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J. Cell Biol. Vol. 185 No. 4  577–586
www.jcb.org/cgi/doi/10.1083/jcb.200810035 JCB 577
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M.S. Luijsterburg and C. Dinant contributed equally to this paper.
Correspondence to Roel van Driel: r.vandriel@uva.nl
Abbreviations used in this paper: CD, chromodomain; CSB, Cockayne syndrome
protein B; CSD, chromo shadow domain; DDR, DNA damage response;
DSB, double-strand break; FLIP, fluorescence loss in photobleaching;
FP, fluorescent protein; GGR, global genome NER; HP1, heterochromatin
protein 1; IR, ionizing radiation; mRFP, monomeric RFP; NER, nucleotide
excision repair; NHEJ, nonhomologous end joining; PCNA, proliferating cell
nuclear antigen; SCFP, super cyan FP; TCR, transcription-coupled NER; XP, xeroderma
pigmentosum.
Introduction
DNA can be damaged by various agents, including ionizing
radiation (IR) and UV radiation. Cells respond to genotoxic stress
by activating DNA damage response (DDR) systems, including
DNA repair, cell cycle arrest, senescence, and apoptosis (Bartek
and Lukas, 2007). DNA repair pathways each deal with specific
types of lesions (Hoeijmakers, 2001). Nucleotide excision repair
(NER) removes bulky adducts and UV-induced photoproducts
such as cyclobutane pyrimidine dimers and 6-4 photoproducts
from the genome, whereas DNA double-strand breaks (DSBs)
are removed by homologous recombination or nonhomologous
end joining (NHEJ; Hoeijmakers, 2001).
The heterochromatin protein 1 (HP1) isoforms HP1-,
HP1-, and HP1- are versatile epigenetic regulators with functions
in chromatin organization, transcription regulation, and
DNA replication (Maison and Almouzni, 2004). Recent studies
suggested that HP1 may respond to chromosomal breaks in
heterochromatin (Ayoub et al., 2008; Goodarzi et al., 2008).
HP1 proteins bind to histone H3 that is methylated at lysine 9
(H3K9me) via their N-terminal chromodomain (CD; Jacobs and
Khorasanizadeh, 2002). Additionally, HP1 interacts with a large
number of nuclear proteins via its C-terminal chromo shadow
domain (CSD), including epigenetic regulators and chromatin
remodelling complexes (Nielsen et al., 2002; Fuks et al., 2003;
Eskeland et al., 2007).
In this study, we show that all three HP1 proteins are recruited
to UV-induced lesions and DSBs in living human cells.
H
eterochromatin protein 1 (HP1) family members
are chromatin-associated proteins involved in transcription,
replication, and chromatin organization.
We show that HP1 isoforms HP1-, HP1-, and HP1- are
recruited to ultraviolet (UV)-induced DNA damage and
double-strand breaks (DSBs) in human cells. This response
to DNA damage requires the chromo shadow domain of
HP1 and is independent of H3K9 trimethylation and proteins
that detect UV damage and DSBs. Loss of HP1 results
in high sensitivity to UV light and ionizing radiation in the
nematode Caenorhabditis elegans, indicating that HP1
proteins are essential components of DNA damage response
(DDR) systems. Analysis of single and double HP1
mutants in nematodes suggests that HP1 homologues have
both unique and overlapping functions in the DDR. Our
results show that HP1 proteins are important for DNA
repair and may function to reorganize chromatin in response
to damage.
Heterochromatin protein 1 is recruited to various
types of DNA damage
Martijn S. Luijsterburg,1,7
Christoffel Dinant,3,4,8
Hannes Lans,4
Jan Stap,2
Elzbieta Wiernasz,1,5
Saskia Lagerwerf,6
Daniël O. Warmerdam,4
Michael Lindh,7
Maartje C. Brink,1
Jurek W. Dobrucki,5
Jacob A. Aten,2
Maria I. Fousteri,6
Gert Jansen,4
Nico P. Dantuma,7
Wim Vermeulen,4
Leon H.F. Mullenders,6
Adriaan B. Houtsmuller,3
Pernette J. Verschure,1
and Roel van Driel1
1
Swammerdam Institute for Life Sciences and 2
Center for Microscopical Research, Department of Cell Biology and Histology, Academic Medical Center,
University of Amsterdam, 1012 WX Amsterdam, Netherlands
3
Department of Pathology, Josephine Nefkens Institute and 4
Department of Cell Biology and Genetics, Medical Genetics, Erasmus Medical Center,
3000 CA Rotterdam, Netherlands
5
Division of Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland
6
Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands
7
Department of Cell and Molecular Biology, Karolinska Institutet, S-17177 Stockholm, Sweden
8
Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark
© 2009 Luijsterburg et al.  This article is distributed under the terms of an Attribution–
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THEJOURNALOFCELLBIOLOGY
JCB • VOLUME 185 • NUMBER 4 • 2009 578
579HP1 IS RECRUITED TO DAMAGED DNA • Luijsterburg et al.
