Research Article 2605

IntroductionHutchinson-Gilford progeria syndrome (HGPS) is a premature-aging syndrome which affects one in 4-8 million children withsymptoms resembling normal adult aging that include very thinskin, loss of subcutaneous fat, alopecia, stiff joints, osteoporosisand heart disease (Hennekam, 2006; Merideth et al., 2008). Theage of onset is within 2 years of life, with death at a mean age of13 as a result of heart attack or stroke (Hennekam, 2006; Meridethet al., 2008). HGPS is caused by a silent G608G mutation withinthe LMNA gene that encodes lamin A (De Sandre-Giovannoli etal., 2003; Eriksson et al., 2003). This mutation exposes a crypticsplice site in exon 11 that leads to deletion of 50 amino acidsrequired for normal lamin A processing, resulting in an aberrantand permanently farnesylated lamin A protein termed progerin (DeSandre-Giovannoli et al., 2003; Eriksson et al., 2003). Laminshave a structural role in supporting the nuclear envelope and havebeen implicated in many nuclear functions, including mitosis, DNAsynthesis and repair, RNA transcription and processing, apoptosis,organization of chromatin structure and regulation of geneexpression (Dechat et al., 2008; Goldman et al., 2002; Gruenbaumet al., 2005; Stuurman et al., 1998; Taddei et al., 2004).

Cellular defects associated with HGPS include a reducedlifespan in culture, irregular nuclear phenotypes such as blebbingof the nuclear envelope, altered chromatin organization, reducedtelomere lengths, and a chronic DNA-damage response (Allsoppet al., 1992; Decker et al., 2009; Goldman et al., 2004; Huang etal., 2008; Liu et al., 2005; Liu et al., 2006; Shumaker et al.,2006). Previous studies have shown that the catalytic subunit ofhuman telomerase (TERT) extends HGPS cellular lifespan andrescues proliferative defects associated with progerin (Kudlow etal., 2008; Ouellette et al., 2000; Wallis et al., 2004). However,progerin has been reported to induce increased DNA damagedespite the presence of exogenous telomerase expression (Scaffidi

and Misteli, 2008), suggesting that telomerase immortalizationdoes not involve amelioration of the DNA-damage phenotype.We undertook the present studies in an effort to elucidate themechanism by which telomerase overcomes the HGPS premature-senescence phenotype.

ResultsTERT rescues HGPS premature senescence throughinhibition of tumor-suppressor pathway activationPrevious studies have provided evidence that ectopic telomeraseexpression can extend the lifespan of HGPS fibroblasts (Kudlowet al., 2008; Ouellette et al., 2000; Wallis et al., 2004). To confirmand extend these findings, we infected HGPS fibroblasts that werenear the end of their proliferative lifespan [two remainingpopulation doublings (PDs)] with a retroviral construct expressingTERT. HGPS fibroblasts transfected with a control vector ceasedproliferation within two additional PDs (Fig. 1A). However, HGPSfibroblasts expressing TERT propagated continuously for over 70PDs without any evidence of a decline in their proliferative capacity(Fig. 1A). This proliferative ability was correlated with an increasein S-phase and decrease in G0-G1-phase fractions of TERT-expressing HGPS cells (Fig. 1B). We also analyzed cultures forsenescence-associated -galactosidase (SA--gal), an empiricalmarker of cellular senescence (Dimri et al., 1995). Althoughessentially all control HGPS fibroblasts were positive for SA--galactivity, more than 90% of HGPS fibroblasts expressing TERTwere negative for SA--gal at 2 weeks after selection (Fig. 1C). Ofnote, exogenous TERT expression did not result in decreasedprogerin expression, and even after many PDs in the presence ofTERT, progerin protein levels remained unchanged (Fig. 1D).

The p53 and Rb tumor-suppressor pathways have beenimplicated in the cellular senescence of normal fibroblasts andcan be activated by DNA damage, including damage leading to

Role of progerin-induced telomere dysfunction inHGPS premature cellular senescenceErica K. Benson1, Sam W. Lee2 and Stuart A. Aaronson1,*1Department of Oncological Sciences, Mount Sinai School of Medicine, Box 1130, One Gustave L. Levy Place, NY 10012, USA2Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charleston, MA 02129, USA*Author for correspondence (stuart.aaronson@mssm.edu)

Accepted 5 May 2010Journal of Cell Science 123, 2605-2612 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.067306

SummaryHutchinson-Gilford Progeria Syndrome (HGPS) is a premature-aging syndrome caused by a dominant mutation in the gene encodinglamin A, which leads to an aberrantly spliced and processed protein termed progerin. Previous studies have shown that progerin inducesearly senescence associated with increased DNA-damage signaling and that telomerase extends HGPS cellular lifespan. We demonstratethat telomerase extends HGPS cellular lifespan by decreasing progerin-induced DNA-damage signaling and activation of p53 and Rbpathways that otherwise mediate the onset of premature senescence. We show further that progerin-induced DNA-damage signalingis localized to telomeres and is associated with telomere aggregates and chromosomal aberrations. Telomerase amelioration of DNA-damage signaling is relatively rapid, requires both its catalytic and DNA-binding functions, and correlates in time with the acquisitionby HGPS cells of the ability to proliferate. All of these findings establish that HGPS premature cellular senescence results fromprogerin-induced telomere dysfunction.

