From the Department of Oncology, Mayo Clinic and Foundation, Rochester, Minnesota 55905
Received for publication, February 10, 2003, and in revised form, February 25, 2003
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ABSTRACT |
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BRCA1 is a tumor suppressor involved in DNA
repair and damage-induced checkpoint controls. In response to DNA
damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions.
However, little is known about the regulation of BRCA1 relocalization
following DNA damage. Here we show that mediator of
DNA damage checkpoint protein 1
(MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of
DNA damage responses that regulates BRCA1 function. MDC1 regulates
ataxia-telangiectasia-mutated (ATM)-dependent
phosphorylation events at the site of DNA damage. Importantly
down-regulation of MDC1 abolishes the relocalization and
hyperphosphorylation of BRCA1 following DNA damage, which coincides
with defective G2/M checkpoint control in
response to DNA damage. Taken together these data suggest that MDC1
regulates BRCA1 function in DNA damage checkpoint control.
FHA1 and BRCT domains
are functional modules that are involved in protein-protein interaction
(1-3). Many proteins involved in the DNA damage response
pathway, such as mammalian BRCA1, 53BP1, Chk2, NBS1, yeast Rad9, and
Rad53, contain FHA or BRCT domains. Furthermore mutations within FHA
and BRCT domains have been associated with tumorigenesis
(4).2 These findings suggest
important roles of FHA and BRCT domains in DNA damage response pathways.
Kiaa0170 or NFBD1 (nuclear factor with
BRCT domains protein 1) is a nuclear protein
that contains both FHA and BRCT domains. Our initial studies on
Kiaa0170 and studies from other laboratories have shown that Kiaa0170
forms nuclear foci at the sites of DNA damage and is phosphorylated in
an ATM-dependent manner (5-7). Furthermore Kiaa0170
functions as a critical regulator in DNA damage signaling pathways (7,
8).3 Therefore, Kiaa0170 has
been renamed as mediator of DNA damage checkpoint protein 1 (MDC1) to better reflect
its role in DNA damage checkpoint
controls.4 In this study, we
further explored the role of MDC1 in ATM-dependent DNA
damage response pathways.
Plasmids and Small Interfering RNAs (siRNAs)--
MDC1 cDNA
was kindly provided by Dr. T. Nagase from Kazusa DNA Research Institute
(Chiba, Japan). MDC1 siRNAs were synthesized by Xerogon Inc.
(Huntsville, AL). The siRNA duplexes were 21 base pairs including a
two-deoxynucleotide overhang. The coding strand of MDC1 siRNA1 was
UCCAGUGAAUCCUUGAGGUdTdT, and the coding strand of MDC1
siRNA2 was ACAACAUGCAGAGAUUGAAdTdT. The coding strand of BRCA1 siRNA was GGAACCUGUCTCCACAAAGdTdT, and the control
siRNA was UUCAAUAAAUUCUUGAGGUdTdT.
Antibodies and Cell Lines--
MDC1 antibodies were raised
against glutathione S-transferase fusion proteins
containing N-terminal residues 1-150 or 151-484 of MDC1.
Anti-phospho-H2AX ( Immunoprecipitation, Immunoblotting, and
Immunostaining--
Cell lysate preparation, immunoprecipitation,
immunoblotting, and immunostaining were performed as described before
(8).
siRNA Transfection--
siRNA transfection was performed as
described previously (9). Briefly, cells were grown in six-well plate
to 30% confluence and immediately before transfection washed with
serum-free medium, and 800 µl of serum-free medium were added per
well. For each well, 200 nM siRNA was mixed with 5 µl of
Oligofectamine (Invitrogen) in 200 µl of serum-free medium.
The mixtures were incubated for 20 min at room temperature and then
added to cells. Serum was added 4 h later to a final concentration
of 10%. 24 h after the initial transfection, a second
transfection was performed in the same way as the previous one. 72 h after initial transfection, cells were treated and harvested as indicated.
