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Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut, USA
1Correspondence: Department of Pathology, School of Medicine, Yale University, 310 Cedar St., BML342, New Haven, CT 06510, USA. E-mail: df.stern{at}yale.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to -H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to -H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, -H2AX, and possible ATM/ATR substrates recognized by anti-phospho-SQ/TQ antibody. These results suggest that NFBD1 is an ATM/ATR-dependent organizer that recruits DNA checkpoint signaling and repair proteins to the sites of DNA damage.—Xu, X., Stern, D. F. NFBD1/MDC1 regulates ionizing radiation-induced focus formation of DNA checkpoint signaling and repair factors.
Key Words: 53BP1 • -H2AX • MRE11 complex • ATM/ATR substrates • assembly of ionizing radiation-induced foci
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2)
. Several proteins phosphorylated by ATM or activated indirectly by ATM translocate to common intranuclear sites, where they colocalize. The resulting IR-induced nuclear foci (IRIF) accumulate at the sites of DNA damage (3)
. For example, a form of H2AX modified by ATM-dependent phosphorylation (-H2AX) translocates to and forms IRIF at sites of DNA damage (4)
. -H2AX may mark a chromatin region at or near the DNA damage site and serves as a platform for recruitment of DNA checkpoint signaling and repair factors including the DSB repair MRN (MRE11-RAD50-NBS1) (Nijmegen breakage syndrome) complex, 53BP1 (p53 binding protein 1), and BRCA1 (breast cancer susceptibility gene-1) (4
, 5)
. The MRN complex localizes to the sites of DSBs in vivo and plays a vital role in DNA repair (6)
.
In budding yeast, DNA damage induces phosphorylation of Rad9 in a MEC1 (ATR ortholog) and/or TEL1 (ATM ortholog) -dependent manner. Phosphorylated Rad9 in turn recruits the CHK2 ortholog Rad53 to the Mec1 complex for activation or directly activates Rad53 (7
8
9
10)
. Rad9 also regulates activation of Chk1 (11)
. Both 53BP1 and BRCA1 are candidate orthologs of budding yeast Rad9 in mammals since these proteins are involved in DNA damage responses and share limited sequence homology, with a tandem repeat of carboxyl-terminal BRCT domains (12)
. BRCA1 regulates CHK1 activation in the G2/M checkpoint (13)
. 53BP1 is required for damage-dependent phosphorylation of ATM target proteins including p53, BRCA1, and the cohesion protein SMC1 (structural maintenance of chromosomes) (14
, 15)
. Some reports, but not others, implicate 53BP1 in IR-induced phosphorylation of CHK2 (14
, 16
, 17)
.
NFBD1, a "nuclear factor with an amino-terminal FHA domain and a tandem repeat of BRCT domains" (18)
is the newest candidate ortholog for budding yeast Rad9 in mammals. This protein is also called MDC1 (mediator of DNA damage checkpoint 1) (19
20
21)
. NFBD1 is an early participant in DNA damage signaling pathways and is involved in regulation of the intra-S phase checkpoint and the G2/M checkpoint (19
20
21
22
23
24
25)
. Our earlier work had shown that NFBD1 is recruited to IRIF after DNA damage and colocalizes with -H2AX, 53BP1, MRE11, and partially with BRCA1 (22)
. We have now determined whether NFBD1 physically associates with these proteins and whether NFBD1 is important for assembly of IRIF. The results show that NFBD1 interacts with several DNA checkpoint and repair proteins and that overexpression of truncated NFBD1 interferes with formation of IRIF. These data implicate NFBD1 as an important component of DNA checkpoint signaling systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The KN2 fragment (codons 1-568) contains the core homology domain of the FHA domain (codons 55-125) as well as a consensus nuclear localization signal. Deletion of the FHA domain resulted in the KN2
FHA fragment (codons 141-568). KN2ngt was derived by site-directed mutagenesis (26)
of the KN2 fragment with three alanine substitutions at codons 96NGT. The KC1 fragment (codons 1698-2089) includes the BRCT domains (codons 1893-2089) and a putative nuclear localization signal (22)
. KC1mut was derived by site-directed mutagenesis of the KC1 fragment to inactivate the two BRCT domains by the amino acid substitutions (W1952L and F2064L). A BamHI-EcoRI fragment of NFBD1 encoding most of the PST (proline-, serine-, and threonine-rich repeat) domain (codons 993-1695) and a BamHI fragment encoding the linker region between the KN2 fragment and the PST domain (codons 507-993) were subcloned into the pcDNA-HA vector, resulting in pcDNA-HALink and pcDNA-HAPST, respectively.
