E3 ubiquitin ligase COP1 regulates the stability
and functions of MTA1
Da-Qiang Lia, Kazufumi Ohshiroa, Sirigiri Divijendra Natha Reddya, Suresh B. Pakalaa, Mong-Hong Leeb, Yanping Zhangc,
Suresh K. Rayalaa, and Rakesh Kumara,b,1
aDepartment of Biochemistry and Molecular Biology and Institute of Coregulator Biology, The George Washington University Medical Center, Washington,
DC 20037;bDepartment of Molecular & Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; andcRadiation
Oncology and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
Communicated by Salih J. Wakil, Baylor College of Medicine, Houston, TX, July 17, 2009 (received for review May 28, 2009)
Metastasis-associated protein 1 (MTA1), a component of the nu-
cleosome remodeling and histone deacetylation (NuRD) complex,
is widely upregulated in human cancers. However, the mechanism
for regulating its protein stability remains unknown. Here we
report that MTA1 is an ubiquitinated protein and targeted by the
genesis protein 1 (COP1) for degradation via the ubiquitin–protea-
some pathway. Induced expression of wild-type COP1 but not its
RING motif mutants promotes the ubiquitination and degradation
of MTA1, indicating that the ligase activity is required for the
COP1-mediated proteolysis of MTA1. Conversely, depletion of
endogenous COP1 resulted in a marked decrease in MTA1 ubiq-
uitination, accompanied by a pronounced accumulation of MTA1
protein. MTA1, in turn, destabilizes COP1 by promoting its auto-
ubiquitination, thus creating a tight feedback loop that regulates
both MTA1 and COP1 protein stability. Accordingly, disruption of
stabilization, accompanied by an increased coregulatory function
of MTA1 on its target. Furthermore, we discovered that MTA1 is
required for optimum DNA double-strand break repair after ion-
izing radiation. These findings provide novel insights into the
regulation of MTA1 protein and reveal a novel function of MTA1
in DNA damage response.
coregulator ? DNA repair ? ubiquitination
their coregulators at the target gene chromatin (1, 2), and
deregulation of such processes plays a critical role in the
development of malignant phenotypes. One emerging family of
ubiquitously expressed chromatin modifiers is the metastasis-
associated protein (MTA) family, which has an integral role in
nucleosome remodeling and histone deacetylation (NuRD)
complexes that modify DNA accessibility for cofactors (2, 3).
MTA1, the founding member of the MTA family, is widely
upregulated in human cancers and plays an important role in
tumorigenesis and tumor aggressiveness, especially tumor inva-
sion and metastasis (4–6). MTA1 functions not only as a
transcriptional repressor of estrogen receptor ? (7), but also as
a transcriptional activator on certain promoters, such as the
breast cancer–amplified sequence 3 (BCAS3) promoter (8). In
this context, MTA1 is acetylated at lysine 626 (K626) by histone
acetyltransferase p300; such modification allows MTA1 to re-
cruit RNA polymerase II (Pol II) on the BCAS3 enhancer region
and confers its coactivator function upon BCAS3 (8). MTA1 is
also a mechanistic mediator of c-Myc–regulated transformation
as a downstream target of the oncogene c-Myc (9). Although a
paramount role of MTA1 in cancer and coregulator biology, the
mechanism for regulating its protein stability remains unknown.
Constitutive photomorphogenic 1 (COP1; also known as
RFWD2, RING finger and WD repeat domain protein 2), an
evolutionarily conserved RING-finger ubiquitin–protein ligase,
has been defined as a central regulator of plant development by
egulation of fundamental cellular processes demands dy-
namic coordinated participation of transcription factors and
targeting critical positive regulators and/or the photoreceptors
for ubiquitination and degradation (10–12). In mammals, COP1
COP1 functions as an E3 ligase for the tumor suppressor p53 to
induce its degradation, consequently, regulates cell cycle pro-
gression and cell survival (13). COP1 also regulates lipid me-
tabolism by targeting acetyl-CoA carboxylase (ACC), a rate-
limiting enzyme in fatty acid synthesis, for degradation via its
nase and negative regulator of Akt in muscle and the liver
(14–16). Recently, it was found that COP1 promotes the ubiq-
uitination and degradation of the cAMP responsive coactivator
transducer of regulated CREB activity 2 (TORC2), a key
regulator of fasting glucose metabolism, and thereby regulates
liver glucose metabolism (17, 18). COP1 also inhibits c-Jun
transcriptional activity by recruiting c-Jun to an E3 complex
containing de–etiolated-1, DNA damage binding protein–1,
cullin 4A, and regulator of cullins–1 for c-Jun protein degrada-
tion (19, 20). Because c-Jun is a stress-responsive transcription
factor, it has been speculated that COP1 may be involved in
cellular stress responses (21). Indeed, recent studies revealed
that ionizing radiation (IR) triggers an ataxia telangiectasia
mutated (ATM)–dependent rapid autodegradation of COP1 by
phosphorylating it on Ser 387, thereby stabilizing p53 after DNA
damage (22). In addition to polyubiquitination of its substrates,
COP1 also catalyzes its autoubiquitination for degradation as a
part of an autoregulatory mechanism (19, 23, 24).
