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Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
 
Curr Biol. Author manuscript; available in PMC 2013 March 11.
Published in final edited form as:
PMCID: PMC3466333
HHMIMSID: HHMIMS407953

Inner nuclear envelope proteins SUN1 and SUN2 play a prominent role in the DNA damage response

Summary

The DNA damage response (DDR) and DNA repair are critical for maintaining genomic stability and evading many human diseases [1, 2]. Recent findings indicate accumulation of SUN1, a nuclear envelope (NE) protein, is a significant pathogenic event in Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, both caused by mutations in LMNA [3, 4]. However, roles of mammalian SUN proteins in mitotic cell division and genomic stability are unknown. Here we report that the inner NE proteins SUN1 and SUN2 may play a redundant role in DDR. Mouse embryonic fibroblasts from Sun1−/−Sun2−/− mice displayed premature proliferation arrest in S phase of cell cycle, increased apoptosis and DNA damage, and decreased perinuclear heterochromatin, indicating genome instability. Furthermore, activation of ATM and H2A.X, early events in DDR, were impaired in Sun1−/−Sun2−/− fibroblasts. A biochemical screen identified interactions between SUN1/2 and DNA-dependent protein kinase (DNAPK) complex that functions in DNA nonhomologous end joining repair and possibly in DDR [2, 5, 6]. Knockdown of DNAPK reduced ATM activation in NIH3T3 cells, consistent with a potential role of SUN1/2-DNAPK interaction during DDR. SUN1/2 could affect DDR by localizing certain nuclear factors to the NE or by mediating the communication between nuclear and cytoplasmic events.

Keywords: KASH-SUN complex, Genomic instability, DNA repair, DNAPK, Ku70, Ku80, DDR, H2A.X, ATM, HGPS

Results

Sun1−/−Sun2−/− mouse embryonic fibroblasts (MEFs) exhibited premature proliferative arrest at the S phase of the cell cycle

SUN proteins are inner nuclear membrane proteins with their N-terminal region localized in the nucleoplasm and their C-terminal SUN domain in the lumen of the nuclear envelope (NE) [79]. We have previously used mouse genetics to analyze the physiological functions of SUN1 and SUN2 and found that Sun1−/−Sun2−/− mice died shortly after birth [10, 11]. Although the neonatal death phenotype was partly rescued by expressing SUN1 in the nervous system, the surviving mice still displayed multiple defects including growth retardation [10, 12], prompting us to examine the function of SUN1 and SUN2 in mitotic cell division and genomic stability in MEFs.

The MEFs were isolated from embryos at embryonic day 14.5 (E14.5). MEFs from the Sun1−/−Sun2−/−, but not Sun1−/− or Sun2−/− mice, proliferated significantly slower than wild-type MEFs after passage 5 (Figure S1 and and1A).1A). Cell-cycle analysis on unsynchronized cells from passage 6 via flow cytometry showed that the G0/G1-phase fraction was only slightly increased and the S-phase fraction was slightly reduced in Sun1−/−Sun2−/− MEFs (Figure 1B). In contrast, using bromodeoxyuridine (BrdU) to label the replicative DNA in S-phase cells, we observed that the percentage of proliferative S-phase cells in Sun1−/−Sun2−/− MEFs was less than half that of wild-type MEFs (Figure 1C), suggesting an S-phase arrest in Sun1−/−Sun2−/− MEFs. Furthermore, there were an increased number of annexin V-positive cells in Sun1−/−Sun2−/− MEFs at passage 6 (Figure 1D), indicating an increase in apoptosis. These results raised the possibility that DNA damage accumulated more rapidly in Sun1−/−Sun2−/− MEFs.

Figure 1
Sun1−/−Sun2−/− MEFs exhibit premature proliferative arrest and genomic instability.

Sun1−/−Sun2−/− MEFs exhibit excessive DNA damage

To detect the potential genomic instability in Sun1−/−Sun2−/− MEFs, we carried out single cell electrophoresis to observe the level of DNA damage. In the absence of methyl methanesulfonate (MMS), which induces DNA damage [13], there was no significant difference in the tail moment between wild type and Sun1−/−Sun2−/− MEFs. After treatment with MMS, we observed a significant increase in the number of Sun1−/−Sun2−/− MEFs with prominent comet tails, indicative of DNA fragmentation (Figure 1E). In addition, using transmission electronic microscopy (TEM), we found that the perinuclear heterochromatin was decreased in Sun1−/−Sun2−/− MEFs (Figure 1F). These results suggested that SUN1 and SUN2 have roles in maintaining genomic stability, possibly by affecting DDR and/or DNA repair.

