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instability (1, 2). Epigenetic alterations have been associated
with cellular aging in diverse
model organisms (2–4). In humans, somatic cells derived from
patients with premature aging
syndromes are characterized by
loss of heterochromatin marks
(5–7). However, it is unclear
Weiqi Zhang,1* Jingyi Li,2* Keiichiro Suzuki,3* Jing Qu,4* Ping
whether epigenetic dysregulaWang, Junzhi Zhou, Xiaomeng Liu, Ruotong Ren, Xiuling Xu,
tion is involved in WS pathogen3
Alejandro Ocampo, Tingting Yuan, Jiping Yang, Ying Li, Liang
Shi,6 Dee Guan,1 Huize Pan,1 Shunlei Duan,1 Zhichao Ding,1 Mo
Generation of patient-specific
Li,3 Fei Yi,5 Ruijun Bai,4 Yayu Wang,6 Chang Chen,1 Fuquan Yang,1 induced pluripotent stem cells
(iPSCs) represents a promising
Xiaoyu Li,7 Zimei Wang,8 Emi Aizawa,3 April Goebl,3,9 Rupa Devi
avenue to model and study huSoligalla, Pradeep Reddy, Concepcion Rodriguez Esteban,
man aging and aging-associated
Fuchou Tang,
Guang-Hui Liu,
Juan Carlos Izpisua
disorders (8). WS-specific iPSCs
lines may constitute an ideal
source for in vitro modeling of
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China. 3Gene Expression
WS. However, we found that WS
Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. 4State Key
patient fibroblast lines deposited
Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
in different cell banks presented
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
severe karyotypic abnormalities
Diagnosis and Treatment Center for Oral Disease, the 306th Hospital of the PLA, Beijing, China. 7College of Life Sciences,
Peking University, Beijing 100871, China. 8The Center for Anti-aging and Regenerative Medicine, Shenzhen University,
and secondary DNA mutations
Shenzhen 518060, China. 9 Universidad Católica San Antonio de Murcia, Campus de los Jerónimos s/n, 30107 Guadalupe,
associated with advanced stages
Murcia, Spain. 10Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing 100871, China.
of WS pathology. To create an
Center for Molecular and Translational Medicine (CMTM), Beijing 100101, China. Peking-Tsinghua Center for Life
unbiased human WS cellular
Sciences, Peking University, Beijing 100871, China. 13Beijing Institute for Brain Disorders, Beijing100069, China.
model, we sought to generate an
*These authors contributed equally to this work.
isogenic WS ESC line by knock†Corresponding author. E-mail: (G.-H.L.); (F.T.);
ing out exons 15 and 16 of the
WRN gene encoding for the conWerner syndrome (WS) is a premature aging disorder caused by WRN
served DNA helicase domain (9).
protein deficiency. Here, we report on the generation of a human WS model
Following two rounds of homolin human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to
ogous recombination using helpmesenchymal stem cells (MSCs) recapitulates features of premature
er-dependent adenoviral vector
cellular aging, a global loss of H3K9me3, and changes in heterochromatin
(HDAdV) (10, 11), we successfully
architecture. We show that WRN associates with heterochromatin proteins
generated homozygous WRN
SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein
null ESC lines (ESCs-WRN−/−)
LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type
(Fig. 1A, B and fig. S1A-D). ESCsMSCs recapitulates accelerated cellular senescence, resembling WRNWRN−/− expressed pluripotency
deficient MSCs. Moreover, decrease in WRN and heterochromatin marks
markers, maintained normal
are detected in MSCs from older individuals. Our observations uncover a
karyotype and were able to difrole for WRN in maintaining heterochromatin stability and highlight
ferentiate into all three germ
heterochromatin disorganization as a potential determinant of human
layers (Fig. 1A and fig. S2A-E).
ESCs-WRN−/− lacked detectable
WRN protein, as determined by
Western blot using antibodies
Werner syndrome (WS), also known as adult progeria, recaspecific
terminus of WRN (Fig. 1B).
pitulates certain aspects of human physiological aging (1).
and cell growth rate beWS is caused by mutations in the WRN gene resulting in
was observed (fig. S2Floss of WRN expression or function (1). WRN protein plays
roles in DNA replication, transcription, repair, recombinaWS patients are mainly characterized by premature agtion as well as telomere maintenance, indicating that one of
pathologies associated with degeneration of mesodermal
the major causes for WS pathogenesis relates to genomic
tissues, i.e., osteoporosis, atherosclerosis and grey hair (1).
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A Werner syndrome stem cell model
unveils heterochromatin alterations as a
driver of human aging

We hypothesized that WS patients may suffer from an accelerated exhaustion of the MSC pool. This was tested by
differentiating ESCs-WRN−/− into MSCs. MSCs-WRN−/− expressed MSC-specific cell surface markers CD73, CD90,
CD105, lacked expression of MSC-irrelevant antigens including CD45, CD34 and CD43 (fig. S3A), and were able to differentiate toward osteoblasts, chondrocytes, and adipocytes
(fig. S3B, C) (12).
