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Sirt6 Promotes DNA End Joining in iPSCs Derived from Old Mice Graphical Abstract

Authors Wen Chen, Nana Liu, Hongxia Zhang, ..., Songcheng Zhu, Zhiyong Mao, Jiuhong Kang

Correspondence [email protected] (Z.M.), [email protected] (J.K.)

In Brief Chen et al. find that iPSCs from old mice show lower genomic stability and less efficient NHEJ repair than iPSCs from young mice. This decrease is rescued by overexpression of Sirt6 during reprogramming. Sirt6 binds to Ku80 and facilitates the Ku80/DNA-PKcs interaction, thus leading to efficient NHEJ.

Highlights d

NHEJ is less efficient in iPSCs derived from old mice than from young mice

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Sirt6-deficient iPSCs have impaired NHEJ compared with wild-type controls

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Sirt6 facilitates Ku80/DNA-PKcs interaction to promote NHEJ

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Sirt6 overexpression promotes NHEJ in iPSCs derived from old mice

Chen et al., 2017, Cell Reports 18, 2880–2892 March 21, 2017 ª 2017 The Authors. http://dx.doi.org/10.1016/j.celrep.2017.02.082

Cell Reports

Article Sirt6 Promotes DNA End Joining in iPSCs Derived from Old Mice Wen Chen,1 Nana Liu,1 Hongxia Zhang,1 Haiping Zhang,1 Jing Qiao,1 Wenwen Jia,1 Songcheng Zhu,1 Zhiyong Mao,1,* and Jiuhong Kang1,2,* 1Clinical

and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, Collaborative Innovation Center for Brain Science, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China 2Lead Contact *Correspondence: [email protected] (Z.M.), [email protected] (J.K.) http://dx.doi.org/10.1016/j.celrep.2017.02.082

SUMMARY

Induced pluripotent stem cells (iPSCs) have great potential for treating age-related diseases, but the genome integrity of iPSCs is critically important. Here, we demonstrate that non-homologous end joining (NHEJ), rather than homologous recombination (HR), is less efficient in iPSCs from old mice than young mice. We further find that Sirt6 is downregulated in iPSCs from old mice. Sirt6 directly binds to Ku80 and facilitates the Ku80/DNA-PKcs interaction, thus promoting DNA-PKcs phosphorylation at residue S2056, leading to efficient NHEJ. Rescue experiments show that introducing a combination of Sirt6 and the Yamanaka factors during reprogramming significantly promotes DNA double-strand break (DSB) repair by activating NHEJ in iPSCs derived from old mice. Thus, our study suggests a strategy to improve the quality of iPSCs derived from old donors by activating NHEJ and stabilizing the genome. INTRODUCTION Aging is an irreversible process that often leads to a failing capacity to repair or replace damaged cells or tissues, resulting in numerous age-associated diseases, such as various neurodegenerative disorders (Liu et al., 2012; Vellas et al., 1992). The induced pluripotent stem cell (iPSC) approach, pioneered by Shinya Yamanaka, enables the reprogramming of somatic cells to a pluripotent state by overexpressing Oct4, Sox2, Klf4, and c-Myc and provides a versatile tool for the field of regenerative medicine (Okita et al., 2007; Tabar and Studer, 2014). However, several reports have indicated that iPSCs often carry the information inherited from the somatic cells (Bar-Nur et al., 2011; Kim et al., 2010, 2011).Loss or impairment of essential genetic information greatly hampers the use of iPSCs in potential clinical applications because they could cause incorrect differentiation or even tumorigenesis (Gore et al., 2011; Hussein et al., 2011; Stadtfeld et al., 2010). In particular, the age-related decline of DNA repair capacity in somatic cells causes a rise in genomic

instability (Li et al., 2016), which, in turn, might negatively affect iPSCs. Indeed, recent reports suggest that the reprogramming efficiency of skin fibroblasts derived from old donors is significantly lower than that from young donors (Kim et al., 2010; Li et al., 2009). However, whether the genome integrity in iPSCs derived from old human donors or mice is impaired compared with that from young controls has not been assessed. DNA double-strand breaks (DSBs) are the most deleterious types of DNA damage among all DNA lesions. Unrepaired or improperly repaired DSBs may lead to several types of ramifications, such as cellular senescence and oncogenic transformation. Two repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), have evolved to mend DNA breaks. NHEJ can be further categorized into two sub-pathways: classical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ). DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70/Ku80 heterodimers, Artemis, XRCC4, XLF, and DNA Lig4 participate in c-NHEJ, whereas PARP1 and DNA Lig3 are known factors that participate in alt-NHEJ (Frit et al., 2014; Thompson and Schild, 2001; Waters et al., 2014). c-NHEJ is the dominant pathway for repairing DNA DSBs in mammalian somatic cells (Jeggo, 1998). It is initiated by rapid recruitment of the Ku70/Ku80 heterodimer to DNA damage sites. The donut structure of Ku70/Ku80 binds to the broken ends to provide a platform for the binding of other NHEJ factors, including DNA-PKcs, which subsequently phosphorylates several other factors, such as Artemis, to facilitate completion of the NHEJ process (Lieber, 2010). Dysfunctional NHEJ not only harms the genome integrity of the somatic cells but also impairs iPSCs. Somatic cells with a deletion of DNA Lig4 or DNAPKcs are less efficient at being reprogrammed and are more genetically unstable (Felgentreff et al., 2014; Tilgner et al., 2013), strongly suggesting that efficient NHEJ is very critical to both reprogramming and stabilizing the genome in iPSCs. Sirt6, which is an essential longevity factor that is involved in various biological processes, is a key regulator of genome stability by directly participating in many DNA processes. Sirt6 activates PARP1 by mono-ADP-ribosylating PARP1 at the residue K521 to promote HR, alt-NHEJ, and base excision repair (BER) (Mao et al., 2011; Xu et al., 2015). As a deacetylase, Sirt6 also deacetylates CtIP to facilitate end resection, therefore improving HR (Kaidi et al., 2010). In an enzymatic activity-independent