DDR was impaired in Sun1−/−Sun2−/− MEFs

Phosphorylation of ataxia telangiectasia mutated protein (ATM) and H2A.X, a histone H2A variant, at Ser139 (i.e., γ-H2A.X) are among the earliest events to occur in response to DNA damage [2, 14, 15]. These early DDR events lead to activation of DNA repair factors and cell-cycle checkpoints, ensuring the proper repair of sites of DNA damage [2, 15, 16]. We obtained three pieces of data to indicate that the early events in DDR are affected in Sun1−/−Sun2−/− MEFs. First, the expression level of γ-H2A.X was significantly reduced in Sun1−/−Sun2−/− MEFs (Figure 2A). In addition, the level of phosphorylated Chk1, a cell-cycle checkpoint factor downstream of the DDR pathway, was also reduced (Figure 2A). Second, while ATM was seen to be activated by 0.1 μM of hydroxyurea (HU) in wild-type MEFs, it was not activated by HU in Sun1−/−Sun2−/− MEFs (Figure 2B). Third, we found that the cell-division cycle of Sun1−/−Sun2−/− MEFs was not blocked at the G2/M phase following treatment with 200 ng/μl of mitomycin C (MMC) (Figure 2C), indicating that the mutant cells failed to properly respond to DNA damage. However, due to the lack of a suitable antibody for mouse non-phosphorylated ATM, we could not exclude the possibility that the observed decrease of phosphorylated ATM was partly due to apoptosis induced ATM degradation [17]. However, such an effect of apoptosis is unlikely to be significant because our analysis using Annexin V indicated that apoptosis was not dramatically increased in SUN1/2 DKO MEFs (only 2.2% compared to 1.3% in wild type; Figure 1D). To confirm the defect of Sun1−/−Sun2−/− MEFs in DDR, we examined the sensitivity of Sun1−/−Sun2−/− MEFs to various DNA damaging agents. Although Sun1−/−Sun2−/− MEFs exhibited no significant abnormality in their response to γ-irradiation, they exhibited increased sensitivity to MMS and MMC (Figure S2). These results suggest that SUN1 and SUN2 (SUN1/2) have a prominent role in DDR to specific types of DNA damage.

Figure 2
Sun1−/−Sun2−/− MEFs exhibit defects in DDR.

SUN1/2 interact with the DNAPK holoenzyme

To search for the mechanism of SUN1/2 function in DDR, we screened for SUN1 interacting proteins by applying tandem affinity purification and MALDI-MS/MS proteomic analysis [18]. The effectiveness of this approach was indicated by the identification of, among only 27 candidate proteins (Table S1), three KASH domain proteins (Syne-1/Nesprin-1, Syne-2/Nesprin-2, and Nesprin-3) that have all been well characterized as biochemical and functional partners of SUN1/2 [10, 11, 1921]. In addition, several cytoskeleton proteins and emerin were also identified (Table S1), and this was likely due to their interactions with the Syne/Nesprin proteins [22].

DNAPKcs, the catalytic subunit of the DNAPK holoenzyme, which also includes Ku70 and Ku80 as the regulatory subunits [23], was one of candidate SUN1-associated proteins. The DNAPK holoenzyme has been studied extensively for its role in the nonhomologous end-joining repair pathway [2, 5, 24]. Recently, DNAPKcs was found to interact with the Hutchinson-Gilford progeria syndrome (HGPS) mutant version of Lamin A/C, linking its function to HGPS-related DNA instability and cell aging [25]. Although DNAPKcs has been reported to have a role in the phosphorylation of H2A.X in experiments using DNAPKcs−/− MEFs [6], its function in the early steps of DDR and the potential mechanism of such a role are not clear.