Upon serial passaging, WRN-deficient MSCs recapitulated major phenotypes of premature aging, including premature loss of proliferative potential, increased number of
senescence-associated-β-galactosidase (SA-β-gal) positive
cells, upregulated expression of aging-associated genes
p16Ink4a and p21Waf1, and activation of senescence-associated
secretory phenotype (SASP) (Fig. 1C-E and fig. S3D-G) (13).
Moreover, when WRN-deficient MSCs expressing luciferase
were transplanted into the muscle of NOD/SCID mice, they
underwent an accelerated attrition compared to wild type
MSCs (Fig. 1F and fig. S3H). These results demonstrated
that the loss of WRN promotes premature senescence in
WRN deficiency in MSCs resulted in elevated DNA damage response (DDR), indicated by increased nuclear foci for
53BP1, γ-H2AX and phosphorylated ATM/ATR substrates
(fig. S4A-C). Restoration of WRN activity by lentivirusmediated expression in MSCs-WRN−/− resulted in partial
alleviation of DDR and cellular senescence (fig. S4D, E). To
investigate potential chromosomal abnormalities resulting
from the loss of WRN protein, we performed genome-wide
copy number variation (CNV) analysis by deep sequencing.
In the time frame examined, genomic integrity was minimally affected in MSCs-WRN−/− (fig. S4F).
Epigenetic alteration has been considered as a hallmark
of aging (2). MSCs-WRN−/− showed a distinct nuclear
Hoechst 33342 staining pattern with markedly enlarged
nuclei and high pixel-to-pixel coefficient of variation (C.V.)
value, indicating possible changes in chromatin structure
(Fig. 2A and fig. S5A). Moreover, WRN-deficient MSCs exhibited accelerated diminishment of heterochromatinassociated inner nuclear membrane (INM) proteins LAP2β
and LBR, and reduced heterochromatin structure underneath the nuclear envelope, as indicated by immunostaining
and electron microscopy (Fig. 2B and fig. S5B, C) (14). These
results suggest a progressive disorganization of heterochromatin in WRN-deficient MSCs.
Further investigation of heterochromatin reorganization
at histone and DNA levels revealed significant downregulation of the constitutive heterochromatin mark H3K9me3 in
MSC-WRN−/− (Fig. 2C and fig. S5D, E). In contrast,
H3K27me3 showed slight downregulation, while H3K4me3,
a mark for euchromatin fiber, exhibited comparable levels
between WRN-deficient and wild-type MSCs (Fig. 2C and
fig. S5D, E). We did not observe significant genome-wide
alteration of 5mC in WRN-deficient MSCs (Fig. 2C). Bioinformatic analysis identified 73 H3K9me3-enriched “moun-

tains” throughout the genome in MSCs-WRN+/+, which are
characterized by over 20 kb of consecutive peaks of
H3K9me3 (Fig. 2D). 28 (38%) of these H3K9me3 mountains
were lost in MSCs-WRN−/− (Fig. 2D). Interestingly, 24 (86%)
of these impaired H3K9me3 mountains resided in subtelomeric or sub-centromeric regions (Fig. 2D, E and table
RNA-seq identified 1,047 RefSeq genes that showed differential expression in MSCs-WRN−/− (Fig. 6A, B and table
S2). The most significantly down-regulated genes were centromere-packaging proteins and components of the nuclear
membrane (fig. S6A-E and table S3). These results indicate
alterations in nuclear structure and epigenomic organization potentially leading to a progressive loss of heterochromatin structure in MSCs as a consequence of WRN
In agreement with previous reports describing WRN as a
telomere-associated protein required for telomere maintenance (15), compromised telomerase activity and shorter
telomere length was detected in MSC-WRN−/− (fig. S7A, B).
In addition, quantitative ChIP-PCR (ChIP-qPCR) showed
binding of WRN to the H3K9me3-enriched centromeric loci
α-Satellite (α-Sat) and Satellite 2 (Sat2) (Fig. 3A) (16). Depletion of WRN resulted in an increase in centromeric γ-H2AX
signal and a loss of H3K9me3 from α-Sat and Sat2 loci accompanied by upregulation of transcripts from these sequences (Fig. 3A, B and fig. S7C). Co-immunoprecipitation
(Co-IP) analysis revealed WRN as part of a complex containing the major histone methyltransferase for H3K9me3 SUV39H1, HP1α, and LAP2β, a nuclear envelope component
that recruits heterochromatin via anchoring to HP1α (Fig.
3C and fig. S7D) (17). These observations suggest a role for
WRN, together with SUV39H1 and HP1α, in the stabilization
of heterochromatin.
We next tested whether disorganization of heterochromatin could contribute to accelerated cellular senescence.