Phospho-H3 Staining--
siRNA-transfected cells were To test the role of MDC1 in ATM-dependent
phosphorylation events, we used siRNA technology (9) to down-regulate
MDC1 (Fig. 1A) and monitored
ATM-dependent phosphorylation events by immunofluorescence staining (Fig. 1B). An antibody raised against
phosphoepitope substrates of ATM or ATR has been shown to specifically
recognize ATM/ATR-dependent phosphorylation events (10). As
shown in Fig. 1B, in cells transfected with control siRNA,
ionizing radiation (IR)-induced nuclear foci were present in most
irradiated cells, suggesting the accumulation of phosphorylated ATM
substrates at the sites of DNA breaks. However, down-regulation of MDC1
abolished these IR-induced foci. These results suggest that MDC1
regulates ATM/ATR-dependent phosphorylation events upon DNA
damage. Furthermore down-regulation of MDC1 resulted in increased
sensitivity to IR and camptothecin (Fig. 1C), implying that
MDC1 is required for cell survival following DNA double-stranded
breaks. Together these data suggest that MDC1 is involved in
ATM-dependent DNA damage response pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H2AX) and anti-BRCA1 antibodies were generated as described previously (8). Anti-p1524BRCA1 antibodies were
kindly provided by Dr. KumKum Khanna. Anti-pS317Chk1 antibodies and
anti-pATM/ATR were purchased from Cell Signaling. Anti-Chk1 mAb was
purchased from Santa Cruz Biotechnology. Anti-phospho-histone 3 antibodies were purchased from Upstate Biotechnology. All cells were
obtained from American Type Culture Collection and cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum.
irradiated (2 Gy) or left untreated. Cells were then stained with
anti-phospho-H3 antibodies, and phospho-H3-positive cells were
evaluated by counting or fluorescence-activated cell sorter.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
MDC1 is involved in ATM-dependent
phosphorylation events. A and B, HeLa cells
were transfected with control siRNA (sic) or MDC1 siRNA.
After 72 h, cells were lysed and blotted with anti-MDC1
(upper panel) or anti-actin antibodies (lower
panel) (A), irradiated (10 Gy) or left untreated, and
16 h later stained with anti-phospho-ATM/ATR substrate antibodies
and 4,6-diamidino-2-phenylindole (DAPI)
(B). C, HeLa cells were transfected with control
siRNA (sic) or MDC1 siRNA and treated with IR or
camptothecin (CPT). Colony formation was determined 2 weeks
later. MDC1si, MDC1 siRNA.
In agreement with the role of MDC1 in ATM/ATR-dependent
pathways, down-regulation of MDC1 also partially decreased H2AX foci staining after IR.5 This is
different from a previous report (11) that showed intact
H2AX foci
formation in cells transfected with MDC1 siRNA. This discrepancy could
be due to the extent of MDC1 down-regulation by different siRNAs used.
On the other hand, consistent with the previous report (11), we also
observed that down-regulation of H2AX abolished MDC1 foci formation in
response to DNA damage.5 These findings suggest that
H2AX phosphorylation and MDC1 foci formation depend on each other,
possibly by forming a positive feedback loop:
H2AX recruits MDC1 to
the sites of DNA damage, which in turn enhances the phosphorylation of
H2AX by ATM.
These findings also raise the possibility that H2AX and MDC1
cooperate as early signaling molecules to amplify
ATM-dependent DNA damage signals. In response to DNA
damage, BRCA1 (12) and many other proteins involved in DNA damage
responses, such as MRE11/NBS1/Rad50 (13), 53BP1 (8, 14, 15), and Chk2
(16), form nuclear foci. These nuclear foci colocalize with
H2AX
foci, which reside at the sites of DNA damage (17, 18). MDC1 also redistributes to nuclear foci in response to DNA damage (5-7, 11).
Similarly MDC1 foci colocalized with BRCA1 foci (Fig.
2A). Furthermore we found that
MDC1 co-immunoprecipitated with BRCA1. This interaction is
constitutive, occurring independently of DNA damage (Fig.
2B). Given that BRCA1 forms a stable heterodimeric complex
with BARD1 in vivo (19), we further determined whether MDC1
could also associate with BARD1. As shown in Fig. 2B,
similar to BRCA1, BARD1 also associates with MDC1. These results
suggest that MDC1 interacts with the BRCA1·BARD1 complex and
may be involved in a BRCA1-mediated genome maintenance function.
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The interaction between MDC1 and BRCA1 led us to investigate the
relationship between MDC1 and BRCA1. We used siRNA technology to
down-regulate either BRCA1 or MDC1. Transfection of HeLa cells with
BRCA1 siRNA or MDC1 siRNA resulted in the depletion of BRCA1 and MDC1,
respectively (Figs. 3, A and
B, and 4A). As shown in Fig. 3A,
depletion of MDC1 by siRNA transfection led to a complete loss of BRCA1
foci in response to DNA damage (from 80% of cells containing BRCA1
foci in control samples to 4% in the MDC1 siRNA-transfected sample)
(Fig. 3A). Only cells that retained MDC1 expression still formed BRCA1 foci (data not shown). Conversely, although transfection of HeLa cells with BRCA1 siRNA resulted in a loss of BRCA1 in 90% of
the transfected cells (Fig. 3B and data not shown), the relocalization of MDC1 to nuclear foci following DNA damage was not
affected by the depletion of BRCA1 (Fig. 3B). Furthermore the disappearance of BRCA1 foci in MDC1 siRNA-transfected cells is not
due to a change in kinetics of BRCA1 foci formation since we could not
detect BRCA1 foci either 6 h (data not shown) or 16 h after
DNA damage (Fig. 3A). In unirradiated control cells, there
are a subset of cells that contain BRCA1 foci (30%), which are the
previously reported S-phase BRCA1 foci (12). Interestingly transfection of MDC1 siRNA in these cells led to a dramatic decrease of
cells containing these BRCA1 S-phase foci (2% of cells containing BRCA1 foci) (Fig. 3A), suggesting that BRCA1 S-phase foci
formation may also require MDC1. Transfection with a control siRNA,
which was generated by introducing point mutations into MDC1 siRNA, had
no effect on BRCA1 foci formation (Fig. 3A). Furthermore
transfection with a second, different MDC1 siRNA (siRNA2) also
abolished the localization of BRCA1 to nuclear foci (data not shown).
Taken together these data strongly suggest that MDC1 is required for the recruitment of BRCA1 to the sites of DNA damage.
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Using siRNA technology, we also found that down-regulation of H2AX
significantly decreased IR-induced BRCA1 foci (data not shown). This is
in agreement with early studies using H2AX-deficient mouse cells (20,
21). However, it is unlikely that MDC1 regulates BRCA1 foci formation
solely through influencing H2AX since MDC1 siRNA only partially
reduced
H2AX foci (data not shown) but completely abolished BRCA1
foci in response to IR (Fig. 3A). In addition, while MDC1
regulated BRCA1 S-phase foci formation (Fig. 3A), H2AX does
not seem to be required for the formation of BRCA1 S-phase foci (data
not shown and see Ref. 20). Finally, while H2AX is dispensable for DNA
damage-induced hyperphosphorylation of BRCA1 (data not shown), MDC1 is
required for BRCA1 phosphorylation following DNA damage (see below).
BRCA1 is phosphorylated by ATM at multiple residues including Ser-1387, Ser-1423, Ser-1457, and Ser-1524 after irradiation (22, 23). To further investigate the role of MDC1 in the regulation of BRCA1, we examined the phosphorylation of BRCA1 following DNA damage in MDC1-deficient cells. As shown in Fig. 3C, transfection with MDC1 siRNA resulted in defective hyperphosphorylation of BRCA1 following DNA damage as judged by its mobility shift by SDS-PAGE or by blotting with antibodies against phosphoserine 1524 of BRCA1. However, BRCA1 phosphorylation is normal in cells transfected with H2AX siRNA (data not shown). It is worth mentioning that the defective BRCA1 phosphorylation in MDC1 siRNA-transfected cells is only observed with low doses of irradiation (10 Gy or lower). When higher doses of irradiation are used (30 Gy), phosphorylation of BRCA1 observed in MDC1 siRNA-transfected cells is similar to that in control cells (data not shown), suggesting the possibility that an alternative pathway for BRCA1 phosphorylation is activated at higher doses. Recently 53BP1 has also been reported to regulate BRCA1 foci formation and phosphorylation (24). It is possible that MDC1 and 53BP1 might work together in regulating BRCA1 following DNA damage.
The defective BRCA1 localization and phosphorylation in MDC1
siRNA-transfected cells suggests that MDC1 might regulate
BRCA1-mediated functions. It is well established that BRCA1 is involved
in the maintenance of genome stability. BRCA1 participates in a number of activities following DNA damage including DNA repair (25), Chk1
activation (26), S-phase and G2/M checkpoint control (27, 28), and cell survival (22). This prompted us to investigate how
MDC1 affects BRCA1-mediated downstream events. Using MDC1 siRNA or
BRCA1 siRNA, which specifically down-regulated MDC1 and BRCA1,
respectively (Fig. 4A), we
first examined the Chk1 activation following DNA damage. In response to
DNA damage, Chk1 is phosphorylated at Ser-317 and Ser-345, and the
phosphorylation of these sites is required for Chk1 activation (29).
Down-regulation of either BRCA1 or MDC1 resulted in decreased Chk1
phosphorylation following DNA damage (Fig. 4B). BRCA1 has
recently been shown to regulate the G2/M checkpoint,
probably through its regulation of Chk1 activation (26). Thus, we next
examined the G2/M checkpoint control in cells depleted of
MDC1. As shown in Fig. 4C, HeLa cells transfected with
control siRNA showed a marked decrease in mitotic fraction in response
to irradiation. However, similar to cells transfected with BRCA1
siRNA, cells transfected with MDC1 siRNA showed significantly greater
mitotic fraction after
irradiation, suggesting a G2/M checkpoint defect in cells lacking MDC1 or BRCA1. Taken together these
results suggest that MDC1 is involved in the G2/M
checkpoint, probably through regulating the BRCA1-Chk1 pathway.
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Emerging evidence suggests that MDC1 is an important mediator of DNA damage responses. In addition to its role in BRCA1 foci formation and phosphorylation, MDC1 also interacts with activated Chk2 and regulates S-phase checkpoint, p53 stabilization, and radiation-induced apoptosis (7). A recent report also links MDC1 to the Chk2 pathway (11). Furthermore we and others have shown that MDC1 regulates NBS1 foci formation.3,5 These findings suggest that MDC1 regulates multiple DNA damage signaling pathways. MDC1 contains several protein-protein interaction domains including the FHA domain, BRCT domain, and internal repeated sequences. It is plausible that MDC1 acts as an adaptor protein to recruit downstream signaling molecules to the sites of DNA damage in a role similar to that of Grb2 or Shc in receptor tyrosine kinase-mediated signaling pathways.
In summary, we have shown that MDC1 regulates BRCA1 foci formation,
phosphorylation, and G2/M checkpoint control in response to
DNA damage. These results establish a critical role of MDC1 in the
regulation of DNA damage checkpoint. Given that dysregulation of DNA
damage checkpoints (e.g. mutations of p53 and BRCA1)
frequently leads to tumorigenesis, one may wonder whether MDC1
functions as a tumor suppressor. Future genetic studies of MDC1 will be conducted to test this possibility.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Nagase from Kazusa DNA Research Institute for providing MDC1 (Kiaa0170) cDNA and Dr. KumKum Khanna for providing anti-phospho-BRCA1 antibodies. We also thank the Mayo Protein Core facility for synthesis of peptides. We are extremely grateful to Drs. Larry Karnitz and Scott Kaufmann and members of the Chen and Karnitz laboratories for helpful discussions and ongoing technical support.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants NIH RO1 CA89239 and CA92312 and by the Prospect Creek Foundation and the Breast Cancer Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Recipient of Department of Defense Breast Cancer Career Development Award DAMD17-02-1-0472. To whom correspondence should be addressed: Dept. of Oncology, Mayo Clinic, Guggenheim Bldg., Rm. 1342, 200 First St. S. W., Rochester, MN 55905. Tel.: 507-538-1545; Fax: 507-284-3906; E-mail: chen.junjie@mayo.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.C300060200
2 Breast Cancer Information Core at research.nhgri.nih.gov/bic/ on the World Wide Web.
3 S. Jackson and S. Elledge, personal communication.
4 S. J. Elledge and S. P. Jackson, personal communication.
5 Z. Lou, C. C. S. Chini, K. Minter-Dykhouse, and J. Chen, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
FHA, forkhead-associated;
BRCT, BRCA1 C-terminal;
MDC1, mediator of DNA
damage checkpoint protein 1;
ATM, ataxia-telangiectasia-mutated;
ATR, ATM and Rad3-related;
siRNA, small interfering RNA;
H3, histone 3;
Gy, gray;
IR, ionizing radiation;
H2AX, phospho-H2AX;
BARD1, BRCA1-associated RING domain.
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