Based on co-immunoprecipitation (co-IP) and Western blot analysis, we confirmed that DNAPKcs was associated with both SUN1 and SUN2 (Figure 3A and 3B). Similar experiments showed that both Ku70 and Ku80 also interacted with SUN1 and SUN2 (Figure 3C–3F). We further examined the localization of these proteins by immunofluorescence staining of tagged proteins expressed from transformed plasmids. Consistent with previous studies, DNAPKcs, Ku70, and Ku80 were localized uniformly in the nucleus (Figure 4, S4A–G [25, 26]). Dual-staining analysis with SUN1/2 and these components of the DNAPK complex indicated that these proteins colocalized at a low level along the inner side of the NE (Figure 4A and S4A–G). However, we did not observe an increase in this colocalization after HU treatment (Figure 4B). We further compared the localization of endogenous Ku70 in wild-type and Sun1−/−Sun2−/− MEFs, but also did not observe a significant difference under standard culturing conditions with or without the HU treatment (Figure S4H–K). Since we cannot make a conclusion about the function of the interaction between SUN1/2 and DNAPKc, and their co-localization in DDR, the mechanism by which the DNAPK complex interacts with SUN1/2 remains to be understood.

Figure 3
SUN1 and SUN2 interact with DNAPKcs, Ku70, and Ku80.
Figure 4
The colocalization of SUN2 and the DNAPK complex is not increased after HU treatment.

Given the well-known function of the DNAPK complex in DNA repair, the interaction between SUN1/2 and the DNAPK complex may suggest that SUN1/2 has a function downstream of DDR, especially in DNA repair, which is consistent with the suggestion that Lamin A/C has a role in DNA repair. However, the data presented above indicated a role for SUN1/2 in an early step of DDR. Using shRNA to knockdown the DNAPKcs mRNA level in NIH3T3 cells (Figure 3G), we observed a reduction of ATM and H2A.X phosphorylation when the cells were treated with HU (Figure 3H), suggesting that the interaction between SUN1/2 and the DNAPK complex is potentially involved in mediating the role of SUN1/2 in DDR. However, we cannot exclude the possibility that the reduction of ATM and H2AX phosphorylation in this experiment is solely caused by knocking down DNAPKcs. Due to the lack of an appropriate antibody against the mouse phosphorylated DNAPKcs, we could not examine whether SUN1/2 play roles in activating the DNAPKcs in DDR.

Discussion

The mammalian SUN1 and SUN2 proteins have been studied for their roles in nuclear migration and anchorage as well as in anchoring meiotic telomeres to the NE during animal development [1012, 27, 28]. In this study, we showed that these two inner NE proteins also have a significant function in DDR. Like their roles in anchoring myonuclei and neuronal migration, SUN1 and SUN2 functions in DDR are likely redundant; only MEFs from double knockout mice display obvious defects. We can speculate on a potential model for their function based on our limited observations and the available information.

The identification of the interaction between SUN1/2 and the DNAPK complex provides an important mechanistic clue. Because DNAPK is better known for its function in DNA repair, we can consider two different hypotheses regarding the function of this interaction. One hypothesis is that SUN1/2 may interact with DNAPK for their function in DNA repair, and a defect in this function was masked by the defect in the earlier DDR events in Sun1−/−Sun2−/− MEFs. SUN1/2 function in DDR would thus be mediated by factors that are yet to be determined. An alternative hypothesis is that the DNAPK complex also has a significant role in DDR and its interaction with SUN1/2 is critical for such a function. This hypothesis is consistent with a previous report that DNAPKcs has a role in H2A.X phosphorylation [6] and our result that shRNA knockdown of DNAPKcs compromised ATM and H2A.X activation in NIH3T3 cells (Figure 3H). It is conceivable that SUN1/2 functions in DDR by localizing DNA damage sites or certain DDR factors to the NE. In a yeast study, Ku70, a regulatory subunit of DNAPK, was shown to recognize and recruit the site of DNA damage to the NE in an Mps3-dependent manner [29], but it is not clear whether this NE localization is for DDR or DNA repair. In this study, we observed the colocalization between DNAPK components and SUN1/2 in mammalian cells (Figure 4 and S4A–G), indicating a similar function to their yeast counterparts. However, we did not observe an increase of this colocalization after HU treatment (Figure 4 and S4H–K)). In addition, the localization of endogenous Ku70 was not changed in Sun1−/−Sun2−/− MEFs (Figure S4H–K). Therefore, it is possible that the SUN1/2 interaction with the DNAPK complex is a constitutive cellular event required for proper DDR and the interaction is not required for just the NE localization of DNAPK.