This recruitment of HP1 depends on the CSD but not on H3K9
methylation or on the repair systems that remove these lesion
types. Loss of HP1 proteins renders the nematode Caenorhabditis
elegans highly sensitive to UV and ionizing irradiation.
Our data suggest that HP1 homologues have both distinct and
overlapping essential functions in the DDR.
Results and discussion
HP1 proteins are recruited to UV lesions
by the CSD
To study whether HP1 proteins respond to UV-induced DNA
damage, we locally damaged nuclei of cultured cells with UV-C
light either using a UV-C laser (266 nm; Dinant et al., 2007) or by
irradiation with a UV-C lamp (254 nm) through a polycarbonate
mask (Moné et al., 2001). These methods recruit NER proteins
but not the DSB repair proteins NBS1 (Fig. S1A), Rad50, Rad54,
and Ku80 (Houtsmuller et al., 1999; Dinant et al., 2007). At UVirradiated
sites, we observed recruitment of all three HP1 isoforms
expressed as fluorescent protein (FP)-tagged fusion proteins
(monomeric RFP [mRFP]–HP1-, super cyan FP [SCFP]–HP1-,
and EGFP–HP1-) in human cells (Fig. 1, A–C) and mouse cells
(not depicted). The FPs (CFP, YFP, and GFP) alone do not accumulate
at locally damaged sites, indicating that accumulation is
HP1 dependent (Fig. S1, F–I). Fluorescent immunolabeling with
HP1-–specific antibodies showed that GFP–HP1- is expressed
at 20% of the level of endogenous HP1- (Fig. S1, D and E).
Importantly, endogenous HP1-, HP1-, and HP1- accumulate
at local UV damage in primary human fibroblasts (Fig. 1, D–F).
Comparing the same cells before and after local UV irradiation
(Fig. S1, B and C) showed that SCFP–HP1- accumulated
at damaged sites with a t1/2 of 180 s after local UV irradiation
(Fig. 1 G), which is comparable to the binding kinetics of XPA
(xeroderma pigmentosum [XP] group A) at DNA repair sites
(Fig. S1 J). To further investigate the binding of HP1 at lesions, we
performed photobleaching experiments on mouse cells that express
EGFP–HP1- and that were globally UV-C irradiated with
25 J/m2
. These experiments confirmed that a small but significant
fraction of EGFP–HP1- is immobilized in UV-irradiated cells on
a time scale of several minutes but not in nondamaged control
cells (Fig. 1, H and I). Global UV irradiation did not result in visible
changes in the nuclear distribution of HP1 (Fig. S2 A) nor did
it result in immobilization of GFP-NLS (Fig. S1, K and L).
HP1 proteins contain three distinct domains: the N-terminal
CD, the C-terminal CSD, and the hinge region that separates the
CD from the CSD. Deletion mutants of HP1- lacking the CD,
CSD, or hinge (Fig. 1 J) were tagged with EGFP and tested for
recruitment to UV-irradiated regions. Interestingly, UV lesions
triggered binding of EGFP–HP1- (∆CD) and EGFP–HP1-
(∆hinge), but EGFP–HP1- (∆CSD) failed to accumulate
(Fig. 1, K–M). To test whether the CSD (amino acids 98–185 of
HP1-) is not only necessary but also sufficient for recruitment,
we fused this domain to EYFP and found that EYFP-CSD is
indeed recruited to sites of local UV irradiation (Fig. 1 N). Our
results show that HP1 recruitment to damaged sites is independent
of the CD, suggesting that recruitment of HP1 is independent
of H3K9 trimethylation (H3K9me3). To verify that HP1
recruitment does not require H3K9me3, we examined accumulation
of HP1 in MEFs deficient for Suv39h1 and Suv39h2 (Peters
et al., 2001). After UV irradiation, SCFP–HP1- accumulated
at sites of damage, confirming that binding of HP1 requires neither
the CD nor H3K9me3 (Fig. 1 O). In agreement, we did not
detect recruitment of EYFP-tagged H3K9 methyltransferase
Suv39h1 (not depicted) nor did we detect increased H3K9me3 at
sites of UV lesions (Fig. S2 B). A recent study showed that HP1
is phosphorylated at residue T51 (in the CD) in response to
chromosomal breaks, which was suggested to initiate the DDR
(Ayoub et al., 2008). We show that HP1- lacking the T51 phosphorylation
site (YFP–HP1-T51A
) is recruited to sites of UVinduced
lesions (Fig. 1 P). Together, our results indicate that HP1
proteins are recruited to UV-induced DNA damage through their
CSD independently of the CD and of trimethylation at H3K9.
