SHORT TAKE

Defective ATM-Kap-1-mediated chromatin remodeling impairsDNA repair and accelerates senescence in progeria mouse model

Baohua Liu,1,2* Zimei Wang,1,3* Shrestha Ghosh1 andZhongjun Zhou1,2

1Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University

of Hong Kong, 21 Sassoon Road, Hong Kong2Shenzhen Institute of Research and Innovation, The University of Hong

Kong, 21 Sassoon Road, Hong Kong3Department of Biochemistry and Molecular Medicine, School of Medicine,

Shenzhen University, 3688 Nanhai Ave, Shenzhen 518060, China

Summary

ATM-mediated phosphorylation of KAP-1 triggers chromatin

remodeling and facilitates the loading and retention of repair

proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs)

derived from Zmpste24�/� mice undergo early senescence,

attributable to delayed recruitment of DNA repair proteins. Here,

we show that ATM-Kap-1 signaling is compromised in Zmp-

ste24�/� MEFs, leading to defective DNA damage-induced chro-

matin remodeling. Knocking down Kap-1 rescues impaired

chromatin remodeling, defective DNA repair and early senes-

cence in Zmpste24�/� MEFs. Thus, ATM-Kap-1-mediated chroma-

tin remodeling plays a critical role in premature aging, carrying

significant implications for progeria therapy.

Key words: ATM; KAP-1; chromatin remodeling; DNA repair;

Zmpste24; cellular senescence; progeria.

A de novo G608G mutation in LMNA is the predominant cause of

Hutchinson-Gilford progeria syndrome (HGPS) (Eriksson et al., 2003).

Lamin A is first synthesized as prelamin A and ZMPSTE24 metallopro-

teinase is required for its maturation (Pendas et al., 2002). Mice lacking

Zmpste24 recapitulate many of the progeroid features in HGPS (Pendas

et al., 2002). HGPS cells and Zmpste24�/� MEFs undergo accelerated

senescence, attributable to delayed recruitment of repair proteins and

defective DNA repair (Liu et al., 2005, 2006). However, the underlying

molecular mechanism remains largely unknown.

Upon DNA damage, chromatin opens up for the loading and

retention of repair proteins to DNA lesions (Iijima et al., 2008). To

determine the chromatin accessibility, a micrococcal nuclease sensitivity

assay was employed. In wild-type cells, chromatin accessibility peaked

30 min after c-irradiation, reflected by the highest amount of low

molecular weight oligonucleosomal fragments and the concurrent

disappearance of high molecular weight DNA fragments (Fig. 1A,B).

Thereafter, higher molecular weight DNA fragments gradually increased

whereas the lower molecular weight fragments concurrently decreased,

suggesting recondensation and restoration of chromatin. However,

maximal chromatin accessibility was significantly delayed to 2 h and the

chromatin started to recondense approximately 4 h after c-irradiation in

Zmpste24�/� cells (Fig. 1A,B and Fig. S1A, lane 6-10). Delayed chroma-

tin remodeling in Zmpste24�/� cells was likely a consequence of

accumulated prelamin A, as ectopic prelamin A in HEK293 cells caused

similar defects (Fig. S2).

In response to DNA damage, ATM phosphorylates KAP-1 at Ser

824 (pS824-KAP-1), which weakens the binding of KAP-1 to MNase-

resistant heterochromatin fraction and releases CHD3 from chromatin,

leaving the heterochromatin de-condensed for loading essential repair

proteins (Ziv et al., 2006; Goodarzi et al., 2008, 2011; Noon et al.,

2010). To understand the mechanisms behind the defective chromatin

remodeling in Zmpste24�/� MEFs, pS824-Kap-1 level was examined.

The level of pS824-Kap-1 peaked around 30 min after

c-irradiation and was decreased gradually thereafter in wild-type cells

(Fig. 1C,D), while it was significantly reduced in Zmpste24�/� MEFs.

Consistently, in wild-type cells, the level of Kap-1 associated with

MNase-resistant fraction was significantly reduced at 30 min after c-irradiation in a dose-dependent manner, whereas it was hardly

changed in Zmpste24�/� cells (Fig. 1E). As ATM is the only kinase

responsible for pS824-Kap-1, and loss of ATM in MEFs also leads to

defective chromatin remodeling upon DNA damage (Fig. S3), we

asked whether ATM itself is affected in progeroid cells. Indeed, we

found a significant reduction in the level of pS1981-ATM in

Zmpste24�/� MEFs compared with wild-types (Fig. 1F and Fig. S4).

Consistently, as a direct target of ATM (Shiloh, 2006), the level of

pS343-Nbs1 was also significantly decreased in Zmpste24�/� MEFs in

response to DNA damage (Fig. 1F and Fig. S4). Thus, these data

indicate that defective ATM-Kap-1 signaling might underlie the

defective chromatin remodeling in Zmpste24�/� MEFs.