Antibodies and cell lines
Generation and characterization of rabbit anti-NFBD1 antibody have been described (22)
. Mouse anti-53BP1 monoclonal antibody and rabbit anti-53BP1 polyclonal antibodies were generous gifts from Thanos D. Halazonetis (Wistar Institute) (27)
and Yasuhisa Adachi (University of Edinburgh) (28)
, respectively. Other antibodies used were rabbit and mouse IgG (Sigma, St. Louis, MO, USA), mouse anti-hemagglutinin (HA) monoclonal (16B12, Covance, Princeton, NJ, USA), anti-BRCA1 (Ab-1, CalBiochem, San Diego, CA, USA), anti-ß-actin (clone AC15, Sigma), anti-MRE11 (clone 12D7), anti-RAD50 (clone 13B3), anti-NBS1 (Clone 1C3, GeneTex, San Antonio, TX, USA), anti-phospho-S139 H2AX (clone JBW301, Upstate Biotechnology, Inc., Lake Placid, NY, USA), rabbit anti-NBS1 (Novus Biologicals, Littleton, CO, USA), anti-HA (Y11) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-NBS1 S343 and anti-phospho-(Ser/Thr) ATM/ATR substrate antibody (Cell Signaling Technology, Beverly, MA, USA), and horseradish peroxidase-conjugated rat anti-HA antibody (clone 3F10, Roche Molecular Biochemicals, Nutley, NJ, USA).
Cell lines used in this study were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/mL penicillin, and 50 mg/mL streptomycin.
In vitro-coupled transcription/translation assays, immunoprecipitation, and immunoblotting
Cell lysate preparation, immunoprecipitation, and immunoblotting were performed as described before (22)
. Cell lysates were extracted in high-salt Nonidet P-40 (NP-40) buffer containing 20 mM Tris-HCl (pH 8.0), 0.4 M NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitor mixture. Two micrograms of antibody was used for immunoprecipitation from 400 to 500 µg of total lysate plus an equal volume of buffer containing 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40, and protease inhibitor mixture at 4°C for 4 h. Precipitates were washed with 20 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA, and 0.5% NP-40.
NFBD1 constructs (pcDNA-HAKN2, -HAKN2ngt,-HAKN2FHA, -HAKC1, and -HAKC1mut) were used as templates for in vitro-coupled transcription/translation of NFBD1 fragments. Promega TNT T7 Quick Coupled transcription/translation reticulocyte lysate system was used in a standard 50 µL reaction according to procedures recommended by the manufacturer. Fifteen microliter portions of in vitro translated products (30 µL for the KC1mut and KN2ngt) were mixed with total cell extracts from HEK293 cells. The mixture was used for immunoprecipitation with anti-HA antibody.
Indirect immunofluorescence
Immunostaining was performed as described before (22)
with some modifications. Cells grown on poly-D-lysine-coated, eight-chamber slides (BD Labware, San Jose, CA) were fixed 2 h after gamma radiation (5 Gy) or mock treatment in 4% paraformaldehyde in PBS (phosphate-buffered saline, 0.2 g/L KCl, 0.2 g/L CaCl2, 8 g/L NaCl, and 2.16 g/L Na2HPO4 · 7H2O) for 15 min, followed by permeabilization for 15 min in 0.5% Triton X-100 in PBS. Slides were blocked with 5% bovine serum albumin in PBST for 30 min at 37°C, incubated with primary antibody for 30 min at 37°C, washed with PBST, and incubated with secondary antibody [rhodamine-conjugated donkey anti-mouse IgG (1:1000) or fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:150)] for 30 min at 37°C. Dilutions of primary antibodies were 1:40 for rabbit anti-HA (Y11); 1:200 for anti-MRE11, rabbit anti-53BP1, and anti-phospho-(Ser/Thr) ATM/ATR substrate antibody; 1:400 for anti-Chk2 phospho-T68; 1:500 for anti-NFBD1, rabbit anti-NBS1, and anti--H2AX, and 1:1000 for anti-HA (16B12).
