genes and p16 and loss of H3K27me3 marks (Figures 2E and S3C and S3D). To examine whether upregulation of p21 is required for the induction of senescence by EZH2 depletion, we performed double knockdowns and found that p21 knockdown opposed the appearance of senescent cells (Figures 2F and S3E–S3G). Collectively, these results indicate that p21 is an important and early effector engaged by EZH2 downregulation, and that p16 upregulation occurs later and coincides with the loss of H3K27me3 at the CDKN2A locus.

Because the primary regulator of p21 in senescence is p53, which, in turn, is activated by DNA damage, we examined components of the DDR after knockdown of EZH2. Knockdown of EZH2 led to a strong and rapid induction of 53BP1 DNA damage foci. Quantification of individual cells showed a significant increase in the frequency of both focus-containing cells as well as the number of foci per nucleus (Figure 2G).

To determine whether activation of ATM is required for the induction of senescence by EZH2 downregulation, we performed double knockdowns of ATM and EZH2. We found that ATM knockdown significantly alleviated the onset of senescence (Figures 2H and S3H and S3I). To determine whether the DDR elicited by EZH2 downregulation is localized to telomeres, we performed an assay that colocalizes DNA damage foci with telomeres (Herbig et al., 2006). We did not find significant changes in DNA damage occurring specifically at telomeres upon knockdown of EZH2 (Figures S3J and S3K). Similarly, knockdown of EZH2 in LF1 cells immortalized by expression of telomerase robustly induced senescence, as indicated by SA-β-Gal staining (Figure S3L), suggesting that telomere maintenance is not sufficient to prevent senescence because of EZH2 depletion. Altogether, these data indicate that senescence elicited by EZH2 downregulation is triggered initially by non-telomeric double-strand breaks (DSBs) and mediated by the ATM-p53-p21 DDR pathway.

Induction of DNA Damage by EZH2 Depletion Is Dependent on DNA Replication

Because EZH2 and other PRC2 components localize to sites of DNA replication (Hansen et al., 2008; Leung et al., 2013), and RNA-seq analysis showed the top downregulated ontologies upon knockdown of EZH2 to be associated with DNA strand elongation, DNA replication, and G1-S phase transition (Figure S2A), we examined the effect of cell cycle progression on the ability of EZH2 knockdown to elicit a DDR. We found that arresting the cell cycle, either in G0 by serum withdrawal or in late G1 by a thymidine block, strongly protected against DDR induction, upregulation of p21, and onset of senescence (Figures 2I and S3M and S3N). To determine whether DNA damage localizes to sites of DNA replication, we knocked down EZH2 under proliferative conditions and pulse-labeled with EdU before the cultures became fully senescent. We found that, in many of the cells still capable of EdU incorporation, 53BP1 foci frequently colocalized with sites of EdU labeling (Figure 2J). This is in agreement with recent studies showing that loss of PcG proteins affects the symmetry and progression of DNA replication forks and promotes their collapse (Piunti et al., 2014). Altogether, these data suggest that the DDR and ensuing senescence triggered by EZH2 depletion are dependent on DNA replication.

To further examine the mechanism by which EZH2 depletion induces replication fork stress, we ablated the recognition of the H3K27me3 mark by the PRC2 component EED. EED-H3K27me3 interaction was perturbed in two ways: with a small-molecule inhibitor (EED226) that blocks the H3K27me3-binding pocket of EED (Qi et al., 2017) and by expressing EED mutants unable to bind H3K27me3 marks (Margueron et al., 2009). Treatment with EED226 strongly induced the presence of SA-β-Gal-positive cells, upregulation of both p21 and p16, the appearance of 53BP1 DNA damage foci, and activation of a SASP response (Figures S4A–S4F). Similarly, ectopic expression of a mutant EED (F97A, W364A, or Y365A) unable to bind H3K27me3 elicited a DDR and upregulated SASP genes (Figures S4G and S4H), albeit to a lesser extent than treatment with EED226. In aggregate, these data suggest that the mislocalization or depletion of the PRC2 complex away from the replication fork induces a DDR through replication fork stress.