HP1 recruitment to UV lesions is
independent of NER
Cells from placental mammals are fully dependent on NER for
the removal of UV-induced DNA injuries, involving transcriptioncoupled
NER (TCR) and global genome NER (GGR; Hoeijmakers,
2001). UV lesions trigger several chromatin-related events, such
as recruitment of CAF-1, incorporation of histone H3.1 (Polo
et al., 2006), and ubiquitylation of H2A (Bergink et al., 2006).
These events strictly depend on the binding and activity of
NER proteins that are involved in recognition and subsequent
processing of DNA lesions (Bergink et al., 2006; Polo et al., 2006).
To investigate whether HP1 binding is a late or early event in
NER, we tested accumulation of HP1- in repair-deficient XP-A
cells that have compromised GGR and TCR. SCFP–HP1- accumulated
in XPA mutant cells after UV irradiation (Fig. 2 A),
suggesting that HP1- binding does not occur after DNA repair
is finished. We then considered the possibility that HP1 binding
Figure 1.  Recruitment of HP1s to UV damage depends on the CSD. (A–C, right) Damage-induced accumulation of mRFP–HP1- (A), SCFP–HP1- (B), and
EGFP–HP1- (C) in living HeLa or MRC5 cells 30 min after local irradiation at 100 J/m2
though 5-µm pores. (A–C, left) The site of local DNA damage is
indicated by accumulation of FP-tagged DDB2. (D–F, right) Immunolabeling of endogenous HP1- (D), HP1- (E), and HP1- (F) in locally UV-irradiated
confluent human fibroblasts irradiated at 100 J/m2
through 3- (D and E) or 8-µm pores (F; cells are shown 30 min after irradiation). UV-damaged sites are
visualized by local accumulation of XPA (D and E) or PCNA (F). (G) Accumulation of SCFP–HP1- during the first 15 min after localized UV-C laser damage.
Fig. S1 (B and C) shows the predamage distribution of HP1 in the same cell as shown in G and accumulation of DDB2-mCherry to indicate the site
of damage. (H and I) Combined FLIP/FRAP analysis on NIH/3T3 cells expressing EGFP–HP1-. Cells were either mock treated (H) or globally irradiated
at 25 J/m2
(I). Half of a cell nucleus was bleached, and FLIP was measured in the nonbleached half 30 min after UV irradiation (blue line), whereas FRAP
was measured in the bleached half (red line). (J) HP1- deletion mutants. The CSD is indicated in red, the CD in blue, and the hinge in yellow. Numbers
represent amino acid positions. (K–N) Nuclear localization of EGFP–HP1- (CD) (K), EGFP–HP1- (CSD) (L), EGFP–HP1- (hinge) (M), and EYFP-CSD
(N) in living HeLa or MRC5 cells locally irradiated at 100 J/m2
through 5-µm pores. (O and P) Recruitment of SCFP–HP1- in Suv3-9–deficient mouse cells
(O) and recruitment of EYFP–HP1-T51A
in MRC5 cells (P). The site of local DNA damage is indicated by accumulation of DDB2-mVenus or DDB2-mCherry,
and images were taken 30 min after UV irradiation. Error bars indicate SD.
 
JCB • VOLUME 185 • NUMBER 4 • 2009 580
is caused by replication stress, we determined the cell cycle stage
by expressing mCherry–proliferating cell nuclear antigen (PCNA)
together with SCFP–HP1- and DDB2-mVenus in repair-deficient
XP-A cells and wild-type MRC5 cells. Recruitment of HP1-
was observed in wild-type (not depicted) and NER-deficient cells
(Fig. S2, C and D) in S phase as well as in non–S phase cells,
as shown by the distribution of PCNA. Clear accumulation of
endogenous HP1- was observed in G0 cells (Ki67-negative cells)
at sites of UV irradiation (Fig. S2 E), showing that HP1 accumulation
is not the result of stalled replication.
Together, these results show that HP1 binding to UVdamaged
areas occurs in both cycling and quiescent cells and is
independent of the activity of preincision GGR proteins, the
TCR factor CSB, and ATR kinase.
UV-induced HP1 accumulation persists in
the absence of functional NER
To determine whether loss of HP1 accumulation depends on the
presence of UV-induced DNA lesions, we measured HP1 accumulation
in repair-deficient cells. Binding of HP1- was observed
up to 4 h after local UV irradiation in XP-A cells (Fig. 3 A).
Conversely, in XPA-deficient cells that were transiently transfected
with mVenus-XPA (to restore the repair capacity), bound
HP1- levels gradually decreased and HP1 accumulation had
almost disappeared 4 h after UV irradiation (Fig. 3 B), which
is consistent with the rate of DNA repair (van Hoffen et al.,
1995). Accordingly, accumulation of YFP-tagged CSD became
is an early step after damage detection and tested accumulation
in DDB2-deficient and XPC-deficient cells. Accumulation of
SCFP–HP1- was observed in both cell types (Fig. 2, B–D).