2880 Cell Reports 18, 2880–2892, March 21, 2017 ª 2017 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

manner, Sirt6 stimulates the recruitment of SNF2H, a chromatin remodeler, to DNA damage sites to relax complex chromatin and promote HR (Toiber et al., 2013). Although Sirt6 forms a macromolecular complex with DNA-dependent protein kinase (DNAPK) and stabilizes DNA-PKcs at chromatin flanking the damage sites (McCord et al., 2009), the detailed regulatory mechanisms of c-NHEJ by Sirt6 have not been well elucidated, particularly in the context of the pluripotent state. Here we compare the efficiency of NHEJ and HR between iPSCs from young and old mice and demonstrate that NHEJ, but not HR, in aged mouse-derived iPSCs is less efficient than that in young counterparts. Our further work indicates that decreased Sirt6 expression is responsible for the reduced efficiency. Mechanistically, we demonstrate that Sirt6 directly binds Ku80 and facilitates the Ku80/DNA-PKcs interaction, which, in turn, increases DNA-PKcs/Ser2056 phosphorylation and efficient DSB repair. Overexpressing Sirt6 with the Yamanaka factors during reprogramming rescues the decline of genomic stability by improving NHEJ, providing a simple method for improving the quality of iPSCs derived from old donors. RESULTS Old Mouse-Derived iPSCs Exhibit Less NHEJ Repair Than Young Counterparts To generate iPSCs, we simultaneously transduced skin fibroblasts that were isolated from young mice and old mice using the classic Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]). Consistent with previous reports (Kim et al., 2010; Li et al., 2009), we found that reprogramming of somatic cells to iPSCs was more efficient using cells from young subjects than from old subjects (Figure S1A). The integration of exogenous OSKM factors was examined by PCR (Figure S1B). The introduced exogenous genes were silenced at the transcriptional level (Figure S1C). The iPSC clones from young and old subjects were also positive for pluripotency markers: endo-Sox2, Nanog, and Esrrb (Figure S1D). iPSCs from mice at both ages were able to spontaneously differentiate into derivatives of the three germ layers in vitro (Figure S1E). Consistent with earlier findings (Kim et al., 2010; Li et al., 2009), age did not influence the pluripotency and differentiation capability of the iPS cells. To evaluate and quantify DNA damage in young and old mouse-derived iPSCs, we used the alkaline comet assay to measure single- and double-strand breaks in both lines. Analysis of the two most reliable DNA damage measurements, tail moment and the percentage of DNA content in the tail, showed a significantly elevated level of DNA damage in old mouse iPSCs in comparison with young mouse iPSCs (Figures 1A–1C). To test DSB repair efficiency, we utilized our well established DSB repair reporter system and observed a significant reduction in NHEJ efficiency in old mouse iPSCs compared with young mouse iPSCs (Figure 1D), whereas no difference in HR repair efficiency was observed between the iPSC lines (Figure 1E). Also, we compared the genome stability between young and old fibroblasts used to generate iPSCs and found that old fibroblasts have more DNA damage and lower NHEJ efficiency than young cells (Figures S2A– S2C). Together, these results suggest that iPSCs derived from old mice have more DNA damage, which possibly results from