Knockdown of SUV39H1 or HP1α in wild-type MSCs led to a
significant reduction of overall H3K9me3 and induction of
cellular senescence, as assayed by Western blot, SA-β-gal
staining, and p16 expression (Fig. 3D and fig. S8A-D). On
the contrary, overexpression of HP1α upregulated H3K9me3
levels and repressed cellular senescence in WRN-deficient
MSCs (fig. S8E-H). To confirm these observations, we generated pluripotent ESCs-SUV39H1H324K lines harboring catalytically inactivated endogenous SUV39H1 (fig. S9A-D). Upon
differentiation, MSCs-SUV39H1H324K displayed drastic nuclear structural and chromosomal changes, loss of INM proteins LAP2β and LBR, decreased levels of H3K9me3 and
HP1α, upregulation of centromeric repetitive sequence transcription, and coordinated transcriptional downregulation
of centromere-packaging components (Fig. 4A-C and fig.
S10A, B). MSCs-SUV39H1H324K recapitulated premature aging
phenotypes observed in WRN deficient MSCs, including retarded cell growth and accelerated cellular senescence determined by SA-β-gal staining (Fig. 4D and fig. S10C-E).

/ / 30 April 2015 / Page 2 / 10.1126/science.aaa1356

High expression of SUV39H2, a germline-specific histone
methyltransferase, and/or other factors may functionally
compensate for SUV39H1 deficiency in ESCs (fig. S10F) (18,
19), where upon inactivation of the WRN-SUV39H1 axis, no
discernible heterochromatin change was observed (figs.
S10G and S8A). It should be noted that MSCs-SUV39H1H324K
exhibit neither increased γ-H2AX (P=0.773) and phosphorylated ATM/ATR substrates (P=0.279), nor telomere attrition
(figs. S10H and S7A, B). These results indicate that active
heterochromatin destabilization promotes premature aging
in MSCs.
Finally, we asked whether heterochromatin disorganization could be a common hallmark for physiological human
stem cell aging. For this purpose, we compared the levels of
heterochromatin marks in primary dental pulp MSCs derived from six young (7-26 year old) and six old (58-72 year
old) individuals (fig. S10I and table S4) (20). A marked
downregulation of WRN protein associated with a decrease
in H3K9me3, HP1α, SUV39H1, and LAP2β levels in MSCs
derived from old individuals (Fig. 4E). Therefore, specific
heterochromatin changes may underlie both pathological as
well as physiological mesenchymal stem cell aging.
In summary, we have found that WRN protein, besides
its role in DNA repair, functions to safeguard heterochromatin stability (fig. S11). Our results unveil that the progressive
heterochromatin disorganization observed in WRN deficient
MSCs underlies cellular aging, but more extensive studies
are needed to examine its role during physiological aging.
The methodologies and observations here introduced may
be used and extended toward the systematical study of other
age-associated molecular events with relevance to human
aging and age-related disorders.
1. B. A. Kudlow, B. K. Kennedy, R. J. Monnat Jr., Werner and Hutchinson-Gilford
progeria syndromes: Mechanistic basis of human progeroid diseases. Nat. Rev.
Mol. Cell Biol. 8, 394–404 (2007). Medline doi:10.1038/nrm2161
2. C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The hallmarks of
aging. Cell 153, 1194–1217 (2013). Medline doi:10.1016/j.cell.2013.05.039
3. G. Pegoraro, N. Kubben, U. Wickert, H. Göhler, K. Hoffmann, T. Misteli, Ageingrelated chromatin defects through loss of the NURD complex. Nat. Cell Biol. 11,
1261–1267 (2009). Medline doi:10.1038/ncb1971
4. E. L. Greer, T. J. Maures, A. G. Hauswirth, E. M. Green, D. S. Leeman, G. S. Maro, S.
Han, M. R. Banko, O. Gozani, A. Brunet, Members of the H3K4 trimethylation
complex regulate lifespan in a germline-dependent manner in C. elegans. Nature
466, 383–387 (2010). Medline doi:10.1038/nature09195
5. G.-H. Liu, B. Z. Barkho, S. Ruiz, D. Diep, J. Qu, S.-L. Yang, A. D. Panopoulos, K.
Suzuki, L. Kurian, C. Walsh, J. Thompson, S. Boue, H. L. Fung, I. SanchoMartinez, K. Zhang, J. Yates 3rd, J. C. Izpisua Belmonte, Recapitulation of
premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.
Nature 472, 221–225 (2011). Medline doi:10.1038/nature09879
6. D. K. Shumaker, T. Dechat, A. Kohlmaier, S. A. Adam, M. R. Bozovsky, M. R. Erdos,
M. Eriksson, A. E. Goldman, S. Khuon, F. S. Collins, T. Jenuwein, R. D. Goldman,
Mutant nuclear lamin A leads to progressive alterations of epigenetic control in
premature aging. Proc. Natl. Acad. Sci. U.S.A. 103, 8703–8708 (2006). Medline
7. J. D. Miller, Y. M. Ganat, S. Kishinevsky, R. L. Bowman, B. Liu, E. Y. Tu, P. K.
Mandal, E. Vera, J. W. Shim, S. Kriks, T. Taldone, N. Fusaki, M. J. Tomishima, D.
Krainc, T. A. Milner, D. J. Rossi, L. Studer, Human iPSC-based modeling of lateonset disease via progerin-induced aging. Cell Stem Cell 13, 691–705 (2013).