Because binding of repair proteins involved in GGR is dependent
on XPC (Volker et al., 2001), these results suggest that
HP1 recruitment to damaged sites is independent of the activity
of GGR proteins. Damage detection in TCR requires stalled
RNA polymerase II, and subsequent recruitment of NER factors
depends on the Cockayne syndrome protein B (CSB) protein
(Fousteri et al., 2006). Recruitment of HP1 was also observed in
CSB-deficient cells, indicating that HP1 binding is not dependent
on TCR (Fig. 2 E).
In addition to DNA repair, cells respond to damaged DNA
by activatingATM (ataxia telangiectasia mutated) orATR (ATM
and Rad3-related) kinase signaling pathways, resulting in activation
of cell cycle checkpoints and phosphorylation of a variety
of proteins involved in the DDR (Bartek and Lukas, 2007).
To determine whether ATR is required for the recruitment of
HP1 to UV lesions, we examined the accumulation of HP1-,
HP1-, and HP1- in Seckel cells, which have severely reduced
ATR expression (O’Driscoll et al., 2003). Accumulation of the
HP1 isoforms after local UV irradiation was not affected in
these cells (Fig. 2 F and not depicted), demonstrating that HP1
recruitment to sites of DNA damage does not require DNA
damage-induced signaling mediated by the ATR kinase.
During S phase, stalling of replication forks at UV-induced
lesions can result in DSBs. To exclude that HP1 accumulation
Figure 2.  Recruitment of HP1 in NER-deficient cells. (A–F) Damage-induced accumulation of SCFP–HP1-, EGFP–HP1-, and EGFP–HP1-g in human
fibroblasts deficient for XPA (A), XPC (B), DDB2 (C), MEFs deficient for XPC (D), human fibroblasts deficient for CSB (E), and human Seckel cells (F), which
have severely reduced expression of ATR kinase. Cells were locally UV lamp irradiated at 100 J/m2
through 5-µm pores (A and C) or irradiated using a
UV-C laser (B and D–F). The site of local DNA damage is indicated by accumulation of DDB2-mVenus, DDB2-mCherry, XPC-mVenus (A–C), or by a square
(D and F). Images were taken 30 min after UV irradiation.
581HP1 IS RECRUITED TO DAMAGED DNA • Luijsterburg et al.
mouse cells (Fig. 4 B) and endogenous HP1- in human U2OS
cells (Fig. S3 C) colocalized with the linear H2AX pattern.
In addition, accumulation of GFP–HP1- and GFP–HP1-
(Fig. S3, D and E) was observed in MRC5 cells, showing that
all HP1 isoforms are recruited to DSBs.
Mammalian cells use homologous recombination or NHEJ
to remove DSBs from the genome (Wyman and Kanaar, 2006).
The latter pathway is initiated by the KU70/80 dimer, which
was shown to interact with HP1- (Song et al., 2001). To test
whether HP1 accumulation at DSBs depends on NHEJ, we irradiated
wild-type and KU80-deficient CHO cells with -particles.
Recruitment of endogenous HP1- was observed in both cell
types at all H2A.X tracks, showing that HP1 association is independent
of NHEJ (Fig. 4 C). These results show that HP1
proteins are recruited to DSBs, which is in contrast to the mobilization
of HP1 after DNA damage, as reported recently (Ayoub
et al., 2008). In that study, microscopic analysis showed spreading
of GFP–HP1- into a larger area at sites of laser-assisted
DNA damage inflicted at heterochromatic sites (Ayoub et al.,
2008), which was interpreted as dissociation of HP1- from
heterochromatin. An alternative explanation could be that HP1
does not spread into neighboring chromatin but that the apparent
spreading of HP1 actually reflects accumulation of HP1 at
sites of DNA damage. To explore this, we have used the same
procedure that Ayoub et al. (2008) used to damage DNA locally.
Cells were sensitized with Hoechst, and a narrow strip spanning
the nucleus was irradiated using a 405-nm laser, resulting
in clear accumulation of NBS1-mCherry. At sites marked by
NBS1 accumulation, we observed accumulation of GFP–HP1-
(Fig. 4, D and E). The CD and T51 residue (a protein kinase target
in HP1) of HP1- are dispensable for recruitment to these
damaged sites (Fig. 4, F and G). Similar to what is observed for
UV-induced lesions, we find that the CSD alone is sufficient for
binding and that GFP–HP1- (CSD) does not bind to the
damaged DNA sites (Fig. 4, H and I). We could not detect loss
or dispersal of HP1 in the damage region before accumulation
of HP1. Moreover, monitoring the same cells before and after
damage induction showed that HP1 accumulated at higher levels
in the locally damaged area compared with the predamage
distribution of HP1 (Fig. 4, D and E), showing that the accumulation
of HP1- reflects de novo binding of HP1 molecules at
damaged sites. We subsequently measured the binding kinetics
of GFP–HP1- in cells sensitized with BrdU in which a strip
spanning the nucleus was irradiated using a 337-nm laser
(Fig. 4 J). HP1- rapidly accumulated at damaged sites with a
t1/2 of 85 s. The CSD-dependent accumulation of HP1 is markedly
different from the recently reported reappearance of HP1
at heterochromatic sites after a transient (5 min) dispersal of
HP1 (Ayoub et al., 2008). The latter study showed phosphorylation
of HP1 at T51 (T51P) in response to localized laserassisted
DNA damage, IR, and etoposide treatment. Incubation
of the CD of HP1- with CK2 resulted in a weakened interaction
with the H3K9me2 peptide in vitro (Ayoub et al., 2008).