SUN proteins are known to form the NE complex with outer NE KASH- domain proteins that interact with cytoplasmic factors [1012, 19, 20, 22]. Therefore, an alternative model for the role of SUN1/2 roles in DDR could be that they mediate the communication between nuclear and cytoplasmic events. In our search for SUN1/2 interacting factors, we also identified the Ca2+-binding protein reticulocalcin-2 (Rcn2), which has been suggested to be localized in the lumen of the endoplasmic reticulum (ER) [30] and has been shown to have a role in activating ERK1/2 in a recent report [31]. Interestingly, Sun1−/−Sun2−/− MEFs displayed impaired ERK activation after HU and MMC treatment (Figure S3E–F). Our analysis using co-IP and immunostaining confirmed the interaction between SUN1/2 and Rcn2 and indicated that they co-localized on to the NE (Figure S3G–K). However, when NIH3T3 cells were treated with shRNA against Rcn2, we failed to identify any effect on ATM activation or subsequent after the treatment to induce DDR induction (Figure S3L–N). Though this negative result is not sufficient to exclude a role of for Rcn2 in DDR due to potential redundant functions, the physiological role of the interaction between SUN1/2 and Rcn2 is currently unclear.

Lamin A/C are part of the nuclear lamina located inside the nuclear inner membrane, and their functions have been linked to many important cellular events [3234]. Both SUN1 and SUN2 have been shown to interact with Lamin A/C [20, 35, 36], and the HGPS-associated Lamin A/C mutations have been shown to impair the interaction between Lamin A/C and SUN1/2 [37]. These data raise a possibility that SUN1/2 may function in DDR through this interaction with Lamin A/C. However, several studies on Lamin A/C contradict such a model. For example, the HGPS mutant version of Lamin A/C (termed progerin), but not wild type, was found to interact with DNAPKcs in a recent study [25], even though the mutant Lamin A/C cannot bind to SUN1/2 [37]. Furthermore, unlike Sun1−/−Sun2−/−, Lamin A/C mutations were found to cause an increase in γ–H2A.X levels, which were attributed to defective DNA repair [38, 39]. Therefore, the role of the SUN1/2 interaction with Lamin A/C in DDR is still unclear. Chen et al. recently reported that accumulation of SUN1 is a pathogenic event in Emerry-Dreifuss musclular dystrophy and Hutchinson-Gilford progeria, which are caused by mutations in LMNA [3]. Eliminating or reducing SUN1 was found to significantly relieve some pathological phenotypes characterized in mouse models of these diseases. Our results may provide valuable insight into the potential mechanism underlying these observations. We show SUN1 and SUN2 act redundantly to promote DDR, while LMNA mutations were shown to cause potential increases in DDR [39]. Therefore, it is logical to propose that some of the disease phenotypes are caused by hyperactivity in DDR as the result of abnormally high level of SUN1. Mutating SUN1 is expected to only reduce the level of DDR, but the reduction may be sufficient to neutralize the effect of the LMNA mutations. Further studies are needed to uncover the molecular mechanism by which SUN1/2 affect DDR.

Experimental procedures

Cell culture and proliferation assay

We prepared MEFs from E14.5 embryos and cultured them in DMEM (Invitrogen) supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin (Invitrogen). For the continuous passage assay, we plated MEFs at a density of 3×105 cells in a 6-cm plate. We then counted and replated the cell number every 3 days. The BrdU incorporation assay was carried out according to a standard protocol [40]. Briefly, 5×104 MEFs were plated in each well of a 6-well plate. After incubation for 24 hours, they were treated with 10 mg/ml BrdU (Sigma) for 4 hours. The cells were then harvested and stained with a FITC-conjugated anti-BrdU antibody (Caltag) and propidium iodide (PI) (Sigma) or 7-amino-actinomycin D (7AAD). The cell-cycle distribution was analyzed using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest (BD Biosciences) and FlowJo (Tree Star) software.

Statistic methods

Data were calculated using an unpaired two-tailed Student t-test and presented as means ± S.E.M.