Loss of H3K27me3 Marks Induces Senescence by Upregulating p16 and SASP in a DNA Damage-Independent Manner

The temporal correlations between declining H3K27me3 levels, p16 upregulation, and activation of SASP genes suggest a possible contribution of H3K27me3 loss to the phenotypes of senescent cells. We therefore examined the effects of GSK126, a highly selective S-adenosyl-methionine (SAM)-competitive inhibitor of EZH2, on early-passage HDFs (McCabe et al., 2012). GSK126 inhibited EZH2 enzymatic activity within 4 days of treatment, as indicated by the loss of H3K27me3, without altering EZH2 protein levels (Figure 3A). Treatment with GSK126 also elicited senescence in a dose-dependent manner, as evidenced by the inhibition of proliferation and increase in SA-β-Gal staining. These effects were first evident at 8–10 days and continued to increase over a period of 30 days (Figures 3B and 3C and S5A).

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Figure 3

Inhibition of EZH2 Activity Induces Senescence in the Absence of DNA Damage by Depleting H3K27me3 Marks and Activating p16 and SASP Genes

(A) Early passage LF1 cells were grown continuously in the presence of GSK126, and levels of EZH2 protein and H3K27me3 marks were examined by immunoblotting.

(B and C) Cells were treated with GSK126 as in (A), and proliferation (B) or SA-β-Gal staining (C) were determined (**p < 0.01, n = 3).

(D and E) Cells were treated as in (A), and the frequency of p21-positive (D) and p16-positive (E) cells was scored by IF (percent of total cells, random fields, more than 400 cells per condition, *p < 0.05, **p < 0.01).

(F) Cells were treated with GSK126 for 8 days and examined for 53BP1 foci as in Figure 2G (n = 3).

(G) Cells were infected with lentivirus vectors expressing EZH2 cDNA, EZH2 with an in-frame deletion of the SET domain (EZH2ΔSET), or empty vector control, and expression of the indicated genes was determined by real-time qPCR 9 days after infection. Data are represented as heatmaps relative to control cells (n = 3).

(H) Cells were treated with GSK126 (5 μM), JQ1 (100 nM), or both, and expression of the indicated genes was determined by real-time qPCR after 10 days. Data are represented as heatmaps relative to DMSO-treated cells (n = 3).

(I) Cytokine array analysis of conditioned medium from cells treated with GSK126 (5 μM) or GSK126 plus JQ1 (100 nM) for 17 days. For comparison, cells were infected with shRNAs against EZH2 (shEZH2) or GFP (control [CTR]) for 8 days. IL1A and IL1B were secreted at undetectable levels, as reported previously (Orjalo et al., 2009), because they remain cell surface-associated. Data are represented as heatmaps relative to CTR (n = 2). See Table S3 for the measured cytokine concentrations.

(J) Cells were treated with GSK126 (5 μM) or DMSO for 10 days, and H3K27me3 and H3K27ac enrichment at the IL1A-IL1B-IL37 loci was determined by ChIP-qPCR. Primer pairs were chosen in regions of putative enhancers, and their locations are shown in the schematic as red bars. H3K27ac and H3K4me1 tracks were obtained from ENCODE (GEO: {"type":"entrez-geo","attrs":{"text":"GSM469966","term_id":"469966"}}GSM469966 and {"type":"entrez-geo","attrs":{"text":"GSM521895","term_id":"521895"}}GSM521895). Normal rabbit IgG was used as the IP control (*p < 0.05, **p < 0.01, n = 3).

Error bars represent SD. See also Figure S5 and Tables S1, S2, and S3.

To gain further insights into how loss of the H3K27me3 modification induces senescence, we investigated the expression of p21 and p16 proteins using single-cell immunofluorescence (IF) assays. Treatment of cells with GSK126 significantly increased the frequency of p16-positive cells but had no effect on p21 expression (Figures 3D and 3E). Furthermore, in contrast to knockdown of EZH2, inhibition of its activity with GSK126 did not elicit a DDR (Figure 3F), which agrees with the absence of p21 induction. To explore another method of inhibiting EZH2 activity, we used a catalytically inactive EZH2 mutant that lacks the conserved SET domain (EZH2ΔSET). Similar to what was observed after treatment with GSK126, infection with an EZH2ΔSET-expressing lentivirus vector induced senescence, accompanied by upregulation of p16 without triggering a DDR or upregulating p21 (Figures S5B–S5E).