Key words: Cellular senescence, DNA damage, Nuclear lamins, Telomerase, Telomere dysfunction, Tumor suppressors

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telomere dysfunction (Campisi, 2001; Maslov and Vijg, 2009).Furthermore, the p53 pathway has been reported to be chronicallyactivated in HGPS (Kudlow et al., 2008; Liu et al., 2005; Liu etal., 2006; Scaffidi and Misteli, 2006). Previous studies haveindicated that HPV E6, but not E7, suppresses the growthinhibitory effects of ectopic progerin expression in normal diploidhuman fibroblasts (NDFs) (Kudlow et al., 2008). However, thesestudies did not resolve whether this effect was due solely toinactivation of p53 by HPV E6, or whether other E6 activities,such as TERT induction, were involved. Furthermore, lifespanextension in HGPS fibroblasts by direct interference with p53 orRb pathways, or the effect of TERT on the activation of thesepathways has not been studied. Therefore, we analyzed theactivation of several cell cycle inhibitors known to be involvedin these pathways (Vidal and Koff, 2000), and observed thatHGPS fibroblasts transduced with TERT expressed lower levelsof p53, p21 and p16 proteins than control fibroblasts, as well ashigher levels of the phosphorylated (active) form of Rb (Rb-P)(Fig. 1E). Thus, TERT-induced extension of HGPS fibroblastlifespan was associated with decreased activation of p53 and Rbtumor-suppressor pathways.

To directly investigate the involvement of p53 and Rb pathwaysin the premature cellular senescence exhibited by HGPS fibroblasts,we infected HGPS fibroblasts that were near the end of theirproliferative lifespan with retroviral constructs that block either Rbby CDK4 overexpression or p53 by DNp53 expression. Bymeasuring the proliferative lifespan of these fibroblasts, weobserved that CKD4 was able to extend HGPS lifespan by about26 PDs, whereas DNp53 extended HGPS lifespan by about 6 PDs(Fig. 1F). Moreover, the combination of CDK4 and DNp53extended HGPS cellular lifespan by over 68 PDs (Fig. 1F).Although the relative ability of DNp53 or CDK4 to extend lifespanvaried in different experiments, the combination consistentlyextended lifespan for many PDs beyond that of either constructalone. We also observed similar lifespan extension with DNp53and CDK4 in normal senescing fibroblasts (data not shown),indicating that these same effector pathways are involved in HGPSpremature senescence, as well as normal senescence.

TERT blocks progerin-induced DNA-damage signalingDNA damage triggers the phosphorylation and/or activation ofproteins involved in the DNA-damage response. These includeH2AX, a variant of the H2A histone, which is distributed throughoutchromatin and becomes rapidly phosphorylated (H2AX) at nascentdouble-strand breaks, and ATM, which is activated throughautophosphorylation (ATM-P) and recruited to sites of double-strand breaks (Riches et al., 2008). These proteins form discretefoci in cells at sites of DNA damage, and thus are useful markersof such damage. HGPS fibroblasts have been reported to exhibitincreased DNA-damage signaling (Liu et al., 2005; Liu et al., 2006).When we compared the effects of exogenous TERT expression onthe number of H2AX and ATM-P foci observed in these cells, wefound that TERT expression resulted in a striking reduction in thenumber of such foci (Fig. 2A,B). Similarly, the total level of ATM-P detectable by immunoblot analysis was significantly reduced inHGPS fibroblasts expressing TERT (Fig. 2C). The effects of TERTexpression in HGPS fibroblasts were rapid, with H2AX levelssignificantly reduced as early as 7 days after selection, and thisdecrease correlated with the onset of increased proliferative capacityof the same cells (Fig. 2G).

To further investigate the effects of TERT on the HGPS DNA-damage signaling, we ectopically expressed progerin in humanNDFs. In these cells, exogenous progerin was expressed at a levelsimilar to endogenous progerin expression in HGPS fibroblasts(Fig. 2D). Whereas ectopic progerin expression in NDFs inducedDNA-damage signaling (Fig. 2E,F), progerin failed to do so inNDFs previously infected with TERT (Fig. 2E,F; Fig. 3D). Thus,TERT expression was protective against DNA-damage signalinginduced by progerin.

Rescue of progerin-induced growth defects and DNAdamage phenotypes by TERT is specific to its function attelomeresAccumulating evidence indicates that TERT has activities that areindependent of its catalytic function required for telomeremaintenance (Choi et al., 2008; Cong and Shay, 2008; De Semiret al., 2007; Lee et al., 2008; Park et al., 2009; Smith et al., 2003;Zhou et al., 2009). For example, both wild-type and catalyticallyinactive mutant TERT are recruited to the promoters of growth-controlling genes and function indistinguishably to modulate geneexpression and enhance cell proliferation (Park et al., 2009; Zhouet al., 2009). Thus, we compared the abilities of wild-type TERT