Highlights

[arrowhead]
Sun1−/−Sun2−/− MEFs exhibited premature proliferative arrest at S phase
[arrowhead]
Sun1−/−Sun2−/− MEFs exhibit excessive DNA damage
[arrowhead]
DDR was impaired in Sun1−/−Sun2−/− MEFs
[arrowhead]
SUN1/2 interacts with the DNAPK holoenzyme that plays a role in DDR

Supplementary Material

01

Acknowledgements

We thank C. Xu, J. Yao, B. Tan, X. Huang, and the EM facility at Fudan Medical School for assistance and contributions to this study; Y. Jin, Q. Lei, and D. Chen for providing materials; Y. Xiong, K. Guan, T. Su, A. K. Sewell, B. Yin, X. Wu, W. Tao, K. Deng, L. Sun, and members of IDM for valuable comments and discussions. This work was supported by an outstanding graduate student researcher award from the Ministry of Education of China to KL, grants from National Natural Science Foundation of China (No.30871233), National Basic Research Program of China (973-2006CB806700), and the National Hi-Tech Research and Development Program of China (863-2007AA022101). TX and MH are HHMI investigators.

Footnotes

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References

1. Garinis GA, van der Horst GT, Vijg J, Hoeijmakers JH. DNA damage and ageing: new-age ideas for an age-old problem. Nat. Cell Biol. 2008;10:1241–1247. [PMC free article] [PubMed]
2. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Molecular Cell. 2010;40:179–204. [PMC free article] [PubMed]
3. Chen CY, Chi YH, Mutalif RA, Starost MF, Myers TG, Anderson SA, Stewart CL, Jeang KT. Accumulation of the inner nuclear envelope protein sun1 is pathogenic in progeric and dystrophic laminopathies. Cell. 2012;149:565–577. [PMC free article] [PubMed]
4. Capell BC, Collins FS. Human laminopathies: nuclei gone genetically awry. Nature Reviews. Genetics. 2006;7:940–952. [PubMed]
5. Weterings E, Chen DJ. DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys? J. Cell Biol. 2007;179:183–186. [PMC free article] [PubMed]
6. Stiff T, O'Driscoll M, Rief N, Iwabuchi K, Lobrich M, Jeggo PA. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res. 2004;64:2390–2396. [PubMed]
7. Malone CJ, Fixsen WD, Horvitz HR, Han M. UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development. Development. 1999;126:3171–3181. [PubMed]
8. Tzur YB, Wilson KL, Gruenbaum Y. SUN-domain proteins: `Velcro' that links the nucleoskeleton to the cytoskeleton. Nat. Rev. Mol. Cell Biol. 2006;7:782–788. [PubMed]
9. Worman HJ, Gundersen GG. Here come the SUNs: anucleocytoskeletal missing link. Trends Cell Biol. 2006;16:67–69. [PubMed]
10. Lei K, Zhang X, Ding X, Guo X, Chen M, Zhu B, Xu T, Zhuang Y, Xu R, Han M. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc. Natl. Acad. Sci. U. S. A. 2009;106:10207–10212. [PubMed]
11. Zhang X, Lei K, Yuan X, Wu X, Zhuang Y, Xu T, Xu R, Han M. SUN1/2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron. 2009;64:173–187. [PMC free article] [PubMed]
12. Yu J, Lei K, Zhou M, Craft CM, Xu G, Xu T, Zhuang Y, Xu R, Han M. KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development. Hum. Mol. Genet. 2011;20:1061–1073. [PMC free article] [PubMed]
13. Sobol RW, Horton JK, Kuhn R, Gu H, Singhal RK, Prasad R, Rajewsky K, Wilson SH. Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature. 1996;379:183–186. [PubMed]
14. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 2000;10:886–895. [PubMed]
15. Harper JW, Elledge SJ. The DNA damage response: ten years after. Molecular Cell. 2007;28:739–745. [PubMed]
16. d'Adda di Fagagna F. Living on a break: cellular senescence as a DNA-damage response. Nat. Rev. Cancer. 2008;8:512–522. [PubMed]
17. Smith GC, d'Adda di Fagagna F, Lakin ND, Jackson SP. Cleavage and inactivation of ATM during apoptosis. Molecular and Cellular Biology. 1999;19:6076–6084. [PMC free article] [PubMed]
18. Washburn MP, Wolters D, Yates JR., 3rd Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001;19:242–247. [PubMed]