In this study, we provide evidence that the E3 protein-ligase
COP1 targets MTA1 for degradation via the ubiquitin–
proteasome pathway. MTA1, in turn, destabilizes COP1 by
promoting its autoubiquitination, thus creating a feedback loop
that regulates both MTA1 and COP1 protein stability. Further-
more, we observed that IR stabilizes MTA1 by disruption of the
COP1-mediated proteolysis and increases MTA1 coactivator
activity on its target BCAS3, and that MTA1 is required for
optimal DNA double-strand break repair after IR treatment.
These findings provide insights into regulation of MTA1 protein
and its role in cellular response to DNA damage.
Results and Discussion
MTA1 Is an Ubiquitinated Protein. While exploring the role of the
we found that MTA1 protein levels were dramatically increased by
potent and selective proteasome inhibitors, such as MG-132 or
lactacystin (25), in human osteosarcoma U2OS and lung cancer
A549 cells (Fig. 1A and [supporting information (SI) Fig. S1]). To
Author contributions: R.K. designed research; D.-Q.L., K.O., S.D.N.R., S.B.P., and S.K.R.
performed research; M.-H.L. and Y.Z. contributed new reagents/analytic tools; and D.-Q.L.
wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
October 13, 2009 ?
vol. 106 ?
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test the existence of ubiquitination modification of MTA1 in vivo,
U2OS cells were treated with or without MG-132 and subjected to
sequential immunoprecipitation (IP)/Western blot analyses with
the indicated antibodies. We noted the presence of a smear of
with MG-132 (Fig. 1B), suggesting that endogenous MTA1 may be
a target for proteasomal degradation in mammalian cells. To
further test this notion, MTA1-knockout (MTA1?/?) mouse em-
bryonic fibroblasts (MEFs) (26) were transfected with expression
vectors encoding Myc-tagged MTA1 (Myc-MTA1) and hemagglu-
tinin (HA)–tagged ubiquitin (HA-Ub), either alone or in combi-
antibody and immunoblotted with an anti-HA antibody. We found
that Myc-MTA1 was heavily ubiquitinated in the presence of
HA-Ub (Fig. 1C, last lane). This was also true when these studies
were repeated in HEK293 cells (Fig. 1D). These findings suggest
that MTA1 is an ubiquitinated protein within cells.
Ubiquitin-dependent proteolysis occurs after covalent attach-
protein, so the removal or modification of these residues gen-
erally leads to loss of ubiquitin ligation and resistance to
proteasome-mediated degradation (27, 28). Based on our pre-
vious observations that lysine residue 626 (K626) of MTA1 is
acetylated by p300 in breast cancer cells (8), and the fact that
MTA1 contains another lysine residue (K182) which could
potentially be modified, we next tested whether the two lysine
residues are required for the ubiquitination of MTA1. Toward
this aim, we generated various T7-tagged MTA1 expression
vectors in which the lysine residues K182 and K626 were mutated
to alanine (referred to K182A, K626A, or K182A?K626A
mutant) and transfected these expression plasmids into HEK293
cells alone or together with HA-Ub expression vector. Sequen-
tial IP/Western blot analyses revealed that, although ubiquiti-
nation of K182A (lane 4) or K626A (lane 6) mutant was reduced
as compared with its wild-type control (lane 2), a greater
reduction was seen with the double mutant K182A?K626A
(lane 8; Fig. 1E), indicating that K182 and K626 could be two of
ubiquitination sites for MTA1. We further confirmed these
results by measuring MTA1 half-life in the presence of cyclo-
heximide, an inhibitor of protein biosynthesis. As shown in Fig.