impaired NHEJ repair passed from somatic cells to derived iPSCs. Sirt6 / iPSCs Exhibit Impaired NHEJ Repair Sirt6 has been well characterized as a factor participating in the regulation of age-associated decline of DNA repair (Mao et al., 2012; Xu et al., 2015); we therefore hypothesized that potential changes in Sirt6 expression might be responsible for the decreased NHEJ efficiency in iPSCs derived from old mice. Thus, we compared the protein expression levels of Sirt6 and other major NHEJ factors between old and young mouse iPSCs. We observed a significant increase in Sirt6 (p < 0.01) and DNA-PKcs (p < 0.05) expression in the young group compared with the aged group, whereas no significant differences were observed for the other NHEJ factors between the two groups (Figures 2A and 2B; Figures S2D and S2E). Then we programmed OG-MEFs with Oct4, Sox2, and Klf4 (OSK) plus a vector encoding Sirt6 or a control vector, and the integration of OSK and Sirt6 in iPSC lines was examined (Figure S3C). Including Sirt6 had no significant effect on the numbers of alkaline phosphatase (AP)-positive and Oct4-positive colonies (Figures S3A and S3B). In addition, OSK+Sirt6 iPSC lines were pluripotent because they had silenced the expression of exogenous transgenes (Figure S3D) and expressed pluripotent markers at a similar level as that observed in mouse ESCs (Figure S3E). To test the in vivo differentiation potential of these cells, OSK and OSK+Sirt6 iPSCs were injected into the dorsal flanks of athymic nude mice. We observed no significant differences in the weight of teratomas derived from both iPSC lines (Figure S3F), and cells in both groups formed well differentiated teratomas with structures from all three germ layers (representative images can be seen in Figure S3G). However, the analysis of the comet assay indicates that the amount of DNA damage in the OSK+Sirt6 iPSC group is significantly lower than that in the OSK iPSC group (Figures 2C–2E). Next, we infected wild-type (Sirt6+/+) and Sirt6 knockout (Sirt6 / ) MEFs with retroviral vectors expressing OSK to generate iPSCs. Interesting, Sirt6 knockout improved the reprogramming efficiency in MEFs (Figure S4A). Additionally, we found that pluripotent genes (endo-Oct4 and Nanog) were significantly upregulated by Sirt6 knockout during the first 5 days of OSK reprogramming, whereas the mesenchymal-to-epithelial transition (MET)-related genes were not affected by loss of Sirt6 (Figure S4B). An increase in these core pluripotent genes in Sirt6 knockout may partly explain why a lack of Sirt6 could increase the reprogramming efficiency. After characterizing the iPSCs by testing whether OSK integrated and examining the expression levels of exogenous transgenes and pluripotent genes (Figures S4C–S4E), we compared the DNA damage and repair ability between wild-type and Sirt6 / iPS cells. Analysis of the comet assay demonstrated that the amount of damage accrued in Sirt6 / iPS cells was significantly greater than in the wild-type (Figures 2F–2H). By comparing DNA repair efficiency between the wild-type and Sirt6 / iPSC lines, we observed that Sirt6 / iPSC lines were less efficient at NHEJ (Figure 2I), but no differences were observed in HR repair efficiency between the iPSC lines (Figure 2J). During the process of HR repair, important factors, such as Rad51, the major

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Figure 1. iPSC Genomic Stability Is Influenced by the Age of the Mice (A) Representative alkaline comet assays of young and old mouse-derived iPSCs. (B) Comparison of genomic instability measured by alkaline comet assay between the two groups. The 15 young mouse-derived iPSC lines and 15 old mousederived iPSC lines (5 3 5 mice) were analyzed at passages 7–10. We performed the comet assay with iPSC lines with no induction of DNA damage. For each cell line, the tail moments of at least 50 cells were quantified using Cometscore software. (C) The percentage of DNA content in tails of 15 young and 15 old mouse iPSC lines. (D) NHEJ efficiency was measured in young and old mouse-derived iPSCs. (E) HR efficiency was measured in two group cells. Bar graphs represent mean ± SEM. p Values are according to Student’s t tests in (B)–(E). See also Figures S1 and S2.

recombinase in this pathway, are recruited to the lesion sites in response to DNA damage. Failed recruitment of essential factors to the damage sites would lead to the decline in HR efficiency. We therefore tested the recruitment of Rad51 to DSB sites at different time points (0, 8, 16, and 24 hr) after etoposide treatment and found no significant difference between Sirt6+/+ and Sirt6 / iPS cells (Figures S4F and S4G), confirming that Sirt6 is not involved in regulating HR in iPS cells. Sirt6 Directly Interacts with Ku80 in the NHEJ Pathway During the reprogramming process of converting MEFs to iPSCs, major NHEJ proteins (such as Ku70, Ku80, XRCC4, and PARP1) were upregulated after the transfection of OSK, whereas Sirt6 was decreased after the induction of reprogramming (Figure S5A). Given the protective effect of Sirt6 in genomic stability, the decreased Sirt6 level after OSK reprogramming may be associated with the genomic instability of most iPSC lines. Consistent with this hypothesis, our data in Figure 2 show that Sirt6 deficiency suppresses NHEJ repair, leading to unstable genomes in iPSCs. To gain further insights

2882 Cell Reports 18, 2880–2892, March 21, 2017

into the molecular functions of Sirt6 in the regulation of NHEJ, we first examined whether Sirt6 regulates the expression of proteins involved in NHEJ. Western blot analysis shows that Sir6 overexpression does not affect the expression of NHEJ proteins during the first 6 days of OSK reprogramming (Figure S5B). Next, we found that Sirt6 interacts with Ku80 by immunoprecipitation (IP) following FLAG-Sirt6 expression in iPS cells; this interaction became stronger upon ionizing radiation (IR) treatment (Figure 3A). Moreover, in mouse iPSC, endogenous Ku80 associated with Sirt6, and this interaction increased upon etoposide treatment (Figure 3B; Figure S5C), indicating that these two proteins interact under physiological conditions. Notably, the interaction of Sirt6 with Ku80 was resistant to ethidium bromide (EtBr) and DNase I, which disrupt DNAdependent interactions (Figure 3C). In addition, we took advantage of a catalytic domain mutant form of Sirt6, Sirt6-H133Y. Intriguingly, the interaction between the two proteins was abolished by the H133Y mutation, indicating that the catalytic domain of Sirt6 is essential to the interaction between Sirt6 and Ku80 (Figure 3D). Ku80 contains an a/b domain, a