Small interfering RNA (siRNA) and in situ cell death detection
Transfection of siRNA oligo duplexes (both the specific duplex for NFBD1 and a scrambled duplex) into HeLa cells using Oligofectamine (Invitrogen, San Diego, CA, USA) has been described (22)
. Transfectants were exposed to 5-Gy irradiation 48 h after transfection. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min, then permeabilized in 0.5% Triton X-100 in PBS at room temperature for another 15 min 24 h after irradiation. TUNEL assays were performed on the fixed and permeabilized cells using an in situ cell death detection kit, fluorescein (Roche Molecular Biochemicals), according to the manufacturer’s instructions. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). Images were acquired using a Nikon Microphot-FX microscope with a 25x objective and a SPOT digital camera. Apoptotic cells were counted in 7–30 different fields per experiment encompassing between 500 and 3975 cells. Three independent experiments were performed.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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20
21
22
23
24
25)
. Since these pathways protect cells from the effects of DNA damage, we determined whether NFBD1 is involved in protection from effects of IR. Endogenous NFBD1 protein in HeLa cells was depleted by transfection with a siRNA oligonucleotide duplex specific for NFBD1 (Fig. 1
A). Depletion of endogenous NFBD1 (Fig. 1A
) yielded significantly higher basal (4.8±1.29%) and IR-stimulated (7.0±2.14%) apoptotic rates relative to controls (0.9±0.08% without IR and 1.5±0.12% after IR) (Fig. 1B
). Apparently, NFBD1 has a role in protecting cells from apoptosis under basal culture conditions and possibly after DNA damage.
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To further investigate functions of NFBD1, we identified proteins that copurify with NFBD1 isolated from HEK293T cells by immunoaffinity purification. Among the proteins identified by analysis of specific stained gel bands with MALDI (matrix-assisted laser desorption ionization) -MS (mass spectrometry) protein identification by peptide mass database searching was MRE11 (data not shown). This is consistent with our earlier observation that IR-induced NFBD1 nuclear foci colocalize with MRE11 foci (22)
. In coimmunoprecipitation experiments, endogenous NFBD1 interacted with all three MRN complex constituents (Fig. 2
A). Coimmunoprecipitations occurred in untreated and IR-treated cells. Exposure to IR reduced the amount of MRN constituents coprecipitated with NFBD1. This result prompted us to examine potential physical associations with other checkpoint proteins. Endogenous NFBD1 was coimmunoprecipitated with ATM (Fig. 2B
), p53 (Fig. 2C
), and -H2AX (Fig. 2C
) without and with IR treatment.
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While this work was in progress, similar interactions between NFBD1 and MRN proteins, ATM, and -H2AX were reported by other laboratories (20
, 21)
, but there are some discrepancies. DNA damage did not affect interactions between NFBD1 and the MRN proteins in one of these studies (21)
. We did not observe significant interaction between NFBD1 and BRCA1 or CHK2 in our coimmunoprecipitation assays, although it has been reported that IR-induced NFBD1 foci colocalize with BRCA1 and CHK2 phospho-T68 and that NFBD1 physically associates with BRCA1 and CHK2 (24
, 25)
.
Our novel finding that p53 interacts with NFBD1 is consonant with a report that NFBD1 regulates IR-induced phosphorylation of p53 at serine 20 (19)
. It is noteworthy that all three mammalian proteins with dual BRCT domains (NFBD1, BRCA1, and 53BP1) interact with p53 (29
, 30)
.
We also examined these interactions in cells treated with nocodazole, which activates the mitotic spindle checkpoint by inhibiting microtubule polymerization. Both NFBD1 and 53BP1 are hyperphosphorylated and accumulate after nocodazole treatment (Fig. 2
, Fig. 3
; ref 22
). 53BP1 is extensively phosphorylated in response to spindle disruption with Colcemid and colocalizes with CENP-E to kinetochores in mitosis (28)
. We speculate that NFBD1, like 53BP1, may have a role in mitotic checkpoint signaling.