To investigate the effects of EZH2 inhibition in non-replicating cells, quiescent LF1 cells were treated with GSK126 and monitored for changes in H3K27me3 and p16 levels. Although GSK126 treatment effectively depleted H3K27me3 marks within 4 days in proliferating cells (Figure 3A), it took at least 21 days to observe appreciable changes in quiescent cells (Figures S5F and S5G). Hence, loss of EZH2 methyltransferase activity can deplete H3K27me3 marks and induce p16 independent of cell cycle progression, albeit with much reduced kinetics.

H3K27me3 marks have been reported to decrease at enhancer regions of many SASP genes during senescence (Shah et al., 2013). In contrast, senescence leads to an increase in histone H3 lysine 27 acetyl (H3K27ac) at enhancers adjacent to SASP genes (Tasdemir et al., 2016). SASP factors were broadly upregulated and secreted following treatment with GSK126 or EZH2ΔSET-expressing lentivirus (Figures 3G–S3I). We therefore examined whether loss of H3K27me3 is associated with enrichment of H3K27ac at enhancers of SASP genes. Indeed, we found, using ChIP-qPCR, that treatment of proliferating cells with GSK126 resulted in a decrease of H3K27me3 and increase of H3K27ac marks at annotated enhancers adjacent to IL1A and IL1B (Figure 3J) and MMP3 (Figure S5H) genes.

Members of the bromodomain and extraterminal domain (BET) family of proteins are the “readers” that recognize acetylated lysine residues and facilitate transcriptional activation by recruitment of co-regulatory complexes. Accordingly, treatment with the small molecule BET inhibitor JQ1 antagonized the expression and secretion of GSK126-induced SASP factors (Figures 3H and 3I). However, JQ1 did not affect the GSK126-elicited upregulation of p16 or rescue senescence (Figures S5I and S5J). These data suggest that the H3K27 methylation-to-acetylation switch at enhancers of SASP genes mediates their activation but does not have broader effects on senescence.

SASP gene expression in senescent cells is also regulated, in part, by the mammalian target of rapamycin (mTOR) (Laberge et al., 2015). mTOR activity promotes the translation of the cytokine IL1A, which is then processed at the membrane and binds the IL1R receptor, which, in turn, activates the degradation of IκB, an inhibitor of nuclear factor κB (NF-κB) transcription factors. We found that, in GSK126-treated cells, the mTOR inhibitor rapamycin reduced the transcript levels of some SASP genes upregulated by GSK126 (Figure S5K). Treatment with GSK126 also decreased the levels of IκBα (Figure S5L), and this was rescued by treatment with rapamycin, Hence, NF-κB and mTOR contribute to the regulation of SASP genes during GSK126-induced senescence in a manner that is shared with other forms of senescence. In aggregate, these experiments indicate that inhibition of EZH2 methyltransferase activity is sufficient to induce senescence in the absence of DNA damage and that this response is characterized by the upregulation of p16 and accompanied by SASP.

EZH2 Suppresses Entry into Oncogene-Induced Senescence

We next examined whether preventing the downregulation of EZH2 that normally occurs during senescence could antagonize its establishment. We induced OIS using a 4-hydroxytamoxifen (4-OHT)-inducible estrogen receptor (ER):RAS(G12V) fusion protein and combined this with ectopic expression of EZH2 cDNA delivered with a lentivirus vector (Figure 4A). Expression of EZH2 strongly alleviated entry into OIS, as indicated by cell proliferation, frequency of SA-β-Gal positive cells, and p16 expression (Figures 4B–4D). Additionally, EZH2 also broadly alleviated the induction of SASP genes (Figure 4E). These data indicate that maintenance of EZH2 expression and, hence, H3K27me3 marks suppresses entry into senescence in response to stress.

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Figure 4

Expression of EZH2 Opposes Entry into OIS and Induction of SASP

(A) LF1 cells were engineered using lentivirus vectors to stably express EZH2 cDNA and a 4-OHT-inducible ER:RAS(G12V) protein. Control cell lines were constructed using empty vectors. Cells were treated with 4-OHT (200 nM) or vehicle (ethanol) for the indicated times, and the levels of EZH2 and ER:RAS proteins were determined by immunoblotting.