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Fig. 1. TERT rescues HGPS premature senescence through inhibition oftumor-suppressor pathway activation. (A)Growth curves of HGPSfibroblasts expressing ectopic TERT or vector control. HGPS fibroblasts nearthe end of their proliferative lifespan (two remaining PDs) were transducedand marker selected for 2 weeks with retroviral TERT or control vectors, andthe number of cumulative PDs from the time of selection was calculated.(B)Cell-cycle analysis of HGPS fibroblasts expressing ectopic TERT or vectorcontrol. Propidium iodide was measured by flow cytometry at 2 weeks afterretroviral transduction and selection with vector control or TERT. Error barsindicate s.d. for a representative experiment performed in triplicate. (C)SA--gal staining of HGPS cultures expressing ectopic TERT or vector control at 2weeks after retroviral transduction and selection. (D)Western blot showinglevels of endogenous progerin in HGPS fibroblasts before, 6 PDs after, and 70PDs after TERT transduction and selection. Protein levels of lamin A, lamin Cand -actin are shown as controls. (E)Western blot showing levels of p53,p21, p16 and total Rb (top band indicates Rb-P) in HGPS fibroblastsexpressing TERT compared with vector controls at 2 weeks after infection andselection. -actin levels are shown as a control. (F)Growth curves of HGPSfibroblasts expressing CDK4, DNp53, both CDK4 and DNp53, or vectoralone. HGPS fibroblasts near the end of their proliferative lifespan (tworemaining PDs) were transduced with retroviral vectors encoding the indicatedproteins, and cumulative PDs from the time of infection and selection werecalculated. In the case of CDK4+DNp53, DNp53 was added to cellsexpressing CDK4 at 64 days.

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and either a catalytically inactive TERT mutant, D868A TERT, ora telomere-binding-deficient mutant, N125A+T126A TERT, whichis catalytically active but fails to elongate the telomeres (Goldkornand Blackburn, 2006), to extend lifespan and reduce DNA damage.Although all TERT constructs were expressed at similar levels(Fig. 3C), only wild-type TERT extended the proliferative lifespanof HGPS fibroblasts (Fig. 3A). Likewise, only wild-type TERTreduced the level of DNA-damage signaling in HGPS fibroblasts(Fig. 3B). These results demonstrate that amelioration of DNA-damage signaling and premature senescence in HGPS fibroblastsrequires both the catalytic and the DNA-binding functions of TERTfor telomere maintenance.

To further establish the specificity of telomerase with respect toamelioration of progerin-induced DNA-damage signaling, weinvestigated the effects of exogenous telomerase on DNA damagecaused by doxorubicin (DOX), which induces DNA double-strandbreaks through inhibition of topoisomerase II (Tewey et al., 1984).Under identical conditions in which TERT effectively blockedDNA-damage signaling induced by progerin, DOX treatmentinduced equivalent levels of DNA damage in NDFs with or withoutectopic TERT expression (Fig. 3D). These findings further supportthe conclusion that the ability of TERT to ameliorate progerin-induced DNA-damage signaling and premature senescence isspecific to its functions at the telomere.

Progerin-induced DNA-damage signaling localizes totelomeresWe next sought to determine whether DNA-damage signalinginduced by progerin expression was localized at telomeres. To do

so, we examined progerin-expressing fibroblasts for the presenceof telomere dysfunction-induced foci (TIFs), as defined by the co-localization of H2AX and TRF1, which serves as a telomeremarker (Takai et al., 2003). TRF2 is a telomeric DNA-bindingprotein that is essential for normal telomere protection, and adominant-negative form of TRF2 (TRF2BM) has previously beenshown to induce the formation of TIFs (Takai et al., 2003). Ectopicexpression of progerin induced the formation of TIFs in NDFs ina similar manner to that observed with TRF2BM (Fig. 4A,B). Bycontrast, H2AX foci detected in response to DNA damage inducedby DOX showed no evidence of telomere specificity (Fig. 4A,B).Moreover, progerin-induced TIFs occasionally involved the co-localization of H2AX with several overlapping TRF1 foci(telomere aggregates), which was not observed in any controlcells analyzed (Fig. 4C). These findings further implicate thetelomere as the specific target of progerin-induced DNA-damagesignaling.

To further demonstrate that the DNA-damage signaling observedin response to progerin was localized to telomeres, we performedtelomere chromatin immunoprecipitation (ChIP). Whereas progerininduced a 4.2-fold increase in the amount of H2AX associatedwith a telomere repeat sequence compared with the control, theassociation of H2AX to an internal Alu sequence increased only1.2-fold under the same conditions (Fig. 5). As a positive controlfor telomere-specific DNA damage, TRF2BM induced a 6.2-foldincrease in H2AX associated with telomere repeats, whereasH2AX associated with an Alu sequence showed only a 1.4-foldincrease (Fig. 5). Our confocal imaging of DNA-damage signalingat telomeres, and ChIP data demonstrating increased H2AX