Medline doi:10.1016/j.stem.2013.11.006

8. G. H. Liu, Z. Ding, J. C. Izpisua Belmonte, iPSC technology to study human aging
and aging-related disorders. Curr. Opin. Cell Biol. 24, 765–774 (2012). Medline
9. D. B. Lombard, C. Beard, B. Johnson, R. A. Marciniak, J. Dausman, R. Bronson, J.
E. Buhlmann, R. Lipman, R. Curry, A. Sharpe, R. Jaenisch, L. Guarente, Mutations
in the WRN gene in mice accelerate mortality in a p53-null background. Mol. Cell.
Biol. 20, 3286–3291 (2000). Medline doi:10.1128/MCB.20.9.3286-3291.2000
10. G- H. Liu, J. Qu, K. Suzuki, E. Nivet, M. Li, N. Montserrat, F. Yi, X. Xu, S. Ruiz, W.
Zhang, U. Wagner, A. Kim, B. Ren, Y. Li, A. Goebl, J. Kim, R. D. Soligalla, I.
Dubova, J. Thompson, J. Yates 3rd, C. R. Esteban, I. Sancho-Martinez, J. C.
Izpisua Belmonte, Progressive degeneration of human neural stem cells caused
by pathogenic LRRK2. Nature 491, 603–607 (2012). Medline
11. K. Suzuki, C. Yu, J. Qu, M. Li, X. Yao, T. Yuan, A. Goebl, S. Tang, R. Ren, E. Aizawa,
F. Zhang, X. Xu, R. D. Soligalla, F. Chen, J. Kim, N. Y. Kim, H. K. Liao, C. Benner, C.
R. Esteban, Y. Jin, G. H. Liu, Y. Li, J. C. Izpisua Belmonte, Targeted gene
correction minimally impacts whole-genome mutational load in human-diseasespecific induced pluripotent stem cell clones. Cell Stem Cell 15, 31–36 (2014).
Medline doi:10.1016/j.stem.2014.06.016
12. G. H. Liu, K. Suzuki, M. Li, J. Qu, N. Montserrat, C. Tarantino, Y. Gu, F. Yi, X. Xu, W.
Zhang, S. Ruiz, N. Plongthongkum, K. Zhang, S. Masuda, E. Nivet, Y. Tsunekawa,
R. D. Soligalla, A. Goebl, E. Aizawa, N. Y. Kim, J. Kim, I. Dubova, Y. Li, R. Ren, C.
Benner, A. del Sol, J. Bueren, J. P. Trujillo, J. Surralles, E. Cappelli, C. Dufour, C.
R. Esteban, J. C. Izpisua Belmonte, Modelling Fanconi anemia pathogenesis and
therapeutics using integration-free patient-derived iPSCs. Nat. Commun. 5,
4330 (2014). Medline
13. F. Rodier, J. Campisi, Four faces of cellular senescence. J. Cell Biol. 192, 547–556
(2011). Medline doi:10.1083/jcb.201009094
14. T. Dechat, S. A. Adam, P. Taimen, T. Shimi, R. D. Goldman, Nuclear lamins. Cold
15. A. S. Multani, S. Chang, WRN at telomeres: Implications for aging and cancer. J.
Cell Sci. 120, 713–721 (2007). Medline doi:10.1242/jcs.03397
16. D. Wang, J. Zhou, X. Liu, D. Lu, C. Shen, Y. Du, F. Z. Wei, B. Song, X. Lu, Y. Yu, L.
Wang, Y. Zhao, H. Wang, Y. Yang, Y. Akiyama, H. Zhang, W. G. Zhu, Methylation of
SUV39H1 by SET7/9 results in heterochromatin relaxation and genome
instability. Proc. Natl. Acad. Sci. U.S.A. 110, 5516–5521 (2013). Medline
17. N. Kourmouli, P. A. Theodoropoulos, G. Dialynas, A. Bakou, A. S. Politou, I. G.
Cowell, P. B. Singh, S. D. Georgatos, Dynamic associations of heterochromatin
protein 1 with the nuclear envelope. EMBO J. 19, 6558–6568 (2000). Medline
18. D. O’Carroll, H. Scherthan, A. H. Peters, S. Opravil, A. R. Haynes, G. Laible, S. Rea,
M. Schmid, A. Lebersorger, M. Jerratsch, L. Sattler, M. G. Mattei, P. Denny, S. D.
Brown, D. Schweizer, T. Jenuwein, Isolation and characterization of Suv39h2, a
second histone H3 methyltransferase gene that displays testis-specific
expression. Mol. Cell. Biol. 20, 9423–9433 (2000). Medline
19. W. Zhang, J. Qu, K. Suzuki, G.-H. Liu, J. C. Izpisua Belmonte, Concealing cellular
defects in pluripotent stem cells. Trends Cell Biol. 23, 587–592 (2013). Medline
20. G. B. Tomar, R. K. Srivastava, N. Gupta, A. P. Barhanpurkar, S. T. Pote, H. M.
Jhaveri, G. C. Mishra, M. R. Wani, Human gingiva-derived mesenchymal stem
cells are superior to bone marrow-derived mesenchymal stem cells for cell
therapy in regenerative medicine. Biochem. Biophys. Res. Commun. 393, 377–
383 (2010). Medline doi:10.1016/j.bbrc.2010.01.126
21. B. Li, S. P. Jog, S. Reddy, L. Comai, WRN controls formation of extrachromosomal
telomeric circles and is required for TRF2ΔB-mediated telomere shortening. Mol.