However, it remains unclear whether T51-phosphorylated HP1-
has a lowered affinity for chromatin in vivo, as binding of HP1-
to chromatin is influenced by its dimerization with HP1-, interactions
with the H3 histone fold, and an RNA component
undetectable 4 h after irradiation similar to full-length HP1
(Fig. 3 C), suggesting that the UV lesions directly or indirectly
create a binding site for the CSD of HP1, resulting in its recruitment
to UV lesions.
This is the first example of a protein that is recruited to
sites of UV-induced DNA damage independent of any of the
known UV damage recognition factors, suggesting that HP1 proteins
are involved in the DDR via a novel mechanism that acts
in parallel to the well-studied NER repair pathway.
HP1 is recruited to DSBs
To study the response of HP1 to chromosomal breaks, we
performed photobleaching experiments on human cells that
express EGFP–HP1- and that were globally irradiated with
x rays (5 and 10 Gy). Photobleaching experiments indicated
that a small fraction of HP1 became immobile in response to IR
(Fig. S3 A), suggesting increased binding of HP1 to chromatin
in response to chromosomal breaks. IR did not result in visible
changes in the nuclear distribution of HP1 proteins (Fig. S3 B).
To confirm immobilization of HP1 at sites of double-strand
DNA breaks, we tested HP1 recruitment to locally inflicted
DSBs. Cells were irradiated with a dose of 20 Gy of soft
x rays (extended UV 20 nm) through a nickel filter containing
5-µm pores, resulting in local accumulation of H2AX. At locally
damaged sites, we observed binding of GFP–HP1- (Fig. 4 A),
which is consistent with our fluorescence loss in photobleaching
(FLIP) data (Fig. S3 A). To confirm these results, we irradiated
cells with -particles from a radioactive Americium
(Am-241) source (Aten et al., 2004; Stap et al., 2008). Irradiation
of human U2OS and mouse NIH/3T3 cells with -particles
resulted in linear tracks of H2AX. Binding of EGFP–HP1- in
Figure 3.  Long-term accumulation of HP1- in repair-proficient and
repair-deficient cells. (A) Repair-deficient XP-A cells were transfected
with DDB2-mVenus and SCFP–HP1-. Cells were irradiated at 100 J/m2
,
and accumulation of HP1- was monitored for 4 h after UV irradiation.
(B) Repair-deficient XP-A cells were transfected with mVenus-XPA (to complement
the repair-deficient phenotype), DDB2-mCherry, and SCFP–HP1-.
Cells were irradiated at 100 J/m2
, and accumulation of HP1- was monitored
for 4 h after UV irradiation. (C) Wild-type (MRC5) cells were transfected
with DDB2-mCherry and YFP-CSD, locally irradiated (100 J/m2
),
and accumulation of the CSD was monitored for 4 h after UV irradiation.
The accumulation of DDB2-mVenus or DDB2-mCherry indicates the site
of local damage.
JCB • VOLUME 185 • NUMBER 4 • 2009 582
Loss of HP1 renders C. elegans
highly sensitive to UV irradiation and
chromosomal breaks
Because loss of all HP1 isoforms in mammalian cells is lethal
(Filesi et al., 2002; Schotta et al., 2004), we used the nematode
C. elegans to test whether HP1 is functionally required for the
DDR, as conditional HP1-deficient nematodes are available
(Coustham et al., 2006). Nematodes are a very suitable model
(Nielsen et al., 2001; Maison et al., 2002; Dialynas et al., 2007;
Mateos-Langerak et al., 2007). Our results indicate that neither the
CD nor the T51 residue are required for the binding of HP1 at sites
of DNA damage. It cannot be excluded that phosphorylation of
HP1 bound at damaged sites plays a role in phospho-dependent
interactions with DDR proteins. Interestingly, HP1 is also recruited
to oxidative DNA lesions (unpublished data), indicating that HP1
responds to a variety of DNA lesions in mammalian cells.