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Fig. 2. TERT blocks progerin-induced DNA-damage signaling.(A)Confocal immunofluorescence microscopy of H2AX (green)and ATM-P (red) DNA-damage foci in HGPS fibroblastsexpressing TERT or vector control at 2 weeks after retroviraltransduction and marker selection. The merged images are shownsuperimposed on DAPI (blue) staining of DNA with co-localization of H2AX and ATM-P in yellow. (B)Quantificationof staining shown in A. The percentage of cells with either 0, 1, 2-5 or >5 H2AX and ATM-P foci is shown. For each condition, atleast 300 cells were counted. (C)Western blot showing levels ofATM-P in HGPS fibroblasts expressing TERT or vector control at2 weeks after infection and selection. -actin levels are shown as acontrol. (D)Western blots comparing levels of endogenousprogerin in HGPS fibroblasts with ectopic progerin in NDFs.Lamin A, lamin C, and -actin levels are shown as controls.(E)Confocal immunofluorescence microscopy of H2AX (green)and ATM-P (red) DNA-damage foci in NDFs with or withoutectopic TERT and expressing progerin or vector controlimmediately following lentiviral transduction and selection. Themerged images are shown superimposed on DAPI (blue) stainingof DNA with co-localization of H2AX and ATM-P in yellow.(F)Quantification of staining shown in E. The percentage of cellswith either 0, 1, 2-5 or >5 H2AX and ATM-P foci is shown. Foreach condition, at least 300 cells were counted. (G)Time courseof lifespan extension and DNA-damage reduction in HGPSfibroblasts with ectopic TERT expression. Late-passage HGPSfibroblasts were infected and selected for 2 weeks with a lentivirusexpressing TERT or vector control. Immediately after selection,lifespan was measured by tracking PDs versus time. DNA-damagesignaling was measured by flow-cytometry analysis for H2AXusing G1-gated cells. H2AX positivity was measured on days 7,17, 25 and 39. Error bars indicate s.d. for a representativeexperiment performed in triplicate.

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associated with telomere sequences, provide strong evidence thatimplicates progerin in the induction of telomere dysfunction.

TIF formation caused by TRF2BM is the result of dissociationof TRF2 from telomeric DNA, which leads to a loss of the 3�overhang and DNA-damage signaling (Takai et al., 2003). We usedthe ChIP assay to compare the effects of progerin and TRF2BM

on the association of TRF2 to telomeric DNA. As expected,TRF2BM expression markedly decreased the binding of TRF2 totelomeres while minimally affecting the binding of TRF1, anothermajor regulator of telomere stability (Smogorzewska et al., 2000)(Fig. 6A,B). By contrast, progerin expression was associated withan increase in the binding of both TRF1 and TRF2 to telomericDNA in two independent experiments (Fig. 6A-D). Binding ofTRF1 and TRF2 to telomeric DNA was specific, because nobinding was detected at Alu sequences (Fig. 6C). Thus, unliketelomere dysfunction caused by TRF2BM, progerin-induced TIFsdo not result from a dramatic loss of TRF2 binding to telomeres.

Progerin-induced chromosomal aberrationsThe TIFs observed in response to progerin expression prompted usto investigate whether we could detect any chromosomal aberrationsknown to occur under conditions of telomere dysfunction (Bolzan

and Bianchi, 2006; Davoli et al., 2010; Hande et al., 2001;Michishita et al., 2008). We performed telomere fluorescent in-situhybridization (FISH) on metaphase spreads with progerin-expressing NDFs (IMR90), as well as vector controls. It wasdifficult to identify metaphase cells in progerin-expressing NDFs,which is consistent with their premature-senescence phenotype.However, in those complete or partial progerin metaphasesobserved, we found abnormalities in 3.5% of 1344 chromosomes.These included chromosomal and sister-chromatid fusions, sister-

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Fig. 3. Rescue of progerin-induced growth defects and DNA-damagephenotypes by TERT is specific to its function at telomeres. (A,B)TERTscatalytic and DNA-binding functions are required to rescue the HGPSpremature senescence and DNA-damage phenotypes. (A)Comparison of theproliferative abilities of late-passage HGPS fibroblasts expressing wild-typeTERT, DNA-binding-deficient TERT (N125A+T126A TERT), catalyticallyinactive TERT (D868A TERT), or vector control. Cumulative PDs of eachculture were determined 28 days after infection and selection. Error barsindicate s.e.m. from three separate experiments. (B)Flow-cytometry analysisof H2AX on G1-gated HGPS fibroblasts expressing the indicated TERTconstructs at 2 weeks after infection and selection. Error bars indicate therange for a representative experiment performed in duplicate. (C)Western blotshowing similar protein levels of ectopic wild-type and mutant TERTconstructs in HGPS fibroblasts. -actin was used as a loading control.(D)Effect of TERT on doxorubicin (DOX) treatment. NDFs (IMR90), eitherwith or without ectopic TERT expression, were transduced and markerselected with progerin or vector control. Vector-control fibroblasts were eitheruntreated or treated with 500 nM DOX for 1 hour. H2AX positivity wasmeasured by flow cytometry. Error bars indicate the range for a representativeexperiment performed in duplicate.

Fig. 4. Progerin-induced DNA-damage signaling localizes to telomeres.(A)Telomere-dysfunction-induced foci (TIFs) were detected by confocalimmunofluorescence microscopy of H2AX (green) and TRF1 (telomeremarker) (red) foci at 5 days after infection of NDFs with progerin, TRF2BM

positive control or a vector control that was either untreated or treated with500 nM DOX for 1 hour. The merged images are superimposed on DAPI(blue) staining showing DNA with co-localization of H2AX and TRF1 inyellow. White boxes indicate areas that are enlarged 10� and shown to theright without DAPI. Scale bar: 10m. (B)Quantification of TIFs in cells fromA. TIFs were determined by the co-localization of H2AX and TRF1. Thepercentage of cells with DNA damage containing 0-1, 2-5, 6-10 or >10 TIFsare shown. For each condition, 100 cells were counted. (C)Additional mergedimages of TIFs in progerin-expressing cells, each of which show telomereaggregates. TIFs were detected as in A. White boxes indicate areas that areenlarged 10�. Scale bar: 10m.