Cell. Biol. 28, 1892–1904 (2008). Medline doi:10.1128/MCB.01364-07
22. S. Lachapelle, J. P. Gagné, C. Garand, M. Desbiens, Y. Coulombe, V. A. Bohr, M. J.
Hendzel, J. Y. Masson, G. G. Poirier, M. Lebel, Proteome-wide identification of
WRN-interacting proteins in untreated and nuclease-treated samples. J.
Proteome Res. 10, 1216–1227 (2011). Medline doi:10.1021/pr100990s
23. K. A. Datsenko, B. L. Wanner, One-step inactivation of chromosomal genes in
Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640–
6645 (2000). Medline doi:10.1073/pnas.120163297
24. K. Suzuki, K. Mitsui, E. Aizawa, K. Hasegawa, E. Kawase, T. Yamagishi, Y. Shimizu,
H. Suemori, N. Nakatsuji, K. Mitani; K. Suzukiet, Highly efficient transient gene

/ / 30 April 2015 / Page 3 / 10.1126/science.aaa1356

expression and gene targeting in primate embryonic stem cells with helperdependent adenoviral vectors. Proc. Natl. Acad. Sci. U.S.A. 105, 13781–13786
(2008). Medline doi:10.1073/pnas.0806976105
25. D. J. Palmer, P. Ng, Physical and infectious titers of helper-dependent adenoviral
vectors: A method of direct comparison to the adenovirus reference material.
Mol. Ther. 10, 792–798 (2004). Medline doi:10.1016/j.ymthe.2004.06.1013
26. D. M. Shayakhmetov, Z. Y. Li, A. Gaggar, H. Gharwan, V. Ternovoi, V. Sandig, A.
Lieber, Genome size and structure determine efficiency of postinternalization
steps and gene transfer of capsid-modified adenovirus vectors in a cell-typespecific manner. J. Virol. 78, 10009–10022 (2004). Medline
27. T. Ahfeldt, R. T. Schinzel, Y. K. Lee, D. Hendrickson, A. Kaplan, D. H. Lum, R.
Camahort, F. Xia, J. Shay, E. P. Rhee, C. B. Clish, R. C. Deo, T. Shen, F. H. Lau, A.
Cowley, G. Mowrer, H. Al-Siddiqi, M. Nahrendorf, K. Musunuru, R. E. Gerszten, J.
L. Rinn, C. A. Cowan, Programming human pluripotent stem cells into white and
brown adipocytes. Nat. Cell Biol. 14, 209–219 (2012). Medline
28. D. R. Deyle, I. F. Khan, G. Ren, P. R. Wang, J. Kho, U. Schwarze, D. W. Russell,
Normal collagen and bone production by gene-targeted human osteogenesis
imperfecta iPSCs. Mol. Ther. 20, 204–213 (2012). Medline
29. S. Kaitainen, A. J. Mähönen, R. Lappalainen, H. Kröger, M. J. Lammi, C. Qu,
TiO2 coating promotes human mesenchymal stem cell proliferation without the
loss of their capacity for chondrogenic differentiation. Biofabrication 5, 025009
(2013). Medline doi:10.1088/1758-5082/5/2/025009
30. G. T. Huang, S. Gronthos, S. Shi, Mesenchymal stem cells derived from dental
tissues vs. those from other sources: Their biology and role in regenerative
31. L. Pierdomenico, L. Bonsi, M. Calvitti, D. Rondelli, M. Arpinati, G. Chirumbolo, E.
Becchetti, C. Marchionni, F. Alviano, V. Fossati, N. Staffolani, M. Franchina, A.
Grossi, G. P. Bagnara, Multipotent mesenchymal stem cells with
immunosuppressive activity can be easily isolated from dental pulp.
32. X. Chen, S. Wei, Y. Ma, J. Lu, G. Niu, Y. Xue, X. Chen, F. Yang, Quantitative
proteomics analysis identifies mitochondria as therapeutic targets of multidrugresistance in ovarian cancer. Theranostics 4, 1164–1175 (2014). Medline
33. F. Debacq-Chainiaux, J. D. Erusalimsky, J. Campisi, O. Toussaint, Protocols to
detect senescence-associated beta-galactosidase (SA-βgal) activity, a
biomarker of senescent cells in culture and in vivo. Nat. Protoc. 4, 1798–1806
(2009). Medline doi:10.1038/nprot.2009.191
34. A. Freund, C. K. Patil, J. Campisi, p38MAPK is a novel DNA damage responseindependent regulator of the senescence-associated secretory phenotype.