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DNA-binding domain, a Ku70-binding domain, and a DNAPKcs-binding domain (Koike and Koike, 2008). To identify the specific domains in Ku80 that were required for this interaction, we expressed hemagglutinin (HA)-Ku80 lacking these domains in 293T cells (Figure 3E). The lack of the C terminus DNAPKcs-binding domain completely abolished the interaction, indicating that this domain is necessary for Sirt6 binding (Figure 3F). Further experimentation showed that amino acids (aa) 341–631, but not aa 341–621 of HA-Ku80 could pull down Sirt6, suggesting that aa 622–631Aa of Ku80 contain the Sirt6 binding sites (Figure 3G). Indeed, a truncated form of Ku80 (Ku80D10) that only lacks 622-631Aa could not pull down endogenous Sirt6 (Figure 3H). Together, these data demonstrate that Sirt6 directly interacts with Ku80. Sirt6-Ku80 Binding Is Required for Sirt6 to Promote NHEJ Repair Because Ku80 is an important member of the c-NHEJ pathway, we tested whether Sirt6 regulates NHEJ in a Ku80-dependent manner in iPSCs. We infected wild-type (Ku80+/+) or Ku80 / MEFs with retroviral vectors expressing OSK to generate iPSCs. Ku80 knockout greatly reduced the formation of AP-positive colonies compared with wild-type MEFs (Figure S5D). We picked five independent iPSC lines generated from wild-type and Ku80 / MEFs, respectively; these cell lines had integrated the OSK transgene, silenced exogenous transgenes, and expressed pluripotent genes (Figures S5E–S5G). As expected, the Ku80 / iPSC lines showed a significant increase in DNA breaks (Figures 4A–4C) and a decrease in NHEJ repair (Figure 4D) compared with the wild-type iPSC lines. In the Ku80 / iPSC lines, Sirt6 overexpression failed to activate the NHEJ pathway, indicating that Sirt6 promotes NHEJ in a Ku80-dependent manner in iPSCs (Figure 4E). Then we tested the functional effects of Sirt6/Ku80 binding on NHEJ repair. We overexpressed FLAG-Control, FLAGSirt6 wild-type (WT), and FLAG-Sir6 H133Y in a Sirt6 / iPSC line. As predicted, FLAG-Sir6 WT enhanced NHEJ efficiency compared with the control, whereas the H133Y mutant failed to stimulate it (Figure 4F). This result suggests that binding of the catalytic domain of Sirt6 to Ku80 may be required to enhance NHEJ repair. Furthermore, the restoration of Ku80 WT, but not Ku80D10 lacking the Sirt6 binding sites, in a Ku80 / iPSC line significantly stimulates NHEJ (Figure 4G). Again, deletion of the Sirt6 binding sites from Ku80 resulted in a failure of Sirt6 to enhance NHEJ repair (Figure 4H). These observations reveal that Sirt6 promotes NHEJ via its binding with Ku80.

Figure 2. Sirt6

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Sirt6 Facilitates DNA-PKcs Autophosphorylation via Ku80 The DNA-PK complex is composed of the Ku70/Ku80 heterodimer and DNA-PKcs, which exists as a large functional complex (Lieber, 2010). Because Sirt6 directly interacts with Ku80, Sirt6 may affect the functional DNA-PK complex by binding to Ku80. To directly test this hypothesis, the Ku80/Ku70 or Ku80/DNAPKcs complex was co-immunoprecipitated. As shown in Figure 5A, Sirt6 could not affect the association of Ku80 with Ku70. Interestingly, the association of Ku80 with DNA-PKcs was weakened in Sirt6-deleted cells, whereas overexpressing Sirt6 promoted the association of Ku80 with DNA-PKcs, suggesting that Sirt6 enhances the Ku80/DNA-PKcs association (Figure 5B). Endogenous DNA-PKcs autophosphorylation on Ser2056 represents activated DNA-PKcs (Chan et al., 2002, 2005; Uematsu et al., 2007). DNA-PKcs/Ser2056 phosphorylation was significantly impaired in Sirt6 / compared with Sirt6+/+ iPSC lines following treatment of the cells with etoposide (Figure 5C). Moreover, Sirt6 overexpression increased DNAPKcs/Ser2056 phosphorylation, and the Sirt6-related autophosphorylation of DNA-PKcs was largely blocked by its kinase inhibitor, Nu7026 (Figure 5D). To further test whether Sirt6-mediated activation of DNA-PKcs depends on Ku80, we infected FLAGcontrol or FLAG-Sirt6 viruses into wild-type and Ku80 / iPSCs, respectively. Ku80 deficiency attenuated Sirt6-mediated DNAPKcs phosphorylation of Ser2056 (Figure 5E). As expected, DNA-PKcs/Ser2056 phosphorylation was significantly reduced in Ku80 / compared with Ku80+/+ iPSCs (Figure 5F). Importantly, expression of WT Ku80 in the Ku80 / iPSC line restored the level of DNA-PKcs/Ser2056 phosphorylation. However, Ku80D10 displayed significantly less phosphorylation compared with WT Ku80, indicating that the association of Sirt6 and Ku80 is important for DNA-PKcs phosphorylation (Figure 5G). We propose that Sirt6 binds to Ku80 in the response to DSBs in iPSCs, and promotes the formation of Ku/DNA-PKcs complexes, which facilitates the autophosphorylation of DNA-PKcs. Inclusion of Sirt6 during Reprogramming Improves NHEJ Repair in Old Mouse-Derived-iPSCs Finally, we combined OSKM and Sirt6 in iPSC generation from skin fibroblasts of old mice and analyzed the OSKM+Sirt6 iPSC lines. These cell lines integrated the OSKM and Sirt6 transgenes (Figure S6A) and stained positively for AP and the known pluripotency-associated markers Oct4 and Nanog (Figure 6A). They had also silenced the expression of exogenous transgenes and expressed pluripotent markers (Figures S6B and S6C). The