We next tested whether knocking down Kap-1 could rescue the

impaired chromatin remodeling, defective DNA repair, and early

senescence in Zmpste24�/� MEFs. As shown, knocking down Kap-1

restored chromatin relaxation at 30 min and subsequent recondensa-

tion around 2 h after DNA damage in Zmpste24�/� MEFs (Fig. 2A,B

and Fig. S5). Concurrently, the delayed recruitment of 53BP1 was

restored and the sustained 53BP1 foci staining at 24 h after DNA

damage were substantially reduced in Zmpste24�/� MEFs (Fig. 2C).

Moreover, knocking down Kap-1 rescued the early senescence in

Zmpste24�/� MEFs determined by senescence-associated b-galactosi-dase assay (Fig. 2D,E, 74 � 5% positively stained cells with scramble vs.

35 � 3% with Kap-1 siRNA in Zmpste24�/� cells, Mean � SEM,

P < 0.05). Thus, defective ATM-Kap-1 signaling underlies defective

chromatin remodeling, defective DNA repair, and early senescence in

Zmpste24�/� MEFs.

Collectively, we found that the defective DNA repair in laminopathy-

based progeria was attributable to compromised ATM-Kap-1 signaling

and delayed global chromatin remodeling. Knocking down Kap-1

rescues the defective DNA repair and early senescence in progeroid

cells, suggesting an important role of chromatin remodeling in lamin-

Correspondence

Zhongjun Zhou, PhD, Department of Biochemistry, Li Ka Shing Faculty of Medicine,

University of Hong Kong, 21 Sassoon Road, Hong Kong. Tel. +852 28199542;

fax: +852 28551254; e-mail: zhongjun@hkucc.hku.hk

*B. Liu and Z. Wang have contributed to this work equally.

Accepted for publication 15 November 2012

316 ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland

Aging Cell (2013) 12, pp316–318 Doi: 10.1111/acel.12035Ag

ing

Cell

opathy-based premature aging. The delayed yet completely relaxed

chromatin in Zmpste24�/� MEFs implies the existence of a potential

backup mechanism that mediates late chromatin remodeling in prog-

eroid cells. Indeed, it has been reported that BRIT1 (BRIT-repeat inhibitor

of hTERT expression), Brca1 and cofactor COBRA1, E2F1, and p53

regulate global chromatin relaxation/remodeling (Peng et al., 2009; Ye

et al., 2001). This could be a backup mechanism regulating global

chromatin remodeling in Zmpste24�/� MEFs, in response to the

defective ATM-Kap-1 signaling.

We recently showed that sodium butyrate (NaB) and trichostatin A

(TSA) induced up-regulation of global histone acetylation and feeding

progeria mice with NaB increased the acetylation of H4K16, rescued the

delayed recruitment of 53BP1, and extended lifespan in progeroid mice

(Krishnan et al., 2011). NaB and TSA belong to class I and II HDAC

inhibitors (Bolden et al., 2006). TSA treatment activates ATM upon DNA

damage in addition to increasing histone acetylation (Bakkenist &

Kastan, 2003). Interestingly, preincubation of NaB also rescued defective

chromatin remodeling upon DNA damage in Zmpste24�/� MEFs (see Fig.

S1). Thus, in addition to local H4K16 acetylation surrounding DNA

lesions, NaB treatment may directly enhance ATM activity thus rescuing

defective global chromatin remodeling and DNA repair as well as early

senescence in progeria mice.

Acknowledgments

This work was supported by Hong Kong Research Council (HKU7698/

05M, HKU7655/06M, CRF HKU3/07C) and Progeria Research

Foundation.

References

Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through

intermolecular autophosphorylation and dimer dissociation. Nature 421,499–506.

Bolden JE, Peart MJ, Johnstone RW (2006) Anticancer activities of histone

deacetylase inhibitors. Nat. Rev. Drug. Discov. 5, 769–784.Eriksson M, Brown WT, Gordon LB, Glynn MW, Singer J, Scott L, Erdos MR,

Robbins CM, Moses TY, Berglund P, Dutra A, Pak E, Durkin S, Csoka AB,

Boehnke M, Glover TW, Collins FS (2003) Recurrent de novo point mutations in

lamin A cause Hutchinson-Gilford progeria syndrome. Nature 423, 293–298.Goodarzi AA, Noon AT, Deckbar D, Ziv Y, Shiloh Y, Lobrich M, Jeggo PA (2008)

ATM signaling facilitates repair of DNA double-strand breaks associated with

heterochromatin. Mol. Cell 31, 167–177.

(A) (B)

(C) (D)

(E) (F)

Fig. 1 Defective DNA damage-induced

chromatin remodeling and ATM-Kap-1

signaling in Zmpste24�/� MEFs. (A) A

representative gel photo of MNase

accessibility assay. Arrows show ‘intact’

genomic DNA and oligonucleosomal DNA

fragments. (B) Quantification of ‘intact’

genomic DNA in (A) by Image J®.

(C) Representative immunoblots at various

time points after 5 Gy of c-irradiation.While the level of pS824-Kap-1 was

significantly decreased upon c-irradiation,pS473-Kap-1 was not obviously affected.