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telomere losses, double telomeric signals on single chromatids(telomere doublets), chromosomal breaks, extra-chromosomaltelomeric signals, as well as two metaphases containingdiplochromosomes and a chromatin bridge containing telomericsignals that persisted after reformation of the nuclear envelope(Fig. 7A-H). By contrast, we observed abnormalities in only 0.8%of 1506 control chromosomes analyzed. These results indicate thatprogerin expression promotes chromosomal aberrations in thesetting of telomere dysfunction. The fact that progerin inducespremature senescence probably protects such cells from more-severe chromosomal instability.

DiscussionOur present studies demonstrate that telomerase extends HGPScellular lifespan by decreasing progerin-induced DNA-damagesignaling and activation of p53 and Rb pathways that otherwisemediate the onset of premature senescence. We showed further thatprogerin-induced DNA-damage signaling was localized totelomeres and was associated with telomeric aberrations.Telomerase amelioration of DNA-damage signaling was relativelyrapid, required both its catalytic and DNA-binding functions, andcorrelated in time with the acquisition by HGPS cells of the abilityto proliferate. All of these findings establish that progerin-inducedtelomere dysfunction is responsible for the premature cellularsenescence observed in HGPS fibroblasts as well as in normalhuman fibroblasts in response to exogenous progerin expression.

We observed telomere aggregates in progerin-expressing cellsthat were similar to those reported in cells ectopically expressingother mutant forms of lamin A and lamin B, and in normal senescent

cells (Raz et al., 2008). Telomere aggregates have been suggestedto represent telomere fusions and have been linked to genomicinstability (Amiel et al., 2009; Louis et al., 2005). Consistent withthis, we found several chromosomal aberrations in progerin-expressing cells, including a low level of fusions. In fact, otherevidence of genomic instability in HGPS fibroblasts includesfindings of lagging chromosomes, and binucleated cells, as well asaneuploid cells (Cao et al., 2007; Ly et al., 2000; Mukherjee andCostello, 1998). Such fusions are also consistent with an elevatedlevel of non-homologous end-joining previously reported in HGPScell lines (Liu et al., 2005). Genomic instability is also a hallmarkof tumorigenesis. Thus, the absence of any reported increasedincidence of malignancies in HGPS patients might reflect theseverity of premature aging and early death associated with thissyndrome.

We observed telomeric DNA-damage signaling within 5 days ofexogenous progerin expression in NDFs. Thus, progerin inducestelomere dysfunction rapidly, well before telomere attrition, aspreviously observed in HGPS fibroblasts (Allsopp et al., 1992;Decker et al., 2009; Huang et al., 2008), would be detectable. Wealso observed that exogenous TERT rapidly ameliorated DNA-damage signaling in HGPS fibroblasts. Our findings contrast withthose of a recent report indicating that telomerase fails to protectagainst progerin-induced DNA damage (Scaffidi and Misteli, 2008).

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Fig. 5. Telomeric chromatin immunoprecipitation showing that progerin-induced DNA-damage signaling is enriched specifically at telomeres.(A)NDFs (IMR90) were infected and marker selected for 7 days withprogerin, TRF2BM or vector-control viruses. Antibodies specific for H2AXor IgG negative control were used for immunoprecipitation. DNA was loadedonto a nylon membrane using a slot blot and probed with a DIG-labeledtelomere probe, stripped and re-probed with a DIG-labeled Alu probe. InputDNA represents 0.1% of total DNA. A light and a dark exposure are shown.(B)Quantification of the dark exposure shown in A. Signal density wasmeasured by ImageJ, and histogram values represent the H2AX telomeric orAlu ChIP signal normalized to input signal, and subtracted for backgroundsignal in the IgG control. The amount of DNA in each ChIP is expressed inarbitrary units (a.u.) after vector control sequences were normalized to 1.

Fig. 6. Effect of progerin on the association of telomere-binding proteinswith telomeric DNA. (A)NDFs (IMR90) were infected with virusesexpressing progerin, TRF2BM, or vector control and marker selected for 7days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgGnegative control were used for immunoprecipitation following crosslinkingand DNA shearing. DNA was loaded onto a nylon membrane using a slot blotand hybridized with a DIG-labeled telomere probe. Input DNA represents 1%of total DNA. (B)Quantification of blot shown in A. Signal density wasmeasured by ImageJ, and histogram values represent the TRF1 and TRF2telomeric ChIP signal normalized to input signal. The amount of telomericDNA in each ChIP is expressed in arbitrary units (a.u.) after vector controlsequences were normalized to 1. (C)NDFs (IMR90) were infected withlentiviruses expressing progerin or vector control and marker selected for 5days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgGnegative control were used for immunoprecipitation following crosslinkingand DNA shearing. DNA was loaded onto a nylon membrane using a slot blotand hybridized with a DIG-labeled telomere probe, stripped and re-hybridizedwith a DIG-labeled Alu probe. Input DNA represents 1% of total DNA.(D)Quantification of blot in C performed as described in B.Jo

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A possible explanation for these differences is the use of a GFP-progerin fusion protein in these latter studies, because GFP tagshave been shown to alter protein function, localization and proteininteractions (Limon et al., 2007; Skube et al., 2009; Wang et al.,2008).