Figure 4.  Recruitment of HP1- to DSBs. (A) Mouse cells expressing EGFP–HP1- (green) were locally irradiated with soft x rays through a nickel mask with
pores of 5 µm and subsequently labeled for H2AX (red). (B) Mouse cells expressing EGFP–HP1- (green) were irradiated with -particles and subsequently
labeled for H2AX (red). (C) Hamster cells deficient in Ku80 were irradiated with -particles and subsequently labeled for endogenous HP1- (green) and
H2AX (red). Cells are shown 30 min after irradiation. (D–I) Wild-type U2OS cells expressing various HP1- fusion proteins were sensitized with 10 µg/ml
Hoechst 33342 for 5 min and locally irradiated (five iterations) in a strip spanning the nucleus using a 405-nm laser at 70% output. GFP–HP1- before and
after laser-assisted damage (the damaged area is indicated by arrows; 5 min; D and E), GFP–HP1-CD (F), YFP–HP1-T51A
(G), GFP–HP1-CSD (H), and
YFP-CSD (I). Accumulation of NBS1-mCherry indicates the site of laser-induced DNA damage. (J) GFP–HP1- accumulation in BrdU-sensitized U2OS cells
during the first 800 s after irradiation using a 337-nm laser. (K) Quantification of GFP–HP1- accumulation as described in J. Error bars indicate SD.
583HP1 IS RECRUITED TO DAMAGED DNA • Luijsterburg et al.
with UV-B caused an immediate growth arrest in hpl-2ts
/hpl-1
double-mutant worms, similar to NER-deficient xpa-1 mutant
worms. In contrast, single HP1-like protein mutants exhibit
comparable UV sensitivity as wild-type worms (Fig. 5, A–C).
It should be noted that hpl-2ts
worms displayed considerably
slower growth after UV irradiation, resulting in a smaller size
compared with irradiated hpl-1 worms (Fig. 5 B).An increased
UV-sensitive phenotype was also obtained when juvenile hpl-2ts
/
hpl-1 worms were irradiated instead of eggs (unpublished
data). This indicates that loss of both HP1 proteins renders
C. elegans highly sensitive to UV irradiation. We subsequently exposed
germ cells of single- and double-mutant animals to x rays
system to study the DDR because their response to DNA damage
is similar to that of mammals (O’Neil and Rose, 2006; van Haaften
et al., 2006). Two HP1 homologues (HPL-1 and HPL-2) are
present in C. elegans. To study sensitivity to DNA damage, we
used animals lacking HPL-1 (hpl-1) and carrying a temperaturesensitive
allele of HPL-2 (hpl-2ts
), which is expressed at 20°C
but not at 25°C (Coustham et al., 2006). Eggs of single- and
double-mutant animals were exposed to UV-B radiation (80 J/m2
)
and transferred to 25°C. Wild-type and NER-deficient xpa-1–
null eggs (Stergiou et al., 2007) were assayed in parallel (Fig. 5).
Exposure to UV-B was used because it penetrates nematodes
much better than UV-C light (unpublished data). Irradiation
Figure 5.  Survival of C. elegans HP1 knockout
worms upon UV irradiation and IR. (A) Hatching of
wild-type, hpl-1, hpl-2ts
, and hpl-2ts
/hpl-1 mutant
eggs 8 h after collection. (B) Hatching of wild-type,
hpl-1, hpl-2ts
, and hpl-2ts
/hpl-1 mutant eggs 8 h
after collection and subsequently irradiated with
UV-B at 80 J/m2
. (C) Quantification of hatching
and nonhatching eggs after UV irradiation relative
to nonirradiated eggs. In addition to wild-type,
hpl-1, hpl-2ts
, and hpl-2ts
/hpl-1 mutant eggs, the
survival of xpa mutant eggs was also quantified.
(D) Quantification of hatching and nonhatching
eggs after IR (40–120 Gy) relative to nonirradiated
eggs. Bars show the survival of wild-type eggs and
of hpl-1, hpl-2ts
, and hpl-2ts
/hpl-1 mutant eggs.
Representative assays performed in duplicate or in
quintuplicate are shown. For each assay, at least
120 animals were scored. Error bars indicate SD.
JCB • VOLUME 185 • NUMBER 4 • 2009 584
which is essential for DNA repair. In conclusion, our experiments
reveal an intriguing link between HP1 proteins and DNA
repair systems.
Materials and methods
Cell lines
Cell lines used in this study were HeLa, U2OS, CHOK1, NIH/3T3,
NIH/3T3 EGFP–HP1- (Mateos-Langerak et al., 2007), VH10 hTERT fibroblasts,
ATR-deficient GM18366-hTERT Seckel cells (Bergink et al., 2006),
Suv3-9h double-knockout MEFs (provided by T. Jenuwien, Research Institute
of Molecular Pathology, Vienna, Austria; Peters et al., 2001; Schotta
et al., 2004), and KU80-deficient XR-V15B CHO cells (Mari et al., 2006).