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In the presence of nocodazole, NFBD1 coimmunoprecipitated with MRN proteins, ATM, and -H2AX (Fig. 2A-C
) but not with p53 (Fig. 2C
). (There was somewhat less total p53 under these conditions.) Although p53 does not play a direct role in the spindle checkpoint activated by nocodazole treatment, it does prevent S phase reentry after adaptation to mitotic arrest (31)
. Perhaps the dissociation of NFBD1 from p53 is connected with the p53-dependent block of S phase reentry.
It is surprising that H2AX phosphorylation is significantly increased after nocodazole treatment (Fig. 2
and Fig. 3A
). This increased phosphorylation of H2AX in response to nocodazole treatment was also observed in HeLa and U2OS cells. Whether this is related to DNA damage responses, interactions between spindle checkpoint and DNA damage checkpoint pathways, alterations in chromatin, or other changes that occur remains to be determined.
NFBD1 has three characteristic structural domains: an amino-terminal FHA domain, an internal PST domain, and carboxyl-terminal tandem BRCT domains. The FHA domain-containing fragment KN2 and the BRCT domain-containing fragment KC1 localize to the nucleus (22)
. As shown in Fig. 3A
, the FHA domain-containing fragment KN2 is sufficient to interact with components of the MRN complex without or with IR treatment, and this interaction slightly decreased after exposure to IR. This fragment specifically associated with forms of NBS1 phosphorylated at S343 in response to IR (Fig. 3A
). A control protein HA-CHK2(1-221) (32)
, which contains a functional CHK2 FHA domain, did not associate with components of the MRN complex (data not shown). Similarly, a bacterially produced GST fusion with the FHA domain from NFBD1 is sufficient to pull down the MRE11 complex (ref 20
and data not shown). Full-length NFBD1 associates with the MRN proteins after nocodazole treatment. However, the KN2 fragment dissociates from the MRN complex after nocodazole treatment. The simplest explanation is that a NFBD1 fragment other than or in addition to the KN2 fragment is required for the physical association with the MRN complex. The FHA domain-containing KN2 fragment associated with endogenous NFBD1. This association increased slightly after IR and was disrupted with nocodazole treatment (Fig. 3A
).
The BRCT domain-containing KC1 fragment bound to -H2AX and 53BP1 after IR or nocodazole treatment, and the binding with 53BP1 after nocodazole treatment was significantly increased (Fig. 3A
). Although the IR dependency is consistent with the finding that the interaction between NFBD1 and -H2AX depends upon H2AX phosphorylation (21)
, it is surprising that the BRCT domain-containing fragment, instead of the FHA domain-containing fragment, is involved in this phosphorylation-dependent interaction since FHA domains are phosphopeptide binding modules. Of course, these BRCT-dependent interactions may involve another protein intermediary.
We then determined whether other NFBD1 fragments are involved in binding to the MRN complex and 53BP1. HA-tagged NFBD1 fragments and mutants produced by coupled in vitro transcription/translation were used to pull down endogenous target proteins in HEK293 cells (Fig. 3B
). (We failed to produce the PST domain in bacterial or by in vitro transcription/translation), suggesting that the PST domain alone is labile.) The linker region (Link) did not bind to any targets examined (data not shown). The KN2 fragment bound to the MRN constituents (Fig. 3B
). IR and nocodazole reduced this interaction. Deletion of the FHA domain in the KN2FHA fragment nearly eliminated binding to the MRN complex. Mutation of the conserved FHA domain residues NGT (KN2ngt) reduced, but did not abolish its binding to the MRN complex (Fig. 3B
). A bacterially produced GST-NFBD1 fusion (codons 2-220) with point mutations affecting another conserved FHA domain residue R58 failed to pull down the MRN complex (20)
.
The KC1 fragment, but not the KC1mut fragment that harbors inactivating substitutions within each BRCT domain, bound to 53BP1 (Fig. 3B
). This binding increased dramatically after nocodazole treatment. We conclude that the FHA and BRCT domains are required for quantitative binding of NFBD1 to the MRN complex and 53BP1, respectively.