There is controversy in the literature as to whether the abnormalnuclear shape observed in HGPS fibroblasts is ameliorated bytelomerase (Huang et al., 2008; Kudlow et al., 2008; Scaffidi andMisteli, 2008). Several studies have shown that blocking progerinfarnesylation by farnesyltransferase inhibitors (FTIs) reverses theaberrant nuclear morphology of HGPS fibroblasts (Capell et al.,2005; Columbaro et al., 2005; Toth et al., 2005), whereas FTIshave no effect on progerin-induced DNA-damage signaling (Liu etal., 2006). Thus, studies with FTIs support the concept that nuclear-envelope defects and telomere dysfunction are independentoutcomes of progerin expression and might also explain whyprogerin-induced proliferation defects are only partially improvedby FTIs (Candelario et al., 2008).

The mechanisms involved in progerin-induced telomeredysfunction and its rapid amelioration by telomerase remain to beresolved. Recently, it was shown that knockdown of SIRT6, ahistone H3 lysine 9 (H3K9) deacetylase, which modulateschromatin structure at telomeres, induces an abnormal telomericchromatin state that leads to premature cellular senescence andDNA-damage signaling at telomeres (Michishita et al., 2008),similarly to that observed in response to progerin expression in ourpresent studies. Of note, telomere stabilization by the ectopicexpression of TERT also reversed the premature senescence ofSIRT6-knockdown cells (Michishita et al., 2008). A hallmark ofHGPS cells is decreased heterochromatin (Goldman et al., 2004;

Pegoraro et al., 2009; Shumaker et al., 2006), which might be acontributing factor to progerin-induced telomere dysfunctionreversible by TERT. This is in contrast to the inability of TERT torescue TRF2BM-induced telomere dysfunction (Takai et al., 2003).Also, unlike telomere damage caused by TRF2BM, progerin didnot cause a decreased association of TRF2 with telomeres. Thesefindings help to distinguish telomere dysfunction induced byprogerin from that induced by TRF2BM.

Several mutations in the LMNA gene, which encodes lamins Aand C, result in various laminopathies with overlapping phenotypesto those observed in HGPS patients (Raz et al., 2008). Some ofthese mutations have been reported to result in increased laminbinding to telomeres and altered telomere localization (Raz et al.,2008). In addition, fibroblasts from mice lacking LMNA showaltered telomere localization and telomere dysfunction (Gonzalez-Suarez et al., 2009). Therefore, it is possible that the absence oflamins, or the presence of mutant lamins, might disrupt normallamin-telomere interactions leading to telomere dysfunction bylimiting or changing the ability of proteins that function in normaltelomere maintenance or repair in a manner that can be overcomeby TERT overexpression.

Our results demonstrating that telomere dysfunction, not justreduced telomere lengths, is involved in the premature senescenceof HGPS fibroblasts adds HGPS to the list of premature-agingsyndromes associated with telomere dysfunction such as Werner’s,Bloom’s and ataxia telangiectasia (Callen and Surralles, 2004;Crabbe et al., 2007; Puzianowska-Kuznicka and Kuznicki, 2005).Thus, accumulating evidence indicates that telomere maintenanceis a common target of diverse genetic defects causing premature-aging syndromes and premature cellular aging in culture. Sinceincreased levels of progerin expression through aberrant splicinghave also been observed in normally aging cells (McClintock etal., 2007; Scaffidi and Misteli, 2006), our present findings suggestthat progerin-induced telomere dysfunction also contributes tonormal aging.

Materials and MethodsCell linesFibroblasts from a HGPS patient (AG01972, Coriell Cell Repository) were obtained,and sequencing was performed to confirm a G608G LMNA mutation. Cultures weremaintained in minimum essential medium (MEM; Invitrogen) supplemented with0.2 mM non-essential amino acids (NEAA; Invitrogen), 15% heat-inactivated fetalbovine serum (FBS; Invitrogen) and 50 U/ml penicillin and streptomycin (Pen/Strep;Invitrogen). NDFs used were 501T (derived from adult skin) except where IMR90(fetal lung, ATCC) fibroblasts were used as indicated. NDFs were grown inDulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10%FBS and 50 U/ml Pen/Strep. All cells were cultured at 37°C in 5% CO2. Cellularproliferative lifespan was measured by subculturing fibroblasts 1:4 or 1:2, dependingon growth rate, at 90% confluence and recording total population doublings (PDs)and time in culture. PDs were calculated by the formula PDlog2(1/split ratio)(Harley and Sherwood, 1997).