iPS Cells Show Genomic Instability and Reduction of NHEJ Efficiency

(A) Western blot analysis of Sirt6, c-NHEJ (Ku70, Ku80, Artemis, XLF, Lig4, and DNA-PKcs), and alt-NHEJ factors (PARP1and Lig3) in young and old mousederived iPSCs. (B) Statistical comparison of Sirt6 protein expression between the two groups. Western blot results were analyzed with Gel-Pro Analyzer 4.0 software. (C) Representative alkaline comet assays of the OSK and OSK+Sirt6 iPSC lines. (D and E) Tail moment (D) and percentage of DNA content in tails (E) of six OSK and six OSK+Sirt6 iPSC lines. (F) Representative alkaline comet assays of the Sirt6+/+ and Sirt6 / iPSC lines. (G and H) Tail moment (G) and percent of DNA in tails (H) of six Sirt6+/+ and eight Sirt6 / iPSC lines. (I and J) NHEJ efficiency (I) and HR efficiency (J) were measured in Sirt6+/+ and Sirt6 / iPSCs. The cell lines in (C)-(H) were analyzed at passages 7–10 and at least 50 cells were quantified for each cell line. Bar graphs represent mean ± SEM. p Values are according to Student’s t tests in (B), (D), (E), and (G)–(J). See also Figures S2–S4.




OSKM+Sirt6 iPSC lines spontaneously differentiated into derivatives of the three germ layers in vitro as did OSKM lines, confirming the in vivo differentiation ability of these iPSC lines (Figure S6D). Notably, the OSKM+Sirt6 iPSC lines presented significantly decreased DNA damage (Figures 6B–6D) and higher NHEJ efficiency (Figure 6E) than OSKM iPSC lines from old mice. These results suggest that Sirt6 activates the NHEJ pathway and safeguards genome stability but does not affect the pluripotency of the resultant iPSCs. In addition, to test whether Sirt6 overexpression in established old mouse-derived iPSCs achieves a similar rescue effect, we infected the six independent iPSCs (passage 5) derived from old mice with a lentivirus bearing the Sirt6 gene or a control vector to establish ‘‘old mouse OSKMiPSCs (Sirt6)’’ and ‘‘old mouse OSKM-iPSCs (Control)’’ iPSC lines (Figure S6E). These 12 iPSC lines were tested for NHEJ efficiency and the amount of DNA damage at the passage 10. We found that Sirt6 overexpression could still improve NHEJ efficiency in established old mouse-derived iPSCs (Figure S6F), but the amount of DNA damages did not exhibit any difference between the control and Sirt6-overexpressing groups (Figures S6G–S6I). We reasoned that a short-term restoration of Sirt6 in iPSCs derived from old mice is insufficient to promote genome integrity. Together, these data indicate that, to improve the quality of iPSCs from old mice or future old donors, we should activate Sirt6 at the initial stage of reprogramming. DISCUSSION Mammalian aging is characterized by several hallmarks at the cellular and molecular levels, including genomic instability, decreased telomere length, high levels of mitochondrial reactive oxygen species, and expression of senescence markers. Some age-associated features can be reset during reprogramming, such as increasing telomere length (Agarwal et al., 2010; Marion et al., 2009), mitochondrial fitness (Prigione et al., 2010), and losing senescence markers (Lapasset et al., 2011). However, whether the major features are identical between iPSCs derived from young and old donors has not been investigated. Surprisingly, we found in the present study that the genomes of young mouse iPSCs are more stable than those of old ones, strongly suggesting that, besides reducing the potential therapeutic risks by limiting replication stress (Ruiz et al., 2015), enhancing telomere lengthening (Jiang et al., 2013), or generating integration-