EMBO J. 30, 1536–1548 (2011). Medline doi:10.1038/emboj.2011.69
35. R. M. Cawthon, Telomere measurement by quantitative PCR. Nucleic Acids Res.
30, e47 (2002). Medline doi:10.1093/nar/30.10.e47
36. J. A. Dahl, P. Collas, A rapid micro chromatin immunoprecipitation assay (ChIP).
Nat. Protoc. 3, 1032–1045 (2008). Medline doi:10.1038/nprot.2008.68
37. Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C.
Nusbaum, R. M. Myers, M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIPSeq (MACS). Genome Biol. 9, R137 (2008). Medline doi:10.1186/gb-2008-9-9r137
38. L. Wen, X. Li, L. Yan, Y. Tan, R. Li, Y. Zhao, Y. Wang, J. Xie, Y. Zhang, C. Song, M.
Yu, X. Liu, P. Zhu, X. Li, Y. Hou, H. Guo, X. Wu, C. He, R. Li, F. Tang, J. Qiao, Wholegenome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base
resolution in the human brain. Genome Biol. 15, R49 (2014). Medline
39. F. Krueger, S. R. Andrews, Bismark: A flexible aligner and methylation caller for
Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011). Medline
40. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Ultrafast and memory-efficient
alignment of short DNA sequences to the human genome. Genome Biol. 10, R25
(2009). Medline doi:10.1186/gb-2009-10-3-r25
41. L. R. Meyer, A. S. Zweig, A. S. Hinrichs, D. Karolchik, R. M. Kuhn, M. Wong, C. A.
Sloan, K. R. Rosenbloom, G. Roe, B. Rhead, B. J. Raney, A. Pohl, V. S. Malladi, C.
H. Li, B. T. Lee, K. Learned, V. Kirkup, F. Hsu, S. Heitner, R. A. Harte, M.

Haeussler, L. Guruvadoo, M. Goldman, B. M. Giardine, P. A. Fujita, T. R. Dreszer,
M. Diekhans, M. S. Cline, H. Clawson, G. P. Barber, D. Haussler, W. J. Kent, The
UCSC Genome Browser database: Extensions and updates 2013. Nucleic Acids
Res. 41 (D1), D64–D69 (2013). Medline doi:10.1093/nar/gks1048
42. D. Karolchik, A. S. Hinrichs, T. S. Furey, K. M. Roskin, C. W. Sugnet, D. Haussler,
W. J. Kent, The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 32,
D493–D496 (2004). Medline doi:10.1093/nar/gkh103
43. H. Li, R. Durbin, Fast and accurate long-read alignment with Burrows-Wheeler
44. E. S. Lander, L. M. Linton, B. Birren, C. Nusbaum, M. C. Zody, J. Baldwin, K.
Devon, K. Dewar, M. Doyle, W. FitzHugh, R. Funke, D. Gage, K. Harris, A. Heaford,
J. Howland, L. Kann, J. Lehoczky, R. LeVine, P. McEwan, K. McKernan, J.
Meldrim, J. P. Mesirov, C. Miranda, W. Morris, J. Naylor, C. Raymond, M. Rosetti,
R. Santos, A. Sheridan, C. Sougnez, N. Stange-Thomann, N. Stojanovic, A.
Subramanian, D. Wyman, J. Rogers, J. Sulston, R. Ainscough, S. Beck, D.
Bentley, J. Burton, C. Clee, N. Carter, A. Coulson, R. Deadman, P. Deloukas, A.
Dunham, I. Dunham, R. Durbin, L. French, D. Grafham, S. Gregory, T. Hubbard, S.
Humphray, A. Hunt, M. Jones, C. Lloyd, A. McMurray, L. Matthews, S. Mercer, S.
Milne, J. C. Mullikin, A. Mungall, R. Plumb, M. Ross, R. Shownkeen, S. Sims, R. H.
Waterston, R. K. Wilson, L. W. Hillier, J. D. McPherson, M. A. Marra, E. R. Mardis,
L. A. Fulton, A. T. Chinwalla, K. H. Pepin, W. R. Gish, S. L. Chissoe, M. C. Wendl, K.
D. Delehaunty, T. L. Miner, A. Delehaunty, J. B. Kramer, L. L. Cook, R. S. Fulton, D.
L. Johnson, P. J. Minx, S. W. Clifton, T. Hawkins, E. Branscomb, P. Predki, P.
Richardson, S. Wenning, T. Slezak, N. Doggett, J. F. Cheng, A. Olsen, S. Lucas, C.
Elkin, E. Uberbacher, M. Frazier, R. A. Gibbs, D. M. Muzny, S. E. Scherer, J. B.
Bouck, E. J. Sodergren, K. C. Worley, C. M. Rives, J. H. Gorrell, M. L. Metzker, S. L.