At least three pairs of independently

derived MEFs were examined. (D)

Quantification of experiments in (C). Data

represent mean � SEM, n = 3. *P < 0.05.

(E) Representative Western blotting in

MNase-resistant fraction and total cell

lysate upon c-irradiation. (F) Representativeimmunobots in MEFs at 30 min after 5 Gy

of irradiation. Data are representative of at

least three independent experiments.

Defective chromatin remodeling in progeria, B. Liu et al. 317

ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland

Goodarzi AA, Kurka T, Jeggo PA (2011) KAP-1 phosphorylation regulates CHD3

nucleosome remodeling during the DNA double-strand break response. Nat.

Struct. Mol. Biol. 18, 831–839.Iijima K, Ohara M, Seki R, Tauchi H (2008) Dancing on damaged chromatin:

functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to

DNA damage. J. Radiat. Res. 49, 451–464.Krishnan V, Chow MZ, Wang Z, Zhang L, Liu B, Liu X, Zhou Z (2011) Histone H4

lysine 16 hypoacetylation is associated with defective DNA repair and premature

senescence in Zmpste24-deficient mice. Proc. Natl Acad. Sci. USA 108, 12325–12330.

Liu B, Wang J, Chan KM, Tjia WM, Deng W, Guan X, Huang JD, Li KM, Chau PY,

Chen DJ, Pei D, Pendas AM, Cadinanos J, Lopez-Otin C, Tse HF, Hutchison C,

Chen J, Cao Y, Tryggvason K, Zhou Z (2005) Genomic instability in laminopathy-

based premature aging. Nat. Med. 11, 780–785.Liu Y, Rusinol A, Sinensky M, Wang Y, Zou Y (2006) DNA damage responses in

progeroid syndromes arise from defective maturation of prelamin A. J. Cell Sci.

119, 4644–4649.Noon AT, Shibata A, Rief N, Lobrich M, Stewart GS, Jeggo PA, Goodarzi AA

(2010) 53BP1-dependent robust localized KAP-1 phosphorylation is essential

for heterochromatic DNA double-strand break repair. Nat. Cell Biol. 12,177–184.

Pendas AM, Zhou Z, Cadinanos J, Freije JM, Wang J, Hultenby K, Astudillo A,

Wernerson A, Rodriguez F, Tryggvason K, Lopez-Otin C (2002) Defective

prelamin A processing and muscular and adipocyte alterations in Zmpste24

metalloproteinase-deficient mice. Nat. Genet. 31, 94–99.Peng G, Yim EK, Dai H, Jackson AP, Burgt I, Pan MR, Hu R, Li K, Lin SY (2009)

BRIT1/MCPH1 links chromatin remodelling to DNA damage response. Nat. Cell

Biol. 11, 865–872.

Shiloh Y (2006) The ATM-mediated DNA-damage response: taking shape. Trends

Biochem. Sci. 31, 402–410.Ye Q, Hu YF, Zhong H, Nye AC, Belmont AS, Li R (2001) BRCA1-induced large-

scale chromatin unfolding and allele-specific effects of cancer-predisposing

mutations. J. Cell Biol. 155, 911–921.Ziv Y, Bielopolski D, Galanty Y, Lukas C, Taya Y, Schultz DC, Lukas J, Bekker-Jensen

S, Bartek J, Shiloh Y (2006) Chromatin relaxation in response to DNA double-

strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.

Nat. Cell Biol. 8, 870–876.

Supporting Information

Additional Supporting Information may be found in the online version of this

article at the publisher’s web-site.

Fig. S1 NaB treatment rescues chromatin remodeling in Zmpste24�/� MEFs.

Fig. S2 Ectopic prelamin A leads to defective chromatin remodeling.

Fig. S3 ATM mediates KAP-1 phosphorylation and chromatin remodeling.

Fig. S4 Defective ATM-Kap-1 signaling in Zmpste24 null MEFs.

Fig. S5 Knocking down Kap-1 in MEFs.

(A) (B)

(C)

(D) (E)

Fig. 2 Knocking down Kap-1 rescues

defective chromatin remodeling, impaired

DNA repair and early senescence in

Zmpste24�/� MEFs. (A) Representative gel

photo of MNase assay in cells treated with

Kap-1 or scramble siRNA after c-irradiation.(B) Quantification of lower molecular

weight nucleosomal fragments in (A) by

Image J®. (C) Time course of the number of

53BP1 foci per cell in MEFs after c-irradiation. Insert is the number of

spontaneous 53BP1 foci per cell prior to

c-irradiation. At least 200 cells were

counted. Data represent mean � SEM.

*P < 0.05. (D) Senescence-associated

b-galactosidase staining in MEFs at passage

6. Scale bar, 200 lm. (E) Percent

senescence-associated b-galactosidasepositive cells out of at least 200 from (D).

Data represent mean � SEM. *P < 0.001.

Data are representative of at least three

independent experiments.

Defective chromatin remodeling in progeria, B. Liu et al.318

ª 2012 The AuthorsAging Cell ª 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland