Molecular Cell
Article
A Cell Cycle-Dependent Regulatory Circuit
Composed of 53BP1-RIF1 and BRCA1-CtIP
Controls DNA Repair Pathway Choice
Cristina Escribano-Dı́az,1 Alexandre Orthwein,1 Amélie Fradet-Turcotte,1 Mengtan Xing,2 Jordan T.F. Young,1,3
,1,3 Michael A. Cook,1,4 Adam P. Rosebrock,4 Meagan Munro,1 Marella D. Canny,1 Dongyi Xu,2,*
Ján Tkác
and Daniel Durocher1,3,*
1Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada
Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, 100871 Beijing, China
3Department of Molecular Genetics
4Donnelly Centre for Cellular and Biomolecular Research
University of Toronto, Toronto, ON M5S 3E1, Canada
*Correspondence: [email protected] (D.X.), [email protected] (D.D.)
http://dx.doi.org/10.1016/j.molcel.2013.01.001
2State
SUMMARY
DNA double-strand break (DSB) repair pathway
choice is governed by the opposing activities of
53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes
end resection and homologous recombination (HR).
Here we show that 53BP1 is an inhibitor of BRCA1
accumulation at DSB sites, specifically in the G1
phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites,
and we identify RIF1 as the critical effector of 53BP1
during DSB repair. Remarkably, RIF1 accumulation
at DSB sites is strongly antagonized by BRCA1 and
its interacting partner CtIP. Lastly, we show that
depletion of RIF1 is able to restore end resection
and RAD51 loading in BRCA1-depleted cells. This
work therefore identifies a cell cycle-regulated
circuit, underpinned by RIF1 and BRCA1, that
governs DSB repair pathway choice to ensure that
NHEJ dominates in G1 and HR is favored from
S phase onward.
INTRODUCTION
DNA double-strand breaks (DSBs) are highly deleterious lesions.
Their inaccurate repair can lead to mutations, chromosome
translocations, or the generation of repair products that are toxic
to the cell (Chapman et al., 2012b). To avoid these adverse
outcomes, cells must choose from a group of mutually exclusive
DSB repair processes. This decision process, referred to as
DSB repair pathway choice, is critical for the maintenance of
genomic stability and must integrate information about cell cycle
position and the nature of the DNA end (Chapman et al., 2012b;
Symington and Gautier, 2011).
DSBs are repaired primarily by nonhomologous end joining
(NHEJ), homologous recombination (HR), or microhomology872 Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc.
mediated end joining (MMEJ) (Symington and Gautier, 2011).
Canonical NHEJ involves the Ku70/Ku80-dependent ligation of
broken DNA ends by DNA ligase IV. NHEJ operates throughout
the cell cycle but is particularly important during the G1 phase
of the cell cycle. NHEJ is also important for the development
of the immune system, as it is critical for V(D)J recombination
and immunoglobulin class switching (Soulas-Sprauel et al.,
2007).
NHEJ is only active on minimally processed DNA ends. As
a consequence, NHEJ is inhibited by end resection, the process
by which 50 -30 nucleolytic degradation generates singlestranded DNA (ssDNA) (Chapman et al., 2012b). Initiation of
DNA end resection by the CtIP/MRE11-RAD50-NBS1 (MRN)
complex removes Ku from DNA ends to generate ssDNA overhangs that are refractory to canonical NHEJ. End resection is
necessary for homology searching, which is central to HR, and
thus the decision to resect is fundamental to DSB repair pathway
choice (Symington and Gautier, 2011). Extensive end resection
is stimulated in the S/G2 phase of the cell cycle in a manner
that depends on CDK1 activity (Symington and Gautier, 2011).
CtIP is a major target for the cell cycle-dependent regulation of
resection, as the CDK-dependent phosphorylation of its T847
and S327 residues increases CtIP activity and interaction with
BRCA1, respectively (Huertas and Jackson, 2009; Yu and
Chen, 2004). CtIP and MRN are sufficient for short-range resection, but for the generation of longer ssDNA tracts, the activities
of the nucleases EXO1 and DNA2 are required, in collaboration
with BLM helicase (Symington and Gautier, 2011). The resulting
ssDNA is rapidly bound by replication protein A (RPA), which
is then replaced by RAD51 to form a nucleofilament that is
essential for homology searching and the subsequent steps of
the HR process (Symington and Gautier, 2011).
BRCA1 plays a critical, yet enigmatic role in HR. BRCA1
participates in HR by promoting DNA end resection (Schlegel
et al., 2006), but this activity is not universally observed. A major
clue as to the function of BRCA1 emerged when it was found that
the lethality, tumorigenesis, and genome instability associated
with BRCA1 deficiency can be rescued by the concomitant
loss of 53BP1 (Bouwman et al., 2010; Bunting et al., 2010).
Loss of 53BP1 promotes end resection and HR, which has led
Molecular Cell
RIF1 Is the 53BP1 Effector in DSB Repair
to a model speculating that the function of BRCA1 in DSB repair
is to antagonize 53BP1-dependent end protection.
Genetic studies in the avian DT40 cell line have firmly placed
53BP1 in the NHEJ pathway by demonstrating that 53bp1/
cells display G1-specific sensitivity to ionizing radiation (IR)
that is epistatic to a Ku70 gene deletion (Nakamura et al.,
2006). This role of 53BP1 in NHEJ is consistent with the observation that 53BP1 deletion in mouse results in a severe defect in
class-switch recombination (CSR), an NHEJ-dependent
process, which is accompanied by increased DNA end resection
at the IgH locus (Bunting et al., 2012; Manis et al., 2004; Ward
et al., 2004).
The accumulation of 53BP1 and BRCA1 at DSB sites is dependent on a common biochemical pathway controlled by the RNF8
and RNF168 E3 ubiquitin ligases (Al-Hakim et al., 2010). How
a common pathway promotes the recruitment of two seemingly
antagonistic factors to DSB sites is unknown. However, BRCA1
does not form IR-induced foci in G1 (Jin et al., 1997), leaving
53BP1 free to promote NHEJ during this stage of the cell
cycle. In S/G2, BRCA1 modulates the morphology of 53BP1
IR-induced foci (Chapman et al., 2012a), suggesting that the
molecular underpinnings of DSB repair pathway choice might
reside in the regulation of 53BP1 and BRCA1 accumulation at
DSB sites (Chapman et al., 2012b).
In the present study, we reexamined how cells restrict BRCA1
accumulation at DNA break sites in G1. We made the surprising
observation that BRCA1 recruitment to DSB sites is suppressed
by 53BP1 in the G1 phase of cycling cells. The mechanism of
BRCA1 inhibition by 53BP1 involves the phosphorylationdependent recruitment of RIF1 to DSB sites, which we identify
as the critical 53BP1 effector in NHEJ and DSB repair pathway
choice. Remarkably, the accumulation of RIF1 is G1-restricted
and is suppressed by BRCA1 and its interacting protein CtIP in
S/G2. RIF1 suppresses end resection and RAD51 nucleofilament
formation in BRCA1-deficient cells, identifying RIF1 as a candidate genetic modifier of BRCA1-deficient malignancies. Our
results suggest a model in which DSB repair pathway choice is
controlled by a cell cycle-regulated inhibitory circuit composed
of 53BP1-RIF1 and BRCA1-CtIP, the former winning the tussle
in G1 and the latter prevailing in S/G2. These results provide
a glimpse of the molecular logic underpinning DSB repair
pathway choice.
RESULTS
BRCA1 Is Present in the G1 Phase of Cycling Cells
Since BRCA1 is largely absent in the G0/G1 phase of contactinhibited cells (e.g., Chen et al., 1996) it had been assumed
that the regulation of BRCA1 protein levels explains the absence
of BRCA1 IR-induced foci in this phase of the cell cycle.
However, as these studies examined BRCA1 levels in cells that
had exited cycling (or that had resumed cycling after a prolonged
arrest), we sought to revisit the control of BRCA1 levels in the
G1 phase of an otherwise unperturbed cell cycle.
To purify G1 cells in cycling cultures, we took advantage of
the Fucci system, which is based on fluorescent proteins with
fragments of CDT1 and Geminin, for the G1 and S/G2 reporters,
respectively (Sakaue-Sawano et al., 2008) (Figure 1A). We
subjected HeLa-Fucci cells to fluorescence-activated cell sorting (FACS) to purify G1 and S/G2 cells (Figures 1B and S1A ).
Whole-cell extracts from asynchronously dividing and sorted
cells were prepared and probed for BRCA1 and cyclin A.
Analysis of cyclin A expression, which is restricted to S/G2
(Figure 1C), indicated that we prepared a G1 cell fraction largely
devoid of S/G2 cells. We found that BRCA1 is present in G1 at
levels similar to those of S/G2 and asynchronously dividing cells
(Figure 1C). Despite its presence, BRCA1 was unable to form
IR-induced foci in HeLa-Fucci cells in G1, even though g-H2AX
foci were readily observed (Figures 1D, 1E, and S1B, siCTRL
condition). These data suggest that the accumulation of
BRCA1 at DSB sites in G1 is inhibited in cycling cells. We
surmised that identifying the mechanisms leading to inhibition
of BRCA1 IR-induced foci in G1 would yield new clues as to
how cells regulate DSB repair pathway choice.
53BP1 Inhibits BRCA1 in G1
The inhibition of BRCA1 in G1 was reminiscent of the proposed
negative regulation of 53BP1 accumulation at DSB sites by
BRCA1 in S/G2. This raised the intriguing possibility that
53BP1 and BRCA1 may mutually inhibit each other. Therefore,
we tested whether 53BP1 acted as an inhibitor of BRCA1 IRinduced focus formation in G1. Strikingly, 53BP1 depletion
resulted in ectopic BRCA1 IR-induced foci in G1 cells in HeLaFucci, U2OS, and RPE1 cells (Figures 1D–1F and S2A–S2D).
The ectopic BRCA1 G1 foci were not dependent on the Fucci
markers since they could be seen by selecting cyclin A-negative
cells, which are enriched for G1 cells (Figure 1F); the phenotype
was seen with an independent BRCA1 antibody (Figure S2A) and
was observed with multiple siRNAs against 53BP1 (Figures S2A–
S2D). Importantly, the ectopic BRCA1 IR-induced foci caused
by 53BP1 depletion were dependent on RNF8 (Figure S2E),
and we could restore the inhibition of BRCA1 accumulation at
DSBs by reintroducing siRNA-resistant 53BP1 into the depleted
cells (Figures 1G, S2F, and S2G), ruling out the possibility that
our observations were caused by off-target effects of the
siRNAs. We conclude that 53BP1 antagonizes BRCA1 accumulation at DSB sites in G1.
To gain insight into the mechanism of BRCA1 inhibition by
53BP1, we undertook structure-function studies (Figure 1G).
Cells were first depleted of endogenous 53BP1 and reconstituted with various siRNA-resistant 53BP1 vectors that expressed proteins at similar levels (Figures S2F and S2G). We
found that the suppression of BRCA1 foci in G1 was independent
of the 53BP1 C terminus (i.e., residues 1712–1972), which
comprises the tandem BRCT domains, but was dependent on
the ability of 53BP1 to accumulate at DSB sites since the
D1521R mutant, which mutates the Tudor domain, was unable
to restore inhibition of BRCA1 foci in G1 (Figures 1G and S2G
and Table S1). Interestingly, deletion of the N-terminal half of
the protein up to residue 1220, in the context of the C-terminal
deletion, failed to inhibit BRCA1 foci in G1 despite being able
to form IR-induced foci (Figures 1G and S2G). The N terminus
of 53BP1 is phosphorylated by ATM and is rich in its S/TQ
consensus sites. 53BP1 phosphorylation by ATM plays an
important role in promoting DSB repair during immunoglobulin
class switching (Bothmer et al., 2011). Strikingly, we found that
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RIF1 Is the 53BP1 Effector in DSB Repair
Figure 1. BRCA1 Accumulation at DSB
Sites Is Suppressed by 53BP1 in G1
(A) Schematic representation of the Fucci system.
(B) Strategy for isolation of G1 and S/G2 cells by
fluorescence-activated cell sorting (FACS) of
Fucci cells. See Figure S1 for details.
(C) BRCA1 is expressed in the G1 and S/G2 phases of cycling cells. Whole-cell extracts were
prepared from sorted Fucci cells and probed for
BRCA1, cyclin A (S/G2 marker), and tubulin
(loading control) by immunoblotting.
(D) 53BP1 inhibits BRCA1 foci in G1. HeLa-Fucci
cells were first transfected with a nontargeting
siRNA (siCTRL) or an siRNA targeting 53BP1
(si53BP1) and then processed for BRCA1
immunofluorescence 1 hr postirradiation (10 Gy).
Indicated is the phase of the cell cycle. Scale
bar = 5 mm.
(E) Quantitation of BRCA1 and g-H2AX foci according to cell cycle position. HeLa-Fucci cells
were transfected with the indicated siRNAs and
irradiated with a 10 Gy IR dose. Cells were then
fixed and processed for BRCA1 and g-H2AX
immunofluorescence. Shown is the quantitation of
IR-induced foci in G1 and S/G2 cells. Data are
represented as the mean ± SEM (n = 3).
(F) 53BP1 inhibits BRCA1 G1 foci in multiple cell
lines. U2OS and RPE1 cells were transfected with
the indicated siRNAs, fixed 1 hr postirradiation
(10 Gy), and processed for BRCA1 and cyclin A
immunofluorescence. Shown in the left panel is
the quantitation of foci in the cyclin A-negative
cells. Data are represented as the mean ± SEM
(n = 2). In the right panel are representative
micrographs. Scale bar = 5 mm.
(G) Top: schematic representation of 53BP1.
Bottom: HeLa-Fucci cells were first transfected
with a single siRNA targeting 53BP1. After transfection, cells were transfected either with an
empty vector control (ctrl) or the indicated siRNAresistant 53BP1 expression vectors. Cells were
irradiated (10 Gy dose) and processed for BRCA1
immunofluorescence. Data are represented as the
mean ± SEM (n > 3). Representative micrographs
are shown in Figure S2.
the ATM site-deficient 53BP128A mutant (Bothmer et al., 2011)
was unable to inhibit BRCA1 accumulation at DSB sites in G1
despite being able to form IR-induced foci (Figures 1G and
S2G). These results suggested that 53BP1 phosphorylation by
ATM plays a key role in blocking BRCA1 accumulation at DSB
sites. Together, the domains of 53BP1 necessary to inhibit
BRCA1 foci in G1 are identical to those required to promote
DSB repair (Bothmer et al., 2011) (Table S1). Interestingly, the
ectopic BRCA1 foci seen upon 53BP1 depletion occurred independently of CtIP (Figure S2H), suggesting that 53BP1 blocks
BRCA1 accumulation at DSB sites in G1 independently of its
ability to regulate DNA end resection.
RIF1 Acts Downstream of 53BP1 to Inhibit BRCA1 in G1
The importance of the ATM sites in 53BP1 suggests that 53BP1
may carry out its function via an effector protein, as these phosphorylation sites are dispensable for the accumulation of 53BP1
at DNA damage sites. In a search for effectors of 53BP1, we
874 Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc.
considered RIF1 as a candidate since it is a recognized but
poorly understood genome stability factor that accumulates at
DSB sites in a manner that depends on both 53BP1 and ATM
kinase activity (Silverman et al., 2004; Xu and Blackburn,
2004). We first confirmed that RIF1 accumulation at DSB sites
is dependent on the RNF8, RNF168, and 53BP1 proteins and
independent of BRCA1 (Figures S3A and S3B).
To test the contribution of RIF1 to the inhibition of BRCA1 in
G1, we depleted RIF1 with four independent RIF1 siRNAs in
HeLa-Fucci cells and monitored BRCA1 IR-induced focus
formation. We found that RIF1 depletion with all four siRNAs resulted in ectopic BRCA1 foci in G1 to levels approaching that of
53BP1 knockdown (Figures 2A, 2B, and S3C). Furthermore, reintroduction of an siRNA-resistant GFP-RIF1 expression construct in RIF1-depleted cells restored the inhibition of BRCA1
recruitment to DSB sites in G1, excluding the possibility that
the ectopic BRCA1 foci in G1 are caused by an off-target effect
of the RIF1 siRNAs (Figures 2C and S3D). The 53BP1-dependent
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RIF1 Is the 53BP1 Effector in DSB Repair
Figure 2. RIF1 Is an Effector of 53BP1 in the
Inhibition of BRCA1 in G1
(A) RIF1 inhibits BRCA1 foci in G1. HeLa-Fucci
cells were transfected with the indicated siRNAs.
At 1 hr postirradiation (10 Gy), cells were processed for BRCA1 immunofluorescence. Shown
is the quantitation of BRCA1 IR-induced foci in
G1 cells. Data are represented as the mean ± SEM
(n = 3). Representative micrographs are shown in
Figure S3C.
(B) Immunoblot analysis of the experiment shown
in (A).
(C) U2OS cells were transfected with the indicated
siRNAs. Cells were transfected with either a GFP
(control) or an siRNA-resistant GFP-RIF1 expression vector 24 hr later. At 1 hr postirradiation (10
Gy), cells were processed for BRCA1 and cyclin
A immunofluorescence. Shown is the quantitation
of BRCA1 foci in the cyclin A-negative (i.e., G1)
cells. Data are represented as the mean ± SEM
(n = 3). Representative micrographs are shown in
Figure S3D.
(D) U2OS cells were transfected with si53BP1 #1
and either an empty vector control (ctrl) or the
indicated siRNA-resistant 53BP1 expression
vectors. At 1 hr postirradiation (10 Gy), cells were
processed for RIF1 immunofluorescence. Shown
are representative micrographs. Scale bar = 5 mm.
(E) Quantitation of the experiments shown in (D).
Data are represented as the mean ± SEM (n = 3).
(F) ATM inhibits RIF1 foci. U2OS cells were treated
either with DMSO or various concentrations of the
ATM inhibitor KU-55933 prior to irradiation. At 1 hr
postirradiation (10 Gy), cells were processed for
53BP1 and RIF1 immunofluorescence. Data are
represented as the mean ± SEM (n = 3).
(G) U2OS cells were transfected with the indicated
siRNAs. Cells were irradiated (10 Gy) at 48 hr
and processed for 53BP1 immunofluorescence
1 hr postirradiation. Data are represented as the
mean ± SEM (n = 3). Representative micrographs
are shown in Figure S3E.
accumulation of RIF1 at DSB sites, coupled with our finding that
its depletion restores BRCA1 IR-induced foci in G1, placed RIF1
as a strong candidate for the 53BP1 effector in the DSB
response.
If RIF1 is a true effector of 53BP1, its accumulation at DSB
sites should be dependent on the same domains of 53BP1
shown to be important for its function as a DSB repair protein.
RIF1 accumulation at DSB sites was independent of the 53BP1
C terminus but dependent on both its N-terminal region and its
Tudor domain (Figures 2D and 2E and Table S1). The 53BP1
ATM consensus phosphorylation sites were also critical for
RIF1 accumulation at DSB sites, thus likely explaining why
ATM is required for RIF1 IR-induced focus formation (Silverman
et al., 2004). In further support of this possibility, RIF1 recruitment to DSB sites was much more sensitive to ATM inhibition
than was that of 53BP1, implying that ATM acts at a step
between 53BP1 and RIF1 (Figure 2F). Finally, RIF1 depletion
by two efficient siRNAs did not impact 53BP1 focus formation
after irradiation (Figures 2G and S3E). Together, these results
suggest that RIF1 is the effector of 53BP1 in the DSB response.
ATM-Phosphorylated 53BP1 Binds to RIF1
RIF1 has previously been detected in 53BP1 immunoprecipitates (Huen et al., 2010), strongly suggesting that 53BP1 might
recruit RIF1 to DSB sites via a physical interaction. We confirmed
the 53BP1-RIF1 interaction in coimmunoprecipitation studies
from 293T cells expressing Flag-53BP1 and GFP-RIF1 proteins
(Figure 3A). The 53BP1-RIF1 interaction was stimulated by IR
treatment and was completely abrogated by the pharmacological inhibition of ATM (Figure 3A). Furthermore, the 53BP1-RIF1
interaction was dependent on the ATM phosphorylation sites
on 53BP1, since the 53BP128A protein failed to retrieve RIF1
from extracts prepared from irradiated cells (Figure 3B).
Together, these results indicate that RIF1 is recruited to DSB
sites through the recognition of ATM-phosphorylated 53BP1.
Inhibition of BRCA1 IR-Induced Foci by RIF1 Is Not Due
to Competition for Binding to DSB Sites
RIF1 is a large protein (Figure 3C) organized in two distinct
regions: an N-terminal domain (residues 1–967) composed
largely of a-helical HEAT repeats and a C-terminal region
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RIF1 Is the 53BP1 Effector in DSB Repair
Figure 3. RIF1 Interacts with ATM-Phosphorylated 53BP1 through Its N-Terminal
HEAT Repeats
(A) 293T cells were transfected with GFP-RIF1 and
Flag-53BP1 expression vectors as indicated. At
24 hr posttransfection, cells were treated with
either DMSO or 10 mM of the ATM inhibitor
KU-55933 for 2 hr prior to irradiation. At 1 hr
postirradiation (10 Gy), nuclear extracts were
prepared and 53BP1-containing complexes
were immunoprecipitated using anti-Flag (M2)
magnetic beads and then analyzed by immunoblotting using RIF1 and 53BP1 antibodies.
(B) 293T cells were transfected with GFP-RIF1 and
either wild-type (WT) Flag-53BP1 or Flag53BP128A expression vectors. After 24 hr cells
were irradiated (10 Gy). At 1 hr postirradiation,
nuclear extracts were prepared, immunoprecipitated with anti-Flag (M2) magnetic beads, and
immunoblotted with RIF1 and 53BP1 antibodies.
(C) Schematic representation of RIF1.
(D) RIF1 requires its N-terminal HEAT repeats to
localize to DSBs. U2OS cells were first transfected
with siRIF1 #2 and 24 hr later were transfected
with the indicated siRNA-resistant GFP-RIF1
expression vectors. Cells were processed for
RIF1 immunofluorescence 1 hr postirradiation
(10 Gy). Data are represented as the mean ± SEM
(n = 3). Representative micrographs are shown
in Figure S4A.
(E) RIF1 C-terminal domain is required to inhibit
BRCA1 foci in G1. U2OS cells were transfected with siRIF1 #2 and with either an empty vector control (GFP) or the indicated siRNA-resistant GFP-RIF1
expression vectors. At 1 hr postirradiation (10 Gy), cells were processed for BRCA1 and cyclin A immunofluorescence. Data are represented as the mean ± SEM
(n = 3). Representative micrographs are shown in Figure S4B.
(residues 2170–2446) that promotes RIF1 oligomerization and
interaction with protein phosphatase 1 (PP1), DNA, and the
BLM helicase (Silverman et al., 2004; Sreesankar et al., 2012;
Xu et al., 2010). To gain insight into the mechanism by which
RIF1 inhibits BRCA1 in G1, we first examined which regions of
RIF1 were involved in its recruitment to DSB sites. We found
that while the C-terminal region was dispensable for accumulation at DSB sites (Figures 3D and S4A), the N-terminal repeats
were required for IR-induced focus formation (Figures 3D and
S4A). From these results, we conclude that RIF1 requires its
N-terminal HEAT repeats to accumulate at DSB sites.
Next, to test whether the accumulation of RIF1 at DSB sites
was sufficient to block BRCA1 IR-induced foci in G1, we introduced various siRNA-resistant RIF1 expression vectors in cells
depleted of the endogenous protein. We found that in addition
to the HEAT repeats, which are required for RIF1 recruitment
to breaks, the extreme C terminus of RIF1, not including the
PP1-binding RVxF motif, was required for inhibiting BRCA1 G1
foci (Figures 3E and S4B). From these results, we conclude
that RIF1 does not simply inhibit BRCA1 accumulation at DSB
sites through competition for binding sites, as sequences
outside its DSB-recruitment domain are necessary to block
BRCA1 in G1.
RIF1 Promotes Class-Switch Recombination
The above results were surprising since 53BP1 plays a welldescribed role in NHEJ, whereas the genetic analysis of RIF1
876 Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc.
suggests a role in replication fork stability (Buonomo et al.,
2009; Xu et al., 2010). We therefore tested whether RIF1 participates in NHEJ. We first examined the role of RIF1 in CSR
since murine 53bp1 deletion causes a profound defect in class
switching. We used two independent cell systems (Figure 4A),
CH12F3-2 cells as well as primary murine B cells isolated from
C57/BL6 mice. The CH12F3-2 cell line is derived from an IgM+
murine B cell lymphoma (Nakamura et al., 1996) and is increasingly used as a model to study CSR. CSR in CH12F3-2 cells
is also dependent on 53BP1 (Ramachandran et al., 2010). We
transduced CH12F3-2 cells with nontargeting (CTRL) and
multiple independent shRNA-containing retroviruses targeting
either RIF1 or 53BP1. The efficiency of RIF1 and 53BP1 knockdown was assessed by immunoblotting and showed varying
degrees of depletion (Figure 4B). Class switching in controltransduced cells was robust in both systems with 27.4% of
CH12F3-2 cells switched to IgA at 24 hr postinduction (Figure 4C)
and 16% of primary B cells switched to IgG1. RIF1 depletion
impaired CSR to an extent that approached that of 53BP1
depletion in both B cell models (Figures 4C–4E). Defective
CSR was seen with all four shRNAs, and the magnitude of
the defect correlated with the extent of RIF1 knockdown,
excluding the possibility of an off-target effect. The CSR defect
caused by RIF1 depletion was not due to a defect in cell proliferation (Figures S5A and S5B) or to a defect in AID (Aicda) or
IgA germline transcript (GLT IgA) expression (Figure 4F).
Together, these results strongly suggest that RIF1 regulates
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RIF1 Is the 53BP1 Effector in DSB Repair
Figure 4. RIF1 Promotes Class-Switch Recombination
(A) Schematic summarizing the strategy used to assay CSR in the murine CH12F3-2 B cell line and in primary B cells.
(B) Whole-cell extracts derived from CHF12F3-2 (left) and primary (right) B cells transduced with the indicated shRNA-expressing retroviral vectors were analyzed
by immunoblotting.
(C) Representative flow cytometric profiles of shRNA-transduced CH12F3-2 cells untreated (no induction) or stimulated with a cocktail of cytokines for 1 day and
stained with anti-IgA antibody. The proportion of IgA+ cells is indicated in each histogram.
(D) Class switching to IgA in shRNA-transduced CH12F3-2 cell lines. The proportion of IgA+ cells was determined by flow cytometry and plotted relative to the
IgA+ proportion in nontargeting shRNA transduced cells (shCTRL, 100%). Data are expressed as the mean ± SD from two independent inductions performed in
duplicate.
(E) Class switching to IgG1 in shRNA-transduced primary B cells. The proportion of IgG1+ cells was determined as above. Data are expressed as mean ± SD from
two independent inductions performed in duplicate.
(F) AID (Aicda) mRNA and IgA germline transcript (GLT IgA) levels were estimated by RT-PCR using 2-fold serial dilutions of cDNA made from activated CH12F3-2
cells transduced with the different shRNA constructs as indicated. Gapdh was used as a control for transcript expression.
CSR at the level of DSB repair, consistent with its potential role
as a 53BP1 effector.
RIF1 Is Involved in Canonical NHEJ
Next, we sought to obtain an orthogonal validation of the
role of RIF1 in NHEJ. We noted that the deletion of Rif1 in
the avian DT40 cell line results in an increase in the efficiency
of gene targeting, consistent with a role for RIF1 in promoting the use of NHEJ over HR (Xu et al., 2010). We first
examined the frequency of random plasmid integration in
DT40 cells containing Rif1/, Ku70/, or the double Rif1/
Ku70/ mutations. We found that deletion of Rif1 reduced
random integration of a linearized plasmid to 22% of wildtype levels (Figure 5A). Random plasmid integration was
strongly suppressed in Ku70/, as expected, but was not
further exacerbated in the Rif1/ Ku70/ double mutant,
consistent with a role for RIF1 in the canonical NHEJ pathway
(Figure 5A).
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RIF1 Is the 53BP1 Effector in DSB Repair
We next employed the same DT40 cell lines to study the
impact of Rif1 deletion on survival following DSB induction (Xu
et al., 2010). We examined the sensitivity of Rif1/, Ku70/,
and the Rif1/ Ku70/ DT40 clones to the radiomimetic agent
bleomycin. We observed that Rif1/ cells were sensitive
to bleomycin, albeit less so than Ku70/ cells (Figure 5B).
However, the cellular sensitivity of Rif1/ cells to bleomycin
was epistatic to Ku70/ (Figure 5B), placing RIF1 in the canonical NHEJ pathway. Next, we tested whether Rif1/ was
epistatic to a 53bp1/ mutation by examining the bleomycin
sensitivity of the single and double mutants. As expected, the
Rif1/ 53bp1/ cells were no more sensitive than the single
53bp1/ mutant, consistent with RIF1 acting downstream of
53BP1 (Figure 5C). Finally, we examined the clonogenic survival
of Rif1/ cells following IR in the G1 phase of the cell cycle since
deletion of 53bp1 in DT40 cells results in a G1-specific hypersensitivity to IR that is epistatic to the Ku70 deletion (Nakamura
et al., 2006). We found that the Rif1 deletion resulted in G1specific hypersensitivity to IR (Figures 5D and S5C), strongly
indicating that RIF1 is critical for the repair of a subset of DSBs
by the canonical, Ku-dependent NHEJ pathway.
53BP1 promotes NHEJ at the expense of HR by gene conversion, MMEJ, and single-strand annealing (SSA), a RAD51independent homologous recombination pathway. We therefore
tested whether depletion of RIF1, by two independent siRNAs,
also led to an increase in SSA, HR, and MMEJ, as monitored
by the SA-, DR-, and EJ2-GFP reporters, respectively. We found
that RIF1 depletion, like that of 53BP1, led to an increase in
SSA (Figure 5E), HR (Figures 5F and S5D), and MMEJ (Figure 5G).
We conclude that RIF1, like 53BP1, promotes the commitment
of ends toward the canonical end-joining pathway over other
available DSB repair pathways.
Figure 5. RIF1 Promotes NHEJ
(A) DT40 cell lines of the indicated genotypes were transformed with the
pLoxBsr plasmid and clonogenic potential was determined in the presence of
blasticidin. Data are expressed as the mean number of blasticidin resistant
(BsdR) colonies ± SEM (n = 3).
(B) DT40 cell lines of the indicated genotypes were treated with increasing
doses of bleomycin and survival (relative to the no-drug condition) was
determined by MTT staining. Data are represented as the mean ± SEM (n = 3).
(C) DT40 cell lines of the indicated genotypes were treated with increasing
doses of bleomycin and survival (relative to the no-drug condition) was
determined by MTT staining. Data are represented as the mean ± SEM (n = 3).
(D) Clonogenic survival of wild-type or Rif1/ DT40 cells after X-irradiation
treatment. Data are represented as the mean ± SEM (n = 4). DNA content
analysis of the G1 culture is shown in Figure S5C.
(E) U2OS SA-GFP cells were transfected with the indicated siRNAs. At 24 hr
posttransfection, cells were transfected with the I-SceI expression plasmid,
and the GFP+ population was analyzed 72 hr post-plasmid transfection. The
percentage of GFP+ cells was determined for each condition and was
normalized with the nontargeting (siCTRL) condition. Data are presented as
the mean ± SEM (n = 3).
(F) HeLa or U2OS DR-GFP cells were transfected with the indicated siRNAs.
At 24 hr posttransfection, cells were transfected with the I-SceI expression
plasmid and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each condition and
was normalized with the nontargeting (siCTRL) condition. Data are presented
as the mean ± SEM (n > 3).
(G) U2OS EJ2-GFP cells were processed as in (E). Data are presented as the
mean ± SEM (n = 3).
878 Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc.
BRCA1-CtIP Inhibits RIF1 in S/G2
Depletion of BRCA1 leads to a noticeable increase in RIF1 IRinduced foci (Figures S3A and S3B), raising the possibility that
BRCA1 may antagonize the localization of RIF1 at break sites.
We therefore irradiated Fucci-HeLa cells and monitored RIF1
focus formation. In control-transfected cells, RIF1 IR-induced
foci were largely limited to G1 cells (Figures 6A and 6B). In stark
contrast, BRCA1 depletion led to an upregulation of RIF1 foci
in S/G2 cells (Figures 6A and 6B). This phenotype was much
more striking than the partial antagonism of BRCA1 on 53BP1,
which is barely seen with conventional microscopy (Chapman
et al., 2012a).
The reintroduction of an siRNA-resistant Flag-BRCA1
expression vector into BRCA1-depleted cells restored the inhibition of RIF1 accumulation at DSB sites in S/G2 (Figures 6C
and S6A). This observation enabled us to probe which activity
of BRCA1 is required to restrict RIF1 recruitment at break sites
in G1. We examined both the I26A mutation of BRCA1, which
disrupts its E3 ligase activity without impairing its interaction
with BARD1 (Brzovic et al., 2003), and the BRCA1 S1655A
mutation, which disrupts its interaction with the CtIP, BACH1/
FANCJ, and Abraxas (ABRA1) proteins. The BRCA1I26A mutant
was fully competent in inhibiting RIF1 accumulation at DSB
sites in S/G2 (Figures 6C and S6A), suggesting that BRCA1
does not use its E3 ligase activity to inhibit RIF1. In stark
Molecular Cell
RIF1 Is the 53BP1 Effector in DSB Repair
Figure 6. RIF1 Is Inhibited by BRCA1-CtIP in S/G2
contrast, reintroduction of BRCA1S1655A failed to inhibit RIF1
foci in S/G2 (Figures 6C and S6A). The importance of BRCTdependent protein interactions for the ability of BRCA1 to
inhibit RIF1 in S/G2 suggests that BRCA1 may require itself
to be concentrated at DSB sites, or that it needs to recruit an
effector protein.
To search for the BRCA1 effector for RIF1 IR-induced foci
inhibition, we depleted HeLa-Fucci cells of the known BRCA1
BRCT-interacting proteins BACH1/FANCJ, CtIP, and ABRA1.
We also depleted PALB2, which interacts with BRCA1 independently of the BRCTs. Remarkably, only the depletion of CtIP
resulted in ectopic RIF1 foci in S/G2 (Figures 6D, S6B, and
S6C). Together, these results suggest that CtIP forms a complex
with BRCA1 to block the recruitment of RIF1 at sites of DNA
lesions from S phase onward.
To test this possibility further, we reintroduced siRNAresistant CtIP and its S327A or T487A mutants. Both are important CDK target sites on CtIP, with the former promoting
interaction with BRCA1 and the latter being involved in upregulating the end-resection activity of CtIP. Remarkably, while
wild-type CtIP largely restored the inhibition of RIF1 foci in
S/G2, cells were insensitive to the reintroduction of the S327A
or T487A mutants (Figures 6E and S6D) indicating that the
formation of the BRCA1-CtIP complex along with its activation
by CDK phosphorylation contribute to RIF1 inhibition in S/G2.
Lastly, we asked whether the expression of the CtIPT847E
mutant, which mimics constitutive phosphorylation of T487,
was able to inhibit RIF1 IR-induced focus formation in G1. As
a control, we used CtIPT847A. We found that CtIPT847E, but not
CtIPT847A, nearly halved the percentage of G1 cells with RIF1
IR-induced foci (Figures 6F and S6E). From these results, we
conclude that the negative regulation of RIF1 accumulation at
DSB sites is stimulated by CDK-dependent phosphorylation of
CtIP and that it likely involves the initiation of end resection.
(A) BRCA1 inhibits RIF1 foci in S/G2. HeLa-Fucci cells were first transfected
with the indicated siRNAs. At 48 hr posttransfection, cells were irradiated
(10 Gy) and then processed for RIF1 immunofluorescence. Left: representative
micrographs. Scale bar = 5 mm. Right: quantitation of the RIF1 foci in G1 and
S/G2. Data are represented as the mean ± SEM (n = 3).
(B) Immunoblot analysis of the cultures in (A).
(C) U2OS cells were first transfected with siBRCA1 and then transfected with
control (ctrl) or siRNA-resistant Flag-BRCA1 expression vectors. At 1 hr
postirradiation (10 Gy), cells were processed for Flag, RIF1, and cyclin A
immunofluorescence. Shown is the quantitation of RIF1 foci in cyclin
A-positive cells (i.e., in S/G2). Data are represented as the mean ± SEM (n > 3).
Representative micrographs are shown in Figure S7A.
(D) HeLa-Fucci cells were transfected with the indicated siRNAs and processed as in (A). Shown is the quantitation of RIF1 foci in S/G2 cells. Data are
represented as the mean ± SEM (n > 3). Representative micrographs and
control immunoblots are shown in Figures S7B and S7C.
(E) U2OS cells were first transfected with a control siRNA (NT) or an siRNA
against CtIP and then with an empty vector control (GFP) or the indicated
siRNA-resistant expression vector. Cells were then irradiated (10 Gy) and
processed for RIF1 and cyclin A immunofluorescence. Shown is the quantitation of RIF1 foci in GFP- and cyclin A-positive cells (i.e., in S/G2). Data are
represented as the mean ± SEM (n = 3). Representative micrographs are
shown in Figure S6D.
(F) U2OS cells were transfected with an empty GFP vector control (GFP) or
the indicated GFP-tagged CtIP mutants. At 24 hr posttransfection, cells were
irradiated (10 Gy) and processed for RIF1 and cyclin A immunofluorescence.
Shown is the quantitation of RIF1 foci in GFP+ G1 cells. Data are represented
as the mean ± SEM (n = 3). See Figure S6E for micrographs.
Loss of RIF1 Suppresses BRCA1 Deficiency Phenotypes
Since RIF1 accumulates at DSB sites in S/G2 when BRCA1 is
depleted, we examined whether the loss of RIF1 could rescue
the impaired end resection imparted by BRCA1 depletion. To
do so, we adapted a cytometric assay that examined RPA32
accumulation on chromatin (Forment et al., 2012), except our
assay was carried out in plates and read by a laser-scanning
cytometer (Figure S7A). Since the role of BRCA1 in promoting
end resection is somewhat controversial, we first examined
whether BRCA1 depletion resulted in an end-resection defect
following treatment with neocarzinostatin (NCS), a radiomimetic
agent. The positive control for this assay was depletion of
CtIP. We observed that BRCA1 depletion resulted in a quantifiable defect in RPA accumulation on chromatin that was
much milder than the effect of CtIP depletion on end resection
(Figure 7A).
Depletion of either 53BP1 or RIF1 (the latter with two independent siRNAs) had little impact on DNA end resection.
Remarkably, we observed that 53BP1 and RIF1 depletion
resulted in a near complete rescue of the end-resection defect
imparted by BRCA1 depletion (Figure 7A). The knockdown of
53BP1 or RIF1 did not rescue the impaired end resection
of CtIP-depleted cells (Figure 7A), indicating that the loss of
Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc. 879
Molecular Cell
RIF1 Is the 53BP1 Effector in DSB Repair
Figure 7. RIF1 Inhibits End Resection and
RAD51 Nucleofilament Formation in
BRCA1-Depleted Cells
(A) Quantitation of end resection in the indicated
siRNA-transfected U2OS cells after NCS treatment. Shown is the quantitation of the percentage
of RPA32-positive cells (see Figure S7A for the
gating scheme). Data are presented as floating
bars with the line at the median. Significance
(***p < 0.001 or ns, no significance) was determined by one-way ANOVA.
(B) U2OS cells were first transfected with the
indicated combination of siRNAs. At 3 hr postirradiation (10 Gy), cells were processed for
RAD51 and g-H2AX immunofluorescence. Shown
is the quantitation of RAD51 foci-positive cells.
Data are represented as the mean ± SEM (n = 3).
(C) Representative micrographs for the experiment shown in (B). Scale bar = 5 mm.
(D) DT40 cell lines of the indicated genotypes
were treated with an increasing concentration of
PARP inhibitor (PARPi), and survival was determined by MTT staining. Data are represented as
the mean ± SEM (n = 3).
(E) Model of the regulatory circuit controlling DSB
repair pathway choice. See Discussion for details.
53BP1-RIF1 does not lead to the upregulation of a CtIP-independent resection activity. We conclude from these results that
the defective resection associated with BRCA1 depletion is at
least in part caused by the maintenance of RIF1 at sites of
DNA damage in S/G2.
Next, we examined whether RIF1 depletion could phenocopy
53BP1 deficiency and rescue the RAD51 nucleofilament
assembly defect observed in BRCA1-deficient cells (Figures
7B, 7C, S7B, and S7C). Using two independent siRNAs, we
found that RIF1 depletion restored RAD51 IR-induced foci in
BRCA1-depleted cells to levels equivalent to that which is seen
with 53BP1 depletion (Figures 7B and 7C). Furthermore, using
DT40 cells, we found that the Rif1/ mutation also restored
RAD51 IR-induced foci in Brca1/ cells (Figures S7B and
S7C). Together, these results are consistent with RIF1 blocking
end resection and RAD51 nucleofilament formation in the
absence of BRCA1.
880 Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc.
Finally, the generation of the single
Rif1/ and Brca1/ and the double
Rif1/ Brca1/ mutant DT40 lines
enabled us to test whether RIF1
promotes toxic DNA repair in the absence
of BRCA1. We therefore tested whether
the Rif1 deletion suppresses the profound PARP inhibitor (PARPi) sensitivity
of BRCA1-deficient cells. As shown in
Figure 7D, the deletion of Rif1 leads to
a mild suppression of PARPi sensitivity.
Since impaired HR is at the root of the
sensitivity to PARP inhibition, these
results indicate that RIF1 not only blocks
end resection and RAD51 nucleofilament
formation in BRCA1-deficient cells, but also inhibits HR to
some extent.
DISCUSSION
The process by which cells commit to a DSB repair pathway is
of paramount importance for the maintenance of genomic
integrity. For example, the inappropriate engagement of the
NHEJ pathway following replication fork collapse is associated
with genome rearrangements and cell death in a variety of
contexts (Saberi et al., 2007). While the stability of DNA ends is
known to be the ultimate arbiter of this process (Chapman
et al., 2012b; Symington and Gautier, 2011), it was not clear if
the initial commitment to resection is controlled at the DSB
site. In this study, we provide evidence of a regulatory circuit,
operating at DSB sites and comprising 53BP1-RIF1 and
BRCA1-CtIP, which influences DSB repair pathway choice.
Molecular Cell
RIF1 Is the 53BP1 Effector in DSB Repair
In particular, we identify RIF1 as the effector of 53BP1 during
DSB repair. Our results indicate that RIF1 is recruited to DSB
sites by interacting with ATM-phosphorylated 53BP1. Furthermore, loss-of-function studies in human, mouse, and avian cells
indicate that RIF1 plays a critical role during CSR and in the
repair of a subset of DSBs by the canonical NHEJ pathway,
acting downstream of 53BP1.
RIF1 is also the effector of 53BP1 with respect to restricting
BRCA1 accumulation at DSB sites to the S/G2 phases of the
cell cycle. Interestingly, the ectopic BRCA1 IR-induced foci in
G1 cells are formed independently of CtIP and therefore independently of end resection. We conclude that the ability of
53BP1-RIF1 to oppose BRCA1 does not reflect a loss in end
protection per se, but rather that those DNA ends are no longer
committed to end joining. This activity of RIF1 requires both its
recruitment to DSBs and its extreme C terminus, suggesting
that RIF1 does not antagonize BRCA1 simply via competition.
It will be interesting in the future to determine exactly how the
RIF1 C terminus is able to antagonize BRCA1, since it may
instruct us about how chromatin might be marked to channel
some ends toward the NHEJ pathway.
Together, our observations are consistent with a model, which
we refer to as the ‘‘real estate’’ model, where DSB repair pathway
selection is, at least in part, determined by which of RIF1 or
BRCA1-CtIP occupy the chromatin that surrounds the DSB site
(Figure 7E). We propose that RIF1 stimulates canonical NHEJ,
whereas the BRCA1-CtIP complex marks the chromatin to
render it permissive to end resection. The outcome of this battle
for real estate at the break site is ultimately influenced by cell
cycle position. In G1, RIF1 is able to keep BRCA1 at bay. Exactly
how RIF1 is able to oppose BRCA1 is unknown, but one possibility could be that RIF1 modulates the ability of BRCA1 to recognize ubiquitylated chromatin. Progression into S phase enables
the CDK1-dependent formation and activation of the BRCA1CtIP complex, which likely evicts RIF1 from break sites.
Interestingly, our results suggest that the antagonism between
BRCA1 and 53BP1 is particularly important for DSB repair
pathway choice at a subset of ends based on two main observations: first, 53BP1 and RIF1 deletions in the DT40 system
sensitize cells to DSBs less profoundly than does a Ku70 gene
deletion, yet they act in the canonical NHEJ pathway; second,
the impact of BRCA1 deficiency on end resection is similarly
not as profound as the severe resection defect imparted by
CtIP depletion, but the resulting defect in resection is completely
dependent on 53BP1 and RIF1. Why the described 53BP1BRCA1 circuit is important for a subset of ends is mysterious.
It may relate to the observation that 53BP1 participates in
the repair of heterochromatin-associated DSBs, although this
activity was shown to require the 53BP1 BRCT domains (Noon
et al., 2010), which are not involved in the inhibition of BRCA1,
CSR, or end protection. Alternatively, other factors may
contribute to DSB repair pathway choice. Of particular interest
is the observation that XLF acts redundantly with 53BP1 to
protect DNA ends and to promote the repair of DNA breaks
during V(D)J recombination (Oksenych et al., 2012).
Finally, it is important to evaluate the results presented herein
in relation to BRCA1-deficient malignancies since the lethality,
tumorigenesis, and PARP inhibitor sensitivity conferred by
BRCA1 deficiency can be suppressed by 53BP1 deletion
(Bouwman et al., 2010; Bunting et al., 2010). We found that the
loss of RIF1 is able to restore RAD51 IR-induced foci in
BRCA1-deficient human and avian cells and modulate the
PARP inhibitor sensitivity of BRCA1-deficient DT40 cells.
However, this reversal of PARP inhibitor sensitivity was clearly
incomplete, perhaps owing to the fact that RIF1 also plays roles
in the maintenance of DNA replication forks and the regulation
of DNA replication timing (Buonomo et al., 2009; Xu et al.,
2010). It will be important to identify separation-of-function
mutations in RIF1 that selectively disrupt its function downstream of 53BP1. We note that missense mutations in RIF1
have been found in numerous human tumors (http://cancer.
sanger.ac.uk/cosmic/gene/overview?ln=RIF1), suggesting that
some RIF1 variants might specifically impair the function of
RIF1 in NHEJ. It will be important to characterize these variants
and determine whether they can act as genetic modifiers of
BRCA1-deficient malignancies.
EXPERIMENTAL PROCEDURES
Cell Culture and Plasmid Transfection
All culture media were supplemented with 10% fetal bovine serum (FBS). U-2OS (U2OS) cells were cultured in McCoy’s medium (Gibco). HEK293T, HeLa,
and hTERT-RPE1 cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Gibco). Plasmid transfections were generally carried out using
Lipofectamine 2000 Transfection Reagent (Invitrogen) following the manufacturer’s protocol.
DT40 Cells
DT40 cells were grown at 39.5 C, 5% CO2 in RPMI 1640 medium (Gibco)
supplemented with 10% fetal calf serum, 1% chicken serum, 0.1 mM
b-mercaptoethanol. For cell synchronization, G1 populations of DT40 cells
were isolated by centrifugal elutriation using a JE-5.0 elutriation rotor, standard
chamber, and a J6-Mi centrifuge (Beckman, Fullerton, CA) running at
2200 rpm and 16 C. For clonogenic survival assays following irradiation, serial
dilutions of cells were plated on methylcellulose. Viable colonies were counted
15 days after treatment. Sensitivity to the other genotoxins was determined as
described previously (Xu et al., 2010) using MTT staining.
RNA Interference
Unless stated otherwise, all siRNAs employed in this study were SMARTpools
(ThermoFisher). RNAi transfections were performed using Lipofectamine
RNAiMAX (Invitrogen). The individual siRNA duplexes used in rescue experiments were RIF1 (RIF1 #2 [ThermoFisher, D-027983-02] and RIF1 #4
[ThermoFisher, D-027983-04]), CtIP (GCUAAAACAGGAACGAAUC, MWG
Biotech), 53BP1 (ThermoFisher, D-003548-01), and BRCA1 (ThermoFisher,
D-003461-05).
The murine retroviral shRNA vectors for 53BP1 (TF515706) and RIF1
(TF502862), as well as the scrambled shRNA vector, were purchased from
Origene. Retroviral infection of the murine CH12F3-2 B cell line was performed
using RetroNectin-coated plates (Takara) according to the manufacturer’s
guidelines.
DNA End-Resection Assay Utilizing Plate-Based Laser Scanning
Cytometry
U2OS cells were transfected with siRNA in 96-well plates. At 48 hr posttransfection cells were treated with 200 ng/ml of neocarzinostatin (Sigma) for
15 min. After a 3 hr recovery, cells were extracted with 0.2% Triton X-100 in
PBS for 10 min on ice, fixed with 4% paraformaldehyde (PFA), and processed
for RPA32 immofluorescence. Nuclei were counterstained with DAPI. Cells
were imaged using the Celigo laser scanning plate cytometer (Brooks
Automation) and analyzed with the accompanying image analysis software.
Molecular Cell 49, 872–883, March 7, 2013 ª2013 Elsevier Inc. 881
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RIF1 Is the 53BP1 Effector in DSB Repair
Animal Studies
Research involving animals was performed in accordance with protocols
approved by the animal facility at Toronto Centre for Phenogenomics (Toronto).
Full details of all other experimental procedures are given in Supplemental
Experimental Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, one table, and Supplemental Experimental Procedures and can be found with this article online at
http://dx.doi.org/10.1016/j.molcel.2013.01.001.
ACKNOWLEDGMENTS
We are grateful to R. Szilard and S. Panier for critical reading of the manuscript;
to J. di Noia, J. Stark, L. Pelletier, W. Wang, T. Pawson, S. Jackson, J. Lukas,
M. Takata, M. Pagano, K. Hiom, D. Bishop, Y. Taniguchi, and D. Stern for
plasmids and other reagents; and to Ross Chapman and Simon Boulton for
sharing results prior to publication. C.E.-D. was supported by a postdoctoral
fellowship from Fundacion Caja Madrid and is now an Ontario Postdoctoral
Fellow. A.O. and A.F.-T. both received a postdoctoral fellowship from the
CIHR. D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 1) in the Molecular Mechanisms of
Genome Integrity. Work in the D.X. laboratory was supported by grant
31271435 from the China Natural Science Foundation and the Peking University-Tsinghua University Center for Life Sciences. Work in the D.D. laboratory
was supported by CIHR grant MOP84297, grant RE05-037 from the Ontario
Research Fund, and a grant-in-aid from the Krembil Foundation.
Received: October 26, 2012
Revised: December 7, 2012
Accepted: December 28, 2012
Published: January 17, 2013
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