free iPSCs (Okita et al., 2011), promoting the genomic stability of old donor-derived iPSCs is also very critical for their future clinical application. In the past decades, identifying targets to delay the onset of aging or cure age-associated diseases has received great attention in the aging field (Baur et al., 2012; Candore et al., 2010; Ingram et al., 2006; Smigrodzki and Khan, 2005). Sirt6 has been a good candidate for the task of creating a ‘‘fountain of youth’’ because it is involved in numerous age-linked biological processes (Kanfi et al., 2012; Mostoslavsky et al., 2006; Xu et al., 2015). However, most research on Sirt6 has been performed in primary cells or cancer cells (Mao et al., 2011; McCord et al., 2009; Min et al., 2012; Sebastiań et al., 2012; Toiber et al., 2013; Xu et al., 2015). Its roles in rejuvenated iPSCs, particularly those derived from old donors, have not been determined. In addition to the inherited epigenetic or genetic changes in aged somatic cells, one would expect that the ability to maintain genomic stability might be identical in iPSCs derived from young and old donors. Intriguingly, our work here demonstrated that the ability to repair DNA DSBs by NHEJ is impaired in iPSCs derived from old mice, suggesting that repair capacity, and not just the existing DNA mutations or genomic rearrangements, is passed from somatic cells to their iPSCs. More importantly, we determined that Sirt6 plays extremely important roles in the repair process. Restoration of Sirt6 could rescue the reduced NHEJ and destabilized genome integrity in the old iPSCs, indicating that activating Sirt6 can be beneficial to both aged somatic cells and derived iPSCs. Although Sirt6 has been well studied as a regulator of DNA DSB repair, its roles in c-NHEJ have not been clearly elucidated. Our study here greatly advances our knowledge of Sirt6 in DNA repair by delineating a mechanism through which Sirt6 participates in c-NHEJ in the context of iPSCs. A previous study in human 293T cells indicated that Sirt6 directly binds DNA-PKcs and stabilizes it at DSB sites, whereas the association of exogenous Sirt6 and Ku80 is dependent on DNA (McCord et al., 2009). However, our findings in mouse iPSCs showed that Sirt6 interacts with Ku80 independently of DNA, and this interaction is enhanced in response to DNA damage. The discrepancy might result from differences between species, cell lines, or pluripotent stages. Our results also indicate that the catalytically dead mutant Sirt6 H133Y is unable to interact with Ku80 and stimulate NHEJ. There might be two different explanations. One is that the mutation on H133 causes

Figure 3. Sirt6 Directly Interacts with Ku80 (A) FLAG-control or FLAG-Sirt6 viruses were introduced into Sirt6+/+ iPSC clones. Cells were irradiated and immunoprecipitated 30 min after IR exposure (8 Gy). IP was performed using FLAG beads, and FLAG-tagged luciferase was used as a control. Membranes were immunoblotted for Ku80 and FLAG. (B) Endogenous interaction between Ku80 and Sirt6 in iPSCs. iPSCs were treated with etoposide for 0.5 hr. IP was performed using an anti-Ku80 antibody and normal immunoglobulin G (IgG). Membranes were immunoblotted for Sirt6 and Ku80. (C) 293T cells were co-transfected with HA-mouse Ku80 and either FLAG-luciferase (Control) or FLAG-mouse Sirt6. IP was performed with anti-HA beads in the absence or presence of EtBr or DNase I. Membranes were immunoblotted for HA and FLAG. (D) FLAG-control, FLAG-Sirt6, or FLAG-sirt6 mutant (H133Y) viruses were introduced into Sirt6 / iPSC clones and immunoprecipitated with anti-FLAG beads. Membranes were immunoblotted for Ku80 and FLAG. (E) Schematic of wild-type mouse Ku80 and the different truncated Ku80 mutants used in this study. (F) IP of the HA-tagged Ku80 and truncated Ku80 mutants (Ku80-a+b, Ku80-c+d, Ku80-a, Ku80-c, and Ku80-d) in 293T cells. (G) IP of the different HA-tagged Ku80 variants. (H) IP of the HA-tagged control, WT Ku80, and Ku80D10 that only lacks 622-631Aa. Membranes in (F)–(H) were immunoblotted for Sirt6 and HA. Black arrows indicate specific bands. See also Figure S5.


Figure 4. Sirt6-Ku80 Protein Interaction Is Essential for Sirt6 to Improve NHEJ Repair (A) Representative alkaline comet assays of Ku80+/+ and Ku80 / iPSCs. (B and C) Tail moment (B) and percentage of DNA content in tails (C) of five Ku80+/+ and five Ku80 / iPSC lines. (D) NHEJ efficiency was measured between Ku80+/+ and Ku80 / iPSC lines. (E) Loss of Ku80 abolishes the stimulatory effect of Sirt6 on NHEJ. FLAG-control or FLAG-Sirt6 viruses were introduced into Ku80+/+ or Ku80 / iPSC lines. (F) NHEJ efficiency was measured in a Sirt6 / iPSC line infected with FLAG-control, FLAG-Sirt6, or FLAG-sirt6 mutants (H133Y). (G) NHEJ efficiency was measured in a Ku80 / iPSC line infected with HA-control, HA-ku80 or HA-Ku80D10. (H) Ku80D10 abolishes the stimulatory effect of Sirt6 on NHEJ. FLAG-control or FLAG-Sirt6 viruses were introduced into HA-Ku80 rescued Ku80 / or HA-Ku80D10 rescued Ku80 / iPSC lines. The cell lines in (A)–(C) were analyzed at passages 7–10 and at least 50 cells were quantified for each cell line. Bar graphs represent mean ± SEM. *p < 0.05; **p < 0.01; ns, no significant differences according to Student’s t tests in (B)–(D) and one-way ANOVA with Tukey-Kramer post hoc test in (E)–(H) (n = 3 independent experiments). See also Figure S5.

a conformational change on Sirt6 that disrupts the interaction between Sirt6 and Ku80. Indeed, there is a previous report suggesting that H133Y mutation results in an abolished interac-

tion between Sirt6 and G3BP1 (Simeoni et al., 2013). Another possibility is that the interaction between the two proteins is dependent on the enzymatic modification on Sirt6 itself upon