Naylor, R. S. Kucherlapati, D. L. Nelson, G. M. Weinstock, Y. Sakaki, A. Fujiyama,
M. Hattori, T. Yada, A. Toyoda, T. Itoh, C. Kawagoe, H. Watanabe, Y. Totoki, T.
Taylor, J. Weissenbach, R. Heilig, W. Saurin, F. Artiguenave, P. Brottier, T. Bruls,
E. Pelletier, C. Robert, P. Wincker, D. R. Smith, L. Doucette-Stamm, M.
Rubenfield, K. Weinstock, H. M. Lee, J. Dubois, A. Rosenthal, M. Platzer, G.
Nyakatura, S. Taudien, A. Rump, H. Yang, J. Yu, J. Wang, G. Huang, J. Gu, L.
Hood, L. Rowen, A. Madan, S. Qin, R. W. Davis, N. A. Federspiel, A. P. Abola, M. J.
Proctor, R. M. Myers, J. Schmutz, M. Dickson, J. Grimwood, D. R. Cox, M. V.
Olson, R. Kaul, C. Raymond, N. Shimizu, K. Kawasaki, S. Minoshima, G. A. Evans,
M. Athanasiou, R. Schultz, B. A. Roe, F. Chen, H. Pan, J. Ramser, H. Lehrach, R.
Reinhardt, W. R. McCombie, M. de la Bastide, N. Dedhia, H. Blöcker, K.
Hornischer, G. Nordsiek, R. Agarwala, L. Aravind, J. A. Bailey, A. Bateman, S.
Batzoglou, E. Birney, P. Bork, D. G. Brown, C. B. Burge, L. Cerutti, H. C. Chen, D.
Church, M. Clamp, R. R. Copley, T. Doerks, S. R. Eddy, E. E. Eichler, T. S. Furey, J.
Galagan, J. G. Gilbert, C. Harmon, Y. Hayashizaki, D. Haussler, H. Hermjakob, K.
Hokamp, W. Jang, L. S. Johnson, T. A. Jones, S. Kasif, A. Kaspryzk, S. Kennedy,
W. J. Kent, P. Kitts, E. V. Koonin, I. Korf, D. Kulp, D. Lancet, T. M. Lowe, A.
McLysaght, T. Mikkelsen, J. V. Moran, N. Mulder, V. J. Pollara, C. P. Ponting, G.
Schuler, J. Schultz, G. Slater, A. F. Smit, E. Stupka, J. Szustakowski, D. ThierryMieg, J. Thierry-Mieg, L. Wagner, J. Wallis, R. Wheeler, A. Williams, Y. I. Wolf, K.
H. Wolfe, S. P. Yang, R. F. Yeh, F. Collins, M. S. Guyer, J. Peterson, A. Felsenfeld,
K. A. Wetterstrand, A. Patrinos, M. J. Morgan, P. de Jong, J. J. Catanese, K.
Osoegawa, H. Shizuya, S. Choi, Y. J. Chen; International Human Genome
Sequencing Consortium, Initial sequencing and analysis of the human genome.
Nature 409, 860–921 (2001). Medline doi:10.1038/35057062
45. W. Huang, B. T. Sherman, R. A. Lempicki, Systematic and integrative analysis of
large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57
(2009). Medline doi:10.1038/nprot.2008.211
46. W. Huang, B. T. Sherman, R. A. Lempicki, Bioinformatics enrichment tools: Paths
toward the comprehensive functional analysis of large gene lists. Nucleic Acids
Res. 37, 1–13 (2009). Medline doi:10.1093/nar/gkn923
47. M. Krzywinski, J. Schein, I. Birol, J. Connors, R. Gascoyne, D. Horsman, S. J.
Jones, M. A. Marra, Circos: An information aesthetic for comparative genomics.
Genome Res. 19, 1639–1645 (2009). Medline doi:10.1101/gr.092759.109
We are grateful to W. G. Zhu, L. Comai, K. Mitani, P. Ng and A. Lieber for sharing
experimental materials, L. Sun, W. Ding, G. Yuan, and X. Zhu for technical
assistance, and M. Schwarz for administrative help. This work was supported by
National Natural Science Foundation of China (NSFC: 81330008), National Basic
Research Program of China (973 Program, 2015CB964800; 2014CB910500;

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2014CB964600; 2012CB966704), the Strategic Priority Research Program of
the Chinese Academy of Sciences (XDA01020312), NSFC (31222039; 31201111;
81371342; 81300261; 81300677; 81271266; 81471414; 81422017; 81401159;
31322037; 81471407), Beijing Natural Science Foundation (7141005; 5142016),
Key Research Program of the Chinese Academy of Sciences (KJZDEW-TZ-L05),
the Thousand Young Talents program of China, National Laboratory of
Biomacromolecules (012kf02;2013kf05;2013kf11;2014kf02;2015kf10), and
State Key Laboratory of Drug Research (SIMM1302KF-17), and China
Postdoctoral Science Foundation Grant (2013M530751). K.S and M.L. are
supported by a California Institute for Regenerative Medicine Training Grant.
A.O. was partially supported by an NIH Ruth L. Kirschstein National Research
Service Award Individual Postdoctoral Fellowship. The physiological human cell
aging studies were supported by UCAM. J.C.I.B. laboratory was supported by
The Glenn Foundation, The G. Harold and Leila Y. Mathers Charitable Foundation
and The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002).