(B) Proliferation in the experiment shown in (A) was assessed by counting cell numbers (*p < 0.05, **p < 0.01, n = 3).

(C) SA-β-Gal-positive cells were scored after 12 days of 4-OHT treatment in the experiment shown in (A) (**p < 0.01, n = 3).

(D) p16 mRNA expression was determined by real-time qPCR as in (C) (**p < 0.01, n = 3).

(E) Expression of the indicated SASP genes was determined by real-time qPCR as in (C) (*p < 0.05, **p < 0.01, n = 3).

Error bars represent SD. See also Table S1.

Lentiviral Infections

293T cells were co-transfected with plasmid DNAs of a lentiviral vector and the helper vectors pVSV-G and pCMV Δ8.9 using FuGENE HD (Promega). Medium was collected 24, 36, and 48 hr after transfection and used to infect HDF cells. Infected cells were selected with 2 μg/mL puromycin for 2 days, 500 μg/mL G418 for 3 days, or 5 μg/mL blasticidin for 5 days (see Figure S1G for a schematic of the time line and Supplemental Experimental Procedures for details of individual vectors).

Real-Time qPCR

Real-time qPCR was performed using the SYBR Green system (Applied Bio-systems) on the ABI 7900 Fast Sequence Detection instrument as specified by the manufacturer. Total RNA was harvested from cells using Trizol reagent (Invitrogen) and further purified using RNeasy columns (QIAGEN). 1 μg of total RNA was transcribed into cDNA in 50-μL reactions using the TaqMan kit (Applied Biosystems). 1 μL of this reaction was used in subsequent real-time qPCR reactions. GAPDH was used as the normalization control. All reactions were performed in triplicates. The primers used for real-time qPCR are provided in Table S1.

ChIP

The procedure was based on the Magna ChIP protocol (Millipore, 17-10085) and Chromatrap Premium ChIP qPCR (Chromatrap, 500115). Cells (2 × 10⁶) were cross-linked in 1% formaldehyde for 10 min at room temperature, harvested by scraping, centrifuged, and resuspended in SDS lysis buffer. After 10 min on ice, lysed cells were diluted 2-fold in ChIP dilution buffer and disrupted with the S220 Focused Ultrasonicator (Covaris) to yield genomic DNA fragments of 300 bp. The samples were immunoprecipitated overnight at 4°C with the indicated antibodies. Immunocomplexes were pulled down with 20 μL of magnetic protein A beads as described in the Magna ChIP protocol. The DNA was recovered by 2 hr digestion with proteinase K at 65°C, followed by a 10-min incubation at 95°C. The DNA was cleaned up by phenol/chloroform extraction and ethanol precipitation and finally resuspended in 30 μL of Tris-EDTA (TE).

The ChIP and input were then used for qPCR with the primers described in Table S2.

Immunofluorescence and Immuno-FISH

Cells were fixed for 20 min with freshly prepared 4% paraformaldehyde in PBS. Permeabilization was performed with 0.2% Triton X-100 in PBS for 20 min at room temperature. After blocking nonspecific binding, antibodies were diluted in the blocking solution and incubated with the specimens overnight at 4°C in a humidified chamber. Samples were washed three times with PBS and incubated for 1 hr at room temperature with secondary antibodies diluted in blocking solution. Nuclei were counterstained with 2 μg/mL DAPI in PBS containing 0.2% Triton X-100 for 15 min. The telomere dysfunction-induced focus (TIF) assay, an immunofluorescence combined with fluores-cence in situ hybridization (Immuno-FISH) procedure (Herbig et al., 2006) is described in detail in the Supplemental Experimental Procedures, as is a listing of the antibodies used. For EdU pulse labeling, cells were incubated with 10 μM of EdU stock solution (Click-iT, Invitrogen) for 1 hr prior to fixation.

This work was supported by NIH/NIA grants R37 AG016694 and P01 AG051449 (to J.M.S.). T.I. was supported in part by NIH grants T32 GM007601 and F31 AG043189. The Brown Genomics Core was supported in part by the COBRE award from NIH/NIGMS P30 GM0103410.