1F, double mutant (K182A?K626A) increases the half-life of
strongly indicates that MTA1 is an ubiquitinated protein in cells
and is subjected to degradation through the ubiquitin–
E3 Ubiquitin–Protein Ligase COP1 Targets MTA1 for Degradation. To
determine which ligase is responsible for MTA1 ubiquitination,
we screened a number of known E3 ligases by cotransfection
experiments. We found that coexpression of Flag-COP1 dra-
matically increased MTA1 ubiquitination (Fig. 2A), whereas
knockdown of endogenous COP1 by a specific siRNA against
COP1 decreased the levels of MTA1 ubiquitination (Fig. 2B),
suggesting that COP1 could be the primary E3 ubiquitin ligase
for MTA1 ubiquitination. To further substantiate a role of COP1
on MTA1 ubiquitination, we generated two E3 ligase–defective
mutants by substitutions of consensus Cys to Ser at the residues
136/139 (C136/139S) and 156/159 (C156/159S) within the RING
domain (residues 136–174), which are required for COP1 E3
ligase activity (24), and tested their effects on MTA1 ubiquiti-
nation in vivo. We found that COP1 but not its E3 ligase-
defective mutants promoted the appearance of inducible MTA1
ubiquitination (Fig. 2C), suggesting that COP1 ligase activity is
required MTA1 ubiquitination.
Because ubiquitination of proteins is usually associated with
their turnover, we next tested whether COP1 could regulate
MTA1 protein abundance. As shown in Fig. 2D, coexpression of
COP1 results in a dose-dependent decrease of MTA1 levels, and
inclusion of proteasome inhibitor MG-132 abolished the effect
of COP1 on the degradation of MTA1 (last lane), indicating a
role of ubiquitin-dependent proteasome pathway in the COP1-
mediated proteolysis of MTA1. In contrast, the E3 ligase–
analysis with an anti-MTA1 antibody. The expression of the ?-actin was used as a loading control. The density of bands was measured using the imageQuest
program and normalized to that of ?-actin. The fold change (MTA1/?-actin) is shown in the bottom of the figure. (B) U2OS cells were treated with or without
MG-132, immunoprecipitated with an anti-MTA1 antibody or control IgG, and immunoblotted with an anti-ubiquitin antibody. (C–E) MTA1?/?MEFs (C) or
HEK293 cells (D and E) were transfected with the indicated expression vectors, immunoprecipitated with an anti-Myc (C and D) or anti-T7 (E) antibody, and
immunoblotted with the indicated antibodies. (F) U2OS cells were transfected with the indicated expression plasmids, treated with cycloheximide after 36 h of
transfection, and collected at the indicated time points for Western blotting analysis using the indicated antibodies. Western blots were subjected to
densitometric analysis and results were normalized based on actin expression levels, and reported in graphical form (lower panel).
MTA1 is an ubiquinated protein. (A) U2OS and A549 cells were treated with or without MG-132 and harvested at the indicated times for Western blot
www.pnas.org?cgi?doi?10.1073?pnas.0908027106Li et al.
defective mutants of COP1 were not able to alter the concen-
tration of MTA1 (Fig. 2E). In support of these findings,
cycloheximide half-life experiments revealed that COP1, but not
its ligase-defective mutants, decreases the half-life of MTA1
(Fig. 2F and Fig. S2). In addition, we consistently demonstrated
that depletion of COP1 using a specific siRNA against COP1
caused a pronounced accumulation of endogenous MTA1 pro-
tein (Fig. 2G). Taken together, these findings establish that
COP1 ligase is a modifier of MTA1 ubiquitination and that
MTA1 stability is regulated by COP1 through a proteasome-
Given that COP1 could ubiquitinate MTA1, we next examined
the possibility of a physical interaction between MTA1 and
COP1. We found that transiently expressed Myc-MTA1 and
Flag-COP1 in the U2OS cells could be coimmunoprecipiated
with Flag or Myc antibodies, respectively (Fig. 2H). Further-
more, endogenous COP1 also coimmunoprecipitated with en-
dogenous MTA1 in the U2OS cells (Fig. 2I), demonstrating that
MTA1 and COP1 proteins interacts with each other in vivo. To
further examine whether the interaction between MTA1 and
COP1 is direct or indirect, we performed in vitro GST pull-down
assays using the immobilized full-length GST-MTA1 and35S-
labeled, in vitro–translated COP1 or NRIF3 as a positive control
(29). Results showed that MTA1 was unable to directly bind to
that COP1 and MTA1 could coexist within a native protein
complex and that the observed MTA1-COP1 interaction in vivo
may be mediated via other proteins. This notion has been already
demonstrated by other studies. For example, a recent study (15)
reported that COP1 regulates lipid metabolism by targeting acetyl-
CoA carboxylase (ACC) for the ubiquitin-dependent degradation
COP1 does not directly interact with ACC. TRB3 associates with
both COP1 and ACC through distinct surfaces and mediates the
interaction between COP1 and ACC and triggers ubiquitination of
ACC by recruitment of COP1 to ACC (15).