Expression constructs, viral production and infectionFull-length cDNA encoding progerin was obtained from total RNA from the AG01972HGPS cell line by RT-PCR amplification with primers specific for LMNA (sense,including a GCCACC Kozak sequence, 5�-GCCACCATGGAGACCCCGTCCCA -GC-3�; and antisense, 5�-GGTCCCAGATTACATGATGCTGC-3�). Progerin wasexpressed in a NSPI-derived lentiviral vector containing a puromycin selectionmarker (Akiri et al., 2009). Wild-type TERT, dominant-negative p53 (R248W) andwild-type CDK4 were expressed in pBabe-derived retroviral vectors containingpuromycin, hygromycin and neomycin selection markers, respectively (Mahale etal., 2008). TERT mutants, N125A+T126A and D868A, in the pBABE retroviralbackbone (kind gift from Elizabeth Blackburn, UCSF, CA) (Kim et al., 2003), alongwith wild-type TERT, were subcloned and expressed in a NSPI-derived lentiviralvector backbone with the blasticidin selection marker. TRF2BM was expressed inthe pLPC retroviral vector with a puromycin selectable marker (Addgene plasmid18008) (Karlseder et al., 2002). To create retroviral stocks, HEK293T cells were co-transfected with the appropriate retroviral expression vector and pCL-ampho

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Fig. 7. Progerin-induced chromosomal aberrations. Telomeric FISH onmetaphases derived from early passage NDFs (IMR90) exogenouslyexpressing progerin for 5, 8 or 13 days. Telomeric PNA hybridization signalsare shown in green and DAPI-counterstained chromosomes in blue.(A)Representative complete metaphase spread from NDFs (IMR90)expressing progerin. Arrow indicates the fusion of two chromosomes at thetelomere, enlarged and shown at the bottom right. (B-H)Additional examplesof progerin-induced chromosomal aberrations. Arrows indicate aberrations.(B)Sister-chromatid fusion. (C)Sister-telomere loss. (D)Telomere doublet.(E)Chromosomal break. (F)Extra-chromosomal telomeric signals.(G)Diplochromosome. (H)Chromatin bridge containing telomeric signalsbetween two interphase nuclei. Scale bar: 2m.

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packaging plasmid. To create lentiviral stocks, HEK293T cells were co-transfectedwith the appropriate lentiviral expression vector, pCMVR8.74 packaging vectorand pMD2 VSVG envelope vector. Titers for each virus stock were determined bycolony formation following marker selection in the same assay cell, HT1080, makingit possible to compare results using similar amounts of virus in different experiments.Retroviral and lentiviral infections were carried out on mass populations of fibroblastsin the presence of 8 g/ml polybrene (Sigma). Cells were subsequently selected forantibiotic resistance (2 g/ml puromycin, 100 g/ml hygromycin, 750 g/mlneomycin and 5 g/ml blasticidin) and expanded as mass populations. In all cases,similar MOIs were used.

AntibodiesAntibodies against the following proteins were used: Lamin A/C (Millipore,MAB3211), p53 (1801, Mount Sinai School of Medicine Hybridoma Center), p21(BD Biosciences, 556431), p16 (Santa Cruz, sc-468), Rb (Cell Signaling, 9309),Ser139 H2AX (Millipore, 05-636), Ser139 H2AX (Millipore, 07-164), Ser-1981phospho-ATM (Rockland, 600-401-400), TERT (abcam, ab32020), TRF1 (#370) (akind gift from Titia de Lange, Rockefeller University, NY), TRF1 (Santa Cruz, sc-6165), TRF2 (Imgenex, IMG-124A), -actin (Sigma, A5441) and IgG (Millipore,12-371).

Flow cytometryCell-cycle analysis was performed using the CycleTEST Plus DNA reagent kit(Beckton Dickinson), according to the manufacturer’s instructions. Detection ofcellular H2AX was performed using the H2AX Phosphorylation Assay Kit forFlow Cytometry (Millipore, 17-344), according to the manufacturer’s instructions.To combine cell-cycle analysis and H2AX staining, cells already stained for H2AXwere incubated with 10 g/ml propidium iodide (Trevigen, 4830-250-3) and 10g/ml RNase A (Invitrogen, 12091-021) for 10 minutes at 37°C. At least 10,000stained cells were sorted by FACS (FACSCalibur, Beckton Dickinson) and analyzedwith Cell Quest 3.2 software (Beckton Dickinson).

Senescence-associated -galactosidase (SA--gal) stainingCells were washed in PBS and fixed with 2% formaldehyde and 0.2% glutaraldehydein PBS for 5 minutes at room temperature and then stained as previously described(Dimri et al., 1995).

ImmunoblottingWhole cell extracts were obtained by solubilizing cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, supplemented with the followingprotease and phosphatase inhibitors: 5 mM EDTA, 50 mM sodium fluoride, 25 mM-glycerophosphate, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonylfluoride, and 10 g/ml aprotinin) or SDS-lysis buffer [50 mM Tris-HCl, pH 8.1, 1%SDS, 10 mM EDTA, supplemented with the Complete Mini Protease InhibitorCocktail (Roche, 11836153001)]. Protein samples (50 g) were subjected to SDS-PAGE, transferred onto an Immobilon-P or Immobilon-FL filter (Millipore) andprobed with the indicated antibodies. Detection was carried out with either an ECLsystem (GE Healthcare) with horseradish peroxidase-conjugated secondary antibodies(GE Healthcare) or an Odyssey Infrared Imaging System (LI-COR Biosciences)with IR-dye-tagged secondary antibodies (LI-COR Biosciences).