IR-induced NFBD1 foci form rapidly and colocalize with and associate with a variety of checkpoint signaling and DNA repair factors (22
, 23)
. The FHA domain- and the BRCT domain-containing fragments are apparently sufficient to mediate several interactions, including interactions with endogenous NFBD1. We sought to determine whether overexpression of these fragments would interfere with production of IRIF by NFBD1-interacting proteins. Ectopic expression of the BRCT domain-containing fragment KC1 in HEK293 cells abolished IR-induced focus formation by NFBD1, MRE11-NBS1, 53BP1, immunoreactive CHK2 phosphorylated at T68, -H2AX, and candidate phospho-ATM/ATR substrates, which are immunoreactive with an anti-phospho-SQ/TQ motif antibody (Fig. 4
). Ectopic expression of KN2 inhibited IR-induced NFBD1 foci and MRE11-NBS1 foci but did not significantly inhibit 53BP1 foci, CHK2 phospho-T68 foci, or -H2AX foci (Fig. 4)
. Ectopic expression of HA-tagged full-length NFBD1 did not interfere with IRIF formation by these proteins (Fig. 4)
. These results suggest that both the FHA domain- and the BRCT domain-containing fragments have dominant-negative effects on NFBD1 functions, presumably mediated by sequestration of endogenous NFBD1 and/or its interacting proteins. The simplest interpretation is that NFBD1 acts before, or in concert with, the MRN complex, 53BP1, CHK2, and -H2AX.
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These results are consistent with other recent observations. Expression of GFP-tagged BRCT domains of NFBD1 (codons 1839-2089) disrupts IR-induced focus formation by NFBD1, -H2AX, and CHK2 phospho-T68 (23)
. Expression of the wild-type FHA domain of NFBD1 (codons 2-220) reduced IR-induced NFBD1 and NBS1 foci, but not -H2AX or 53BP1 foci (20)
. Depletion of endogenous NFBD1 by the siRNA technique interferes with IRIF by NBS1 (20
, 21)
and phospho-ATM/ATR substrates (24)
. However, contradictory results have been obtained for the dependency of 53BP1 and -H2AX IRIF on NFBD1 (20
, 21)
. This discrepancy could arise from differences in residual levels of endogenous NFBD1 protein after siRNA knockdown.
H2AX apparently acts upstream of NFBD1 since H2AX-/- mouse embryonic fibroblasts (MEF) have reduced IR-induced phosphorylation of NFBD1 and do not form NFBD1 IRIF (21)
. Since depletion of endogenous NFBD1 by siRNA reduces IR-induced phosphorylation of H2AX and abolishes H2AX IRIF (21)
, NFBD1 and H2AX are mutually dependent.
Overexpression of the KC1 fragment, but not other portions of NFBD1, increased the modification of -H2AX (Fig. 3A
and data not shown). It has been reported that depletion of endogenous NFBD1 does not reduce ATM-dependent phosphorylation of NBS1, but that IRIF of NBS1 are significantly reduced (20)
. One explanation would be that ATM/ATR-dependent phosphorylation is not sufficient to induce formation of detectable nuclear foci at the sites of damaged DNA, but that focus organizer(s) such as NFBD1, that undergo ATM/ATR-dependent phosphorylation, are essential as platforms for recruitment of DNA checkpoint and repair proteins and/or amplification of the activated ATM/ATR signal at the sites of DNA damage.
In summary, we have demonstrated that NFBD1 protects cells from apoptosis. NFBD1 interacts with ATM, -H2AX, components of the MRN complex, and p53. The FHA domain and the BRCT domains are necessary for NFBD1 to bind to the MRN complex and 53BP1, respectively. Overexpression of the FHA domain- or BRCT domain-containing fragments of NFBD1 interferes with IRIF of several DNA checkpoint signaling and repair proteins. We propose that NFBD1 is an organizer that recruits DNA checkpoint and repair factors to the sites of DNA damage.
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ACKNOWLEDGMENTS |
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Received for publication March 28, 2003. Accepted for publication June 19, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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