The NER-deficient SV40-immortalized cell lines were XP4PA (XP-C), XP20S
(XP-A), XP12RO (XP-A), XP23PV (XP-E), MEFs XPC/
, and CS1AN (CS-B).
All cell lines were cultured as described previously (Luijsterburg et al.,
2007). For immunolocalization experiments, hTERT human fibroblasts
were grown to confluency for 10 d. Subsequently, cells were synchronized
in G0 by keeping them for a minimum of 5 d in medium supplemented
with 0.2% FCS.
DNA constructs
HP1- and HP1- cDNA were ligated in frame with mRFP and SCFP3a.
EGFP–HP1- and EGFP–HP1- were provided by P. Hemmerich (Fritz
Lipmann Institute, Jena, Germany; Schmiedeberg et al., 2004). All constructs
were transiently transfected in various cell lines cells using Lipofectamine
2000 (Invitrogen). EGFP–HP1- was stably expressed in mouse NIH/3T3
cells (Mateos-Langerak et al., 2007), and EGFP–HP1- and EGFP–HP1-
were stably expressed in MRC5-SV cells. HP1- (∆CD) was tagged with
EGFP (Mateos-Langerak et al., 2007). EGFP–HP1- (∆CSD) and EGFP–HP1-
(∆hinge) were provided by T. Misteli (National Cancer Institute, Bethesda,
MD; Cheutin et al., 2003), and EYFP-tagged CSD was provided by Y. Hiraoka
(Osaka University, Osaka, Japan). HP1-T51A
was created by overlap PCR
and fused to EYFP. XPC and DDB2 were fused to mVenus. In addition, DDB2
was fused to mCherry (Luijsterburg et al., 2007). ACF1-EGFP was provided
by P.D. Varga-Weisz (Babraham Institute, Cambridge, England, UK). EYFPSuv3-9H1
was provided by R.W. Dirks (Leiden University Medical Center,
Leiden, Netherlands). NBS1-mCherry was provided by J. Lukas (Institute of
Cancer Biology and Centre for Genotoxic Stress Research, Copenhagen,
Denmark). The cDNAs for SCFP3a and mVenus were provided by J. Goedhart
(University of Amsterdam, Amsterdam, Netherlands), and mCherry and
mRFP cDNA were provided by R.Y. Tsien (University of California, San
Diego, La Jolla, CA).
UV-C irradiation
UV lamp–induced damage was inflicted using a UV source containing four
UV lamps (TUV 9W PL-S; Philips) as described previously (Moné et al.,
2004; Luijsterburg et al., 2007) or using a customized UV cross-linker (CL-
1000; UVP) containing two UV-C lamps. UV laser–induced damage was
inflicted by using a 2-mW pulsed (7.8 kHz) diode-pumped solid-state laser
emitting at 266 nm (Rapp OptoElectronic GmbH) as described previously
(Dinant et al., 2007).
Irradiation with x rays
We used an x-ray generator (150 kV; 15 mA; dose rate, 2.18 Gy/min;
HF160; Pantak) to irradiate cells globally as previously described (Syljuasen
et al., 2004).
Irradiation with soft x rays
Cells were plated in custom-made culture dishes containing an ultra-thin
Mylar bottom (Aten et al., 2004; Stap et al., 2008). The dishes were
placed on a soft x-ray source. We used a modified EG2 electron bombardment
evaporation source (VG Scienta), which was fitted with a carbon
anode to generate 277-eV photons. For detailed information about this type
of source, see Agarwal and Sparrow (1981). The source was operated at
3-kV electron energy and 8-mA electron current. The unit was mounted in
a vacuum chamber (P < 105
Torr) equipped with a Mylar film window
(2-µm thick), which was supported by a stainless steel grid (1-mm maze
size) to withstand atmospheric pressure. Cells were irradiated through a
metal filter (Stork Veco BV) with pores of 5 µm to inflict local damage. Cells
were irradiated for 6 min, corresponding with a dose of 20 Gy.
-Particle irradiation
Cells were cultured in carbon-coated Mylar dishes. Cells were irradiated
using an Americium (Am-241) source with an activity of 140 kBq at an
(40–120 Gy) and determined the survival of eggs. Remarkably,
we observed that hpl-1 animals are more resistant to IR than
wild-type animals (Fig. 5 D), suggesting that loss of HPL-1 is
beneficial for repair of these types of damages. Conversely, hpl-2ts
animals were extremely sensitive to IR, showing that HPL-2 is
essential for the response to IR. Interestingly, double-mutant
worms showed an intermediate phenotype, which is comparable
with wild-type worms. These results suggest that HPL-1 and
HPL-2 have opposing effects on IR sensitivity. It is tempting to
speculate that hpl-1 animals are more resistant to IR as a result
of an altered, possibly more accessible organization of heterochromatin
in these animals. This is reminiscent of a recent study
on HP1 in mammalian cells in which the total HP1 pool was reduced
(Goodarzi et al., 2008). However, our results also indicate
that loss of HPL-2 results in strong IR sensitivity, suggesting an
essential function in the DDR after chromosomal breaks. In conclusion,
it appears that HP1 proteins have partly redundant roles
in response to UV damage, whereas they seem to have unique
functions in response to IR. This reveals an essential role for the
HP1 proteins in response to UV-induced DNA damage and chromosomal
breaks, possibly through different mechanisms.