All materials are available from the Izpisua Belmonte laboratory under a material
transfer agreement with The Salk Institute for Biological Studies. Myc-HP1a
plasmid can be obtained from W. G. Zhu under a material transfer agreement
with Peking University.
Material and Methods
Figs. S1 to S11
Table S1 to S5
References (21–47)
21 October 2014; accepted 15 April 2015
Published online 30 April 2015

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Fig. 1. WRN-deficient MSCs
exhibit phenotypes associated
senescence. (A) Morphology and
immunofluorescence analyses of
pluripotency markers in ESCs.
Scale bar, 100 μm and 10 μm,
respectively. (B) Western blot
analysis of WRN expression in
ESCs using anti-WRN N-terminal
(ab200) and C-terminal (SC5629) antibodies. (C) Growth
curve analyzing the accumulative
population doubling of MSCs. (D)
Senescence-associated (SA)-βgal staining in passage 1 (P1) and
P5 MSCs. Scale bar, 50μm. (E)
Quantitative RT-PCR analysis of
the indicated genes in P1 and P5
MSCs. Transcript levels were
group. Genes with greater mean
value are color coded toward red.
(F) Photon flux from muscle of
NOD-SCID mouse transplanted
with MSCs-WRN+/+ (left) and
MSCs-WRN−/− (right) expressing
represented as mean + SEM.
*P<0.05, **P<0.01, ***P<0.001
by t test; n=3.

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Fig. 2. Epigenomic analyses of WRN-deficient MSCs. (A) Left, Chromatin structure of MSCs
shown by Hoechst 33342 staining of the nucleus. Scale bar, 5μm.; Right, C.V. value of nuclear
Hoechst staining intensity used to evaluate the heterogeneity (pixel-to-pixel variation) of Hoechst
intensity. (B) Immunofluorescence analyses of LAP2β expression in MSC-WRN+/+ and MSC-WRN−/−
at P5. Arrowheads denote abnormal nuclei with decreased LAP2β expression (percentage of
LAP2β−positive nuclei in corner). Scale bar, 10 μm. (C) Enrichment of H3K9me3, H3K27me3,
H3K4me3, and 5mC on the gene bodies and 21kb upstream of TSS and 21kb downstream of TTS
regions in human genome. (D) Sketch map of “H3K9me3 mountain” distribution over 23
chromosomes. The blue lines indicate 73 “H3K9me3 mountains” present in MSCs-WRN+/+ whereas
48 (65.8%) of them are localized within 5 Mb regions around the telomeres or centromeres. The
red arrowheads indicate 28 “H3K9me3 mountains” which are lost in MSCs-WRN−/−. The circles
indicate the centromeres of chromosomes. (E) Representative images showing two “H3K9me3
mountains” on chromosome 2 in the sub-telomere or sub-centromere regions in P5 MSCs-WRN−/−
and MSCs-WRN+/+. Two biological replicates of each sample are presented. Black square denotes
the centromere; red rectangles denote the position of the presented sub-telomere and subcentromere regions, respectively. All data are represented as mean + SEM. ***P<0.001 by t test;

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Fig. 3. WRN associates with centromeric heterochromatin, and forms a
molecular complex with SUV39H1 and HP1α. (A) Enrichment of WRN and
H3K9me3 within the region of α-Sat or Sat2 as measured by ChIP-qPCR. (B)
Quantitative RT-PCR analysis of centromeric repetitive element transcripts
in MSCs at the indicated passages. (C) Left, co-immunoprecipitation of
SUV39H1, HP1α, and LAP2β protein with endogenous WRN protein; Right,
co-immunoprecipitation of WRN and HP1α with endogenous SUV39H1 in
wild-type MSCs. (D) SA-β-gal staining (left) and p16 transcript (right)
analyses in wild-type MSCs transduced with control lentiviral vector (CTRL)
or lentiviral vector encoding for the indicated shRNA (Knock-down). All data
are represented as mean + SEM. *P<0.05, **P<0.01, and ***P<0.001 by t
test; n=3.

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Fig. 4. SUV39H1H324K mutant MSCs exhibit defective nuclear envelope
and heterochromatin, as well as phenotypes of premature cellular
senescence. (A) As described in Fig. 2A, Left, Hoechst staining images of
the nucleus; Right, C.V. value of nuclear Hoechst staining intensity used to
evaluate the heterogeneity (pixel-to-pixel variation) of Hoechst intensity.
(B) Immunofluorescence analyses of LAP2β expression in MSCs.
Arrowheads denote the abnormal nuclei with decreased LAP2β
(Percentages of normal nuclei presented at corner). Scale bar, 20 μm. (C)
Western blot analysis of the indicated proteins in MSCs. (D) SA-β-gal
staining in MSCs at P5. Scale bar, 50 μm. (E) Western blot analysis of the
indicated proteins in human primary MSCs derived from old and young
healthy individuals at P4 (see table S4). All data are represented as mean +
SEM. ***P<0.001 by t test; n=3.

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