19. Padmakumar VC, Libotte T, Lu W, Zaim H, Abraham S, Noegel AA, Gotzmann J, Foisner R, Karakesisoglou I. The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J. Cell Sci. 2005;118:3419–3430. [PubMed]
20. Haque F, Lloyd DJ, Smallwood DT, Dent CL, Shanahan CM, Fry AM, Trembath RC, Shackleton S. SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol. Cell Biol. 2006;26:3738–3751. [PMC free article] [PubMed]
21. Ketema M, Wilhelmsen K, Kuikman I, Janssen H, Hodzic D, Sonnenberg A. Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin. J. Cell Sci. 2007;120:3384–3394. [PubMed]
22. Starr DA, Fridolfsson HN. Interactions Between Nuclei and the Cytoskeleton Are Mediated by SUN-KASH Nuclear-Envelope Bridges. Annu. Rev. Cell Dev. Biol. 2010 [PMC free article] [PubMed]
23. Lee SH, Kim CH. DNA-dependent protein kinase complex: a multifunctional protein in DNA repair and damage checkpoint. Mol. Cells. 2002;13:159–166. [PubMed]
24. Lombard DB, Chua KF, Mostoslavsky R, Franco S, Gostissa M, Alt FW. DNA repair, genome stability, and aging. Cell. 2005;120:497–512. [PubMed]
25. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, et al. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. 2011 [PMC free article] [PubMed]
26. Koike M, Awaji T, Kataoka M, Tsujimoto G, Kartasova T, Koike A, Shiomi T. Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis. J. Cell Sci. 1999;112(Pt 22):4031–4039. [PubMed]
27. Ding X, Xu R, Yu J, Xu T, Zhuang Y, Han M. SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice. Dev. Cell. 2007;12:863–872. [PubMed]
28. Chi YH, Cheng LI, Myers T, Ward JM, Williams E, Su Q, Faucette L, Wang JY, Jeang KT. Requirement for Sun1 in the expression of meiotic reproductive genes and piRNA. Development. 2009;136:965–973. [PubMed]
29. Oza P, Peterson CL. Opening the DNA repair toolbox: localization of DNA double strand breaks to the nuclear periphery. Cell Cycle. 2010;9:43–49. [PubMed]
30. Weis K, Griffiths G, Lamond AI. The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif. J. Biol. Chem. 1994;269:19142–19150. [PubMed]
31. Wei F, Xie Y, He L, Tao L, Tang D. ERK1 and ERK2 kinases activate hydroxyurea-induced S-phase checkpoint in MCF7 cells by mediating ATR activation. Cell Signal. 2011;23:259–268. [PubMed]
32. Burke B, Stewart CL. The laminopathies: the functional architecture of the nucleus and its contribution to disease. Annu. Rev. Genomics Hum. Genet. 2006;7:369–405. [PubMed]
33. Worman HJ, Ostlund C, Wang Y. Diseases of the nuclear envelope. Cold Spring Harb. Perspect. Biol. 2010;2:a000760. [PMC free article] [PubMed]
34. Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, Goldman RD. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 2008;22:832–853. [PubMed]
35. Hasan S, Guttinger S, Muhlhausser P, Anderegg F, Burgler S, Kutay U. Nuclear envelope localization of human UNC84A does not require nuclear lamins. FEBS Lett. 2006;580:1263–1268. [PubMed]
36. Mejat A, Decostre V, Li J, Renou L, Kesari A, Hantai D, Stewart CL, Xiao X, Hoffman E, Bonne G, et al. Lamin A/C-mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy. J. Cell Biol. 2009;184:31–44. [PMC free article] [PubMed]
37. Haque F, Mazzeo D, Patel JT, Smallwood DT, Ellis JA, Shanahan CM, Shackleton S. Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J. Biol. Chem. 2010;285:3487–3498. [PMC free article] [PubMed]
38. Gonzalez-Suarez I, Redwood AB, Perkins SM, Vermolen B, Lichtensztejin D, Grotsky DA, Morgado-Palacin L, Gapud EJ, Sleckman BP, Sullivan T, et al. Novel roles for A-type lamins in telomere biology and the DNA damage response pathway. EMBO J. 2009;28:2414–2427. [PubMed]
39. Liu B, Wang J, Chan KM, Tjia WM, Deng W, Guan X, Huang JD, Li KM, Chau PY, Chen DJ, et al. Genomic instability in laminopathy-based premature aging. Nat. Med. 2005;11:780–785. [PubMed]
40. Sun H, Gulbagci NT, Taneja R. Analysis of growth properties and cell cycle regulation using mouse embryonic fibroblast cells. Methods Mol. Biol. 2007;383:311–319. [PubMed]