HP1 and the DDR
What is the molecular role of HP1 in the DDR? Our data suggest
that HP1 recruitment does not require DNA repair activity because
binding of HP1 at sites of UV lesions and DSBs is independent
of any of the known damage recognition proteins (Fig. 2 and
Fig. 4 C). In TCR, stalled RNA polymerase II initiates NER,
which could trigger binding of HP1 proteins (Mateescu et al.,
2008). However, HP1 proteins also accumulate at damaged sites
in cells in which transcription is blocked with -amanitin (unpublished
data), suggesting that HP1 proteins are recruited through
a damage detection system that is different from that for TCR and
GGR. HP1 binding depends on its CSD but not the CD or H3K9
trimethylation. It is possible that DNA damage-induced changes
in local chromatin structure are recognized by HP1 proteins. In
agreement with this, we show that HP1 accumulation persists in
repair-deficient cells in which lesions are not removed (Fig. 3 A).
ACF1 interacts with the CSD of HP1 and accumulates at UV lesions
(Fig. S3, F and G; Eskeland et al., 2007), suggesting that
this remodelling factor may cooperate with HP1 to modify chromatin
structure in damaged areas. However, HP1 recruitment was
still observed in cells depleted forACF1 (unpublished data). Consistent
with an essential role for HP1 in facilitating DNA repair,
we found that HP1-deficient nematodes are extremely sensitive to
UV-induced DNA damage. HP1 isoforms each distinctly contribute
to the sensitivity to UV- and IR-induced DNA damage, suggesting
divergent functions for HP1 family members to different
types of DNA damage. In support of this idea, neuronal cells derived
from HP1-–deficient mice but not HP1-–deficient animals
display genomic instability (Aucott et al., 2008). Recently,
it was shown that replication of pericentromeric heterochromatin
in mouse cells depends on binding of the p150 subunit of CAF-1
to the CSD of HP1 (Quivy et al., 2008). In analogy to these findings,
we favor a scenario in which HP1 proteins play a role in
reorganizing higher order chromatin structure, as recently suggested
by Kruhlak et al. (2006) and Solimando et al. (2009),
585HP1 IS RECRUITED TO DAMAGED DNA • Luijsterburg et al.
after IR and -particle irradiation and recruitment of ACF1 to local UV
damage. Online supplemental material is available at http://www.jcb
.org/cgi/content/full/jcb.200810035/DC1.
We thank Drs. P. Hemmerich, T. Misteli, R.W. Dirks, Y. Hiraoka, R.Y. Tsien,
J. Goedhart, P.D. Varga-Weisz, and T. Jenuwein for providing constructs and cell
lines and Dr. P. Singh for providing HP1 antibodies. We thank J. Verhoeven and
C. van Oven for technical assistance with soft x-ray experiments. We are grateful
to the Caenorhabditis Genetics Center and the Japanese National Bioresource
Project for hpl-1, hpl-2, and xpa-1 strains. We also thank Drs. E.M.M. Manders
and T.W.J. Gadella (Center for Advanced Microscopy, University of Amsterdam,
Amsterdam, Netherlands) for support.
This work was supported by ZonMW (grant 912-03-012 to R. van
Driel, A.B. Houtsmuller, L.H.F. Mullenders, and W. Vermeulen), the Netherlands
Organisation for Scientific Research (grant 2007/09198/ALW/
825.07.042 to M.S. Luijsterburg), the Danish National Research Foundation
(C. Dinant), the Swedish Research Counsel (N.P. Dantuma), the Dutch Cancer
Society (J. Stap and J.A. Aten), and the Association for International Cancer
Research (grant 08-0084 to H. Lans).
Submitted: 6 October 2008
Accepted: 22 April 2009
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Microscopic analysis
Cells were imaged with a confocal microscope (LSM 510 or LSM 510
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C. elegans UV-B and IR survival assay
The C. elegans strains used were Bristol N2 (wild type), hpl-1(tm1624),
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deletions), and xpa-1(ok698). The hpl-1(tm1624) and xpa-1(ok698) are
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dead eggs. Additional details are available upon request.
Online supplemental material
Fig. S1 shows validation of local UV irradiation and expression of
FP-tagged HP1. Fig. S2 shows that HP1 recruitment is independent of
H3K9me3 and the cell cycle. Fig. S3 shows increased binding of HP1
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