MTA1 Destabilizes COP1 by Promoting Its Autoubiquitination. Like
other RING finger ubiquitin ligases, COP1 catalyzes its auto-
ubiquitination for degradation as a part of an autoregulatory
mechanism (19, 23, 24). We next examined whether MTA1
affects the autoubiquitination activity of COP1. Interestingly,
induced expression of MTA1 in the U2OS cells dramatically
increased the levels of COP1 autoubiquitination (Fig. 3A; com-
pare lane 3 with lane 2), accompanied by decreased COP1
protein level. Furthermore, we found that induced expression of
MTA1 led to a dose-dependent reduction in the protein levels of
endogenous COP1 in the U2OS cells (Fig. 3B). The observed
inhibitory effect of MTA1 on COP1 protein levels was indepen-
dent of p53 and Mdm2, because MTA1 was also able to decrease
the level of exogenous COP1 in the p53?/?/Mdm2?/?double-
of these findings, we further demonstrate that induced expres-
sion of MTA1 decreases the half-life of COP1 (Fig. 3D). These
results suggest that COP1 targets MTA1 for the ubiquitin-
dependent degradation; MTA1, in turn, destabilizes COP1 by
C) or in combination with a specific siRNA against COP1 or control siRNA (B), immunoprecipiated with an anti-Myc antibody, and immunoblotted with the indicated
6 h before harvesting for Western blot analysis with the indicated antibodies. (E) U2OS cells were transfected with expression vector encoding T7-COP1 C136/139S or
T7-COP1 C156/159S, and immunoblotted with the indicated antibodies. (F) U2OS cells were transfected with the indicated expression plasmids, treated with
(H) or untransfected (I) were immunoprecipitated with the indicated antibodies or control IgG, and immunoblotted with the indicated antibodies.
COP1 targets MTA1 for degradation via the ubiquitin–proteasome pathway. (A–C) U2OS cells were transfected with the indicated expression vectors (A and
Li et al. PNAS ?
October 13, 2009 ?
vol. 106 ?
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promoting its autoubiquitination, thereby creating an autoreg-
ulatory feedback loop that regulates the activity of both MTA1
and COP1 proteins.
MTA1 Is Stabilized in Response to Ionizing Radiation. Because IR
triggers an ATM-dependent rapid autodegradation of COP1
(22) and COP1 targets MTA1 for the ubiquitin-dependent
levels were affected by IR in a whole-animal setting. After
exposure of whole-body mice to IR, the expression of MTA1, as
well as p53 (positive control), protein was dramatically increased
(Fig. S4). In support of these observations, we found that
treatment of U2OS cells with IR resulted in a marked increase
in the level of MTA1 protein in a dose-dependent (left panel) and
time-dependent (right panel) manner (Fig. 4B). Consistent with
previous reports (22), the levels of COP1 protein were reduced
by IR in the same cellular lysates from the U2OS cells, which
express high levels of endogenous ATM (31) (Fig. 4B, lower
panels). The increased abundance of MTA1 protein in the U2OS
cells after IR treatment was not attributed to an increase in
MTA1 mRNA levels (Fig. S5), indicating a posttranscriptional
regulation mechanism for MTA1 protein by IR. To further test
this notion, we monitored the effect of cycloheximide on the rate
of decline of the endogenous MTA1 in IR-treated cells versus
controls. We found that IR led to a marked increase in the
half-life of MTA1 in comparison to nonirradiated controls (Fig.
4C and Fig. S6). In contrast, IR decreases the half-life of COP1
(Fig. S6), as reported previously (22). These results suggest that
IR upregulates MTA1 through its posttranslational modifica-
tions, possibly via inhibition of its degradation by COP1. To
further test this hypothesis, U2