Figure 6. Sirt6 Improves the Stability of iPSCs Generated from the Skin Fibroblasts of Old Mice (A) Representative images of AP staining and immunostaining for Oct4 and Nanog in OSKM+Sirt6 iPSCs derived from old mice. Nuclei were stained with Hoechst. Black scale bar, 100 mm; white scale bar, 10 mm. (B) Representative alkaline comet assays of OSKM and OSKM+Sirt6 old mouse iPSC lines. (C and D) Tail moment (C) and percentage of DNA content in tails (D) of six OSKM and six OSKM+Sirt6 old mouse iPSC lines. The 12 cell lines were analyzed at passages 7–10. For each cell line, at least 50 cells were quantified. (E) NHEJ efficiency was measured in OSKM and OSKM+Sirt6 iPSC lines derived from old mice. Bar graphs represent mean ± SEM. p Values are according to Student’s t tests. See also Figure S6.

DNA damage. Indeed, Sirt6 can add an ADP-ribose to itself in vitro (Liszt et al., 2005). It is possible that the modification on Sirt6 itself mediates the interaction between Sirt6 and Ku80. The H133Y mutant lacks both deacetylase and mono-ADP-ribosyltransferase activities (Liszt et al., 2005; Michishita et al., 2008). Which enzymatic activity is important for Sirt6 interaction with

Ku80 and stimulation of NHEJ repair in pluripotent cells remains to be determined in future work. Logically, knocking out Sirt6 would cause reduced NHEJ, leading to a destabilized genome and eventually suppressing reprogramming efficiency. However, previous reports regarding reprogramming efficiency in the absence of Sirt6 are controversial

Figure 5. Ku80 Is Responsible for Sirt6-Induced Phosphorylation of DNA-PKcs on Ser2056 (A) Cells were infected with lentivirus FLAG-Sirt6 and FLAG-Control to establish Sirt6 overexpression and control iPSC lines. Ku80 coIP was performed in Sirt6+/+ and Sirt6 / iPSC lines, and iPSC lines overexpressed control or Sirt6. Membranes were immunoblotted for Ku80 and Ku70. (B) DNA-PKcs coIP was performed in Sirt6+/+ and Sirt6 / iPSC lines, and iPSC lines overexpressed control or Sirt6. Membranes were immunoblotted for Ku80 and DNA-PKcs. (C) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Sirt6+/+ and Sirt6 / iPS lines after treatment with or without etoposide for 0.5 hr. (D) The Sirt6-induced autophosphorylation of DNA-PKcs/S2056 was blocked by the DNA-PKcs inhibitor Nu7026. (E) The Sirt6-induced autophosphorylation of DNA-PKcs/S2056 was attenuated by Ku80 deficiency. (F) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Ku80+/+ and Ku80 / iPSC lines after treatment with or without etoposide for 0.5 hr. (G) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Ku80 / iPSC clones infected with HA-Control, HA-Ku80, or HA- Ku80D10 after treatment with etoposide for 0.5 hr. Black arrows indicate specific bands.


(Etchegaray et al., 2015; Sharma et al., 2013). Interestingly, our data suggest that Sirt6 is a barrier to reprogramming. Why does the reduced NHEJ and genome integrity in the absence of Sirt6 in iPSCs not suppress reprogramming efficiency? One possibility is that HR is probably the dominant pathway over NHEJ in iPSCs because iPSCs are rapidly dividing, and HR occurs mainly in proliferating cells (Tichy et al., 2010). The impairment of NHEJ is probably not severe enough to halt reprogramming by triggering p53 or other signaling pathways. Alternatively, the loss of Sirt6 could positively affect the expression of the core pluripotency genes (Oct4 and Nanog) because of a more open chromatin structure with more acetylated H3K9 and H3K56 (Etchegaray et al., 2015). In summary, our work not only revealed the existence of reduced NHEJ repair efficiency in aged mouse iPSCs compared with iPSCs from young mice but also explored a strategy that can be used to improve this phenomenon: combining Sirt6 with Yamanaka factors. Additionally, we identified a function for Sirt6 in regulating classical NHEJ. A direct interaction of Sirt6 with Ku80 may be required to form Ku/DNA-PKcs complexes and autophosphorylate DNA-PKcs at S2056, which improves DSB repair in iPSCs. Thus, the addition of Sirt6 provides a simple approach to reducing the genomic instability of iPSCs from old subjects for therapeutic purposes. EXPERIMENTAL PROCEDURES Cell Culture and Treatment Skin fibroblasts were obtained from the chest and belly of young (3-month-old) and old (2-year-old) wild-type C57BL/6 mice as described previously (Seluanov et al., 2010) and were cultured in MEM (HyClone) containing 15% fetal bovine serum (FBS), 0.1 mM non-essential amino acids (NEAA, Thermo Fisher Scientific), and 1% penicillin/streptomycin (Gibco) under low-oxygen conditions. OG-MEFs were derived from transgenic OG-2 mice at embryonic day 13.5 (E13.5). OG-MEFs, Sirt6+/+ and Sirt6 / MEFs, and Ku80+/+ and Ku80 / MEFs (a gift from Jun Xu) were cultured in DMEM supplemented with high glucose (Invitrogen), 15% FBS, and 0.1 mM NEAA (Thermo Fisher Scientific) under low-oxygen conditions. All animal care and experimental protocols were reviewed and approved by the Animal Research Committee of Tongji University School of Medicine. MEFs were extracted from embryos using a standard assay and genotyped using PCR and western blot. Mouse iPSC clones and the mouse ESC line (E14.1) were cultured on a feeder layer of inactivated MEFs with knockout DMEM medium (Gibco) containing 20% knockout serum replacement (KOSR, Gibco), 13 penicillin/streptomycin solution (P/S, HyClone), 0.1 mM NEAA (Thermo Fisher Scientific), 1% L-glutamax (Thermo Fisher Scientific), and 55 mM b-mercaptoethanol (Gibco) with leukemia inhibitory factor (LIF, 1,000 U/mL, Millipore). 293T cells and Plat-E cells were grown in DMEM supplemented with high glucose (Invitrogen) and 10% FBS. To induce DNA damage, cells were irradiated with X-rays at a dosage of 8 Gy or treated with 25 mM etoposide (Selleckchem). DNA-PKcs inhibitor NU7026 was purchased from Selleckchem. The plasmid construction steps are described in the Supplemental Experimental Procedures. Generation of Mouse iPSCs Mouse iPS cells were generated with retroviruses as described previously (Okita et al., 2007), with some modifications. Briefly, virus-containing supernatants from the Plat-E cultures were recovered and combined (e.g., to mix viruses for Oct4, Sox2, Klf4, c-Myc, and Sirt6). Fibroblasts were seeded at a density of 1 3 105 cells/well in six-well plates pre-coated with 0.1% gelatin (Sigma) in DMEM supplemented with 10% FBS. The cells were transduced twice in the next 2 days at 24-hr intervals with the retroviral supernatant as indicated. Three days after infection, fibroblasts were digested into single cells and reseeded at the indicated density per well in six-well plates pre-coated


with 0.1% gelatin. Then the culture medium was changed to an optimized culture medium that was changed daily (Chen et al., 2010). The colonies were either scored after alkaline phosphatase staining using the Fast Red alkaline phosphatase kit (Sigma) or picked for further expansion on irradiated feeder fibroblasts. For genomic PCR, real-time qPCR, in vitro differentiation of iPSCs, and teratoma formation experiments, see the Supplemental Experimental Procedures. Western Blotting, Immunoprecipitation, and Immunofluorescence Staining Standard procedures were used throughout (Supplemental Experimental Procedures). Comet Assay Cells were resuspended in PBS to a total cell count of 1.0 3 105 cells/mL, embedded in agarose, and subjected to the alkaline comet assay using a comet assay kit (Trevigen, catalog no. 4250-050-K). The percentage of DNA content in tail and tail moments were later analyzed using Cometscore software. DSB Repair Assays and FACS Analysis A total of 2 3 105 iPSCs were co-transfected with 0.2 mg of NHEJ reporter plasmid or 0.6 mg of HR reporter plasmid to induce DSBs and 0.1 mg of pDsRed2-N1 plasmid to control for transfection efficiency. Transfections were performed using the P3 Primary Cell 4DNucleofector X kit (Lonza) with the mouse ES program on a Lonza 4D machine. Three days after transfection, the numbers of GFP+ and DsRed+ cells were determined by flow cytometry. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACS Verse machine (BD Biosciences). For each treatment, a minimum of 20,000 cells were analyzed. The procedure of testing NHEJ repair of skin fibroblasts was as described previously (Li et al., 2016). Data were analyzed using FlowJo software. The ratio between GFP+ and DsRed+ cells was used as a measure of DSB repair efficiency. Data Analysis GraphPad Prism 4.0 (GraphPad) was used for the data analysis, and the data are presented as the means ± SEM for three independent experiments. Comparisons among means were performed by one-way ANOVA with Tukey-Kramer post hoc test for multiple (more than two) groups or Student’s t test for comparing two means of independent samples. Differences were considered to be significant at *p < 0.05, **p < 0.01, and ***p < 0.001. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2017.02.082. AUTHOR CONTRIBUTIONS W.C., Z.M., and J.K. conceived and designed the experiments. W.C., N.L., Hongxia Zhang, and Haiping Zhang performed the experiments. W.C., Z.M., and J.K. analyzed the data. J.Q., W.J., and S.Z. contributed reagents, materials, and analysis tools. W.C., Z.M., and J.K. wrote the paper. ACKNOWLEDGMENTS This work was supported by grants obtained from the Ministry of Science and Technology (2013CB967600, 2013CB967401, and 2012CB966603), National Natural Science Foundation of China (81530042, 91519320, 31210103905, 31471250, 31371510, 31571519, 31571529, 31571390, 31401257, 31401126, 81622019, 31371396, 31570813, 91519319, and 81502385), Ministry of Education (IRT_15R51), Science and Technology Commission of Shanghai Municipality (15JC1403200 and 15JC1403201), and Fundamental

Research Funds for the Central Universities (2000219138, 20002310002, 2000219136, 1507219042, and 1500219106).

Kaidi, A., Weinert, B.T., Choudhary, C., and Jackson, S.P. (2010). Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science 329, 1348–1353.

Received: November 2, 2016 Revised: January 22, 2017 Accepted: February 28, 2017 Published: March 21, 2017

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