Confocal microscopyCells plated and grown on glass coverslips at least 48 hours were washed twice withPBS followed by fixation for 20 minutes with methanol at –20°C. Cells were thenwashed twice with PBS-T (PBS with 0.1% Tween 20), blocked for 1 hour in PBS-BSA-T (PBS with 1% BSA and 0.1% Tween 20) and incubated overnight at 4°Cwith primary antibodies. Samples were then washed three times with PBS-BSA-Tand incubated with secondary antibodies for 1 hour. Anti-rabbit Cy3 (JacksonImmunoResearch, 711-165-152) or anti-mouse Alexa Fluor 488 (Molecular Probes,A11029) secondary antibodies were used. In some experiments, doxorubicin (DOX)(Sigma, D1515) at 500 nM was added for 1 hour before fixation. Coverslips weremounted using Vectashield Mounting Medium with DAPI (Vector Laboratories, H-1200). Confocal imaging was performed with a Zeiss LSM 510 META confocalmicroscope (Carl Zeiss Microimaging) using the 63� oil objective. Images werecropped and combined in Adobe Photoshop. The amount of DNA damage wasquantified by scoring the percentage of cells containing 0, 1, 2-5 or >5 H2AX andATM-P colocalized foci. At least 300 cells were scored for each variable. For TIFanalysis, cells were scored as having 0-1, 2-5, 5-10 or >10 TRF1 foci that colocalizedwith H2AX staining. At least 100 cells were scored for each variable.

Telomere chromatin immunoprecipitation (ChIP)The ChIP assay was performed using the EZ-Chip assay kit (Millipore, 17-371),according to the manufacturer’s instructions. Sonication was done using a Sonicator3000 (Misonix) under the following conditions: Amp5.5 with six cycles of 20seconds on and 20 seconds off. Immunoprecipitation was performed with eitherSer139 H2AX (Millipore, 07-164) or IgG (Millipore, 12-371) antibodies, andimmunoprecipitated DNA was transferred to a Hybond-N (GE Healthcare,RPN2020N) membrane using a slot-blot apparatus. The membrane was thenhybridized with a DIG-labeled telomeric (TTAGGG)4 probe and detected with the

TeloTAGGG Telomere Length Assay kit (Roche 12209136001). The membrane wasthen stripped by washing twice with 0.2 M NaOH and 0.1% SDS for 30 minutes at52°C and re-hybridized with a DIG-labeled Alu (GGAGGCTGAG -GCAGGAGAATTGCT) probe. DIG labeling was performed using the DIGOligonucleotide 3�-End Labeling Kit (Roche, 03353575910). Quantification of thesignal was performed with ImageJ software (NIH). The amount of telomeric and AluDNA after ChIP was normalized to the total input signal for each condition.

Telomere FISH on metaphase spreadsCells were treated with 0.1 g/ml demecolcine (Sigma, D7385) for 16-22 hours,harvested by trypsinization, and swelled in 0.075 M KCl for 20 minutes at 37°C.Cells were fixed overnight at –20°C in 3:1 methanol:acetic acid, dropped ontohumidified slides, and air-dried overnight. Cells were then rehydrated in PBS andfixed with 4% formaldehyde in PBS for 4 minutes at room temperature. Afterrinsing, cells were dehydrated in a 70%, 90% and 100% ethanol series, and air-dried.Hybridizing solution [70% formamide, 0.5% blocking reagent (Roche, 11096176001),20 mM Tris-HCl, pH 7.5, and 500 nM FAM-OO-(CCCTAAA)3 PNA probe(Panagene)] was added, and the slides were heated for 3 minutes at 80°C, followedby incubation in the dark for 2 hours at room temperature. The slides were washedtwice for 15 minutes each in wash solution 1 (70% formamide, 10 mM Tris-HCl,pH 7.5 and 0.1% blocking solution), and three times for 5 minutes each in washsolution 2 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.08% Tween 20). Slideswere mounted using Vectashield Mounting Medium with DAPI (Vector Laboratories,H-1200) and imaging was performed with a Zeiss Axioplan II microscope (CarlZeiss Microimaging) using the 63� oil objective. Images were cropped and combinedin Adobe Photoshop.

This work was supported by grant number PO1CA80058 from theNCI and the New York State Stem Cell contract #C024313. E.B. wassupported by the NCI Training Program in Cancer Biology (T32CA078207) and the NIGMS Training Program in Cellular andMolecular Biology (T32 GM008553). Confocal laser scanningmicroscopy was performed at the MSSM-Microscopy Shared ResourceFacility, supported with funding from NIH-NCI shared resources grant(5R24 CA095823-04), NSF Major Research Instrumentation grant(DBI-9724504) and NIH shared instrumentation grant (1 S10 RR09145-01). We thank Elizabeth Blackburn (UCSF) for providing uswith TERT mutant constructs, N125A+T126A and D868A, in thepBABE-puro backbone. We thank Titia de Lange (RockefellerUniversity) for the TRF1 (#370) antibody and for a TRF2BM constructobtained via Addgene. We also thank Bo Zhao and Cesar Munoz-Fontela for helpful discussions. Deposited in PMC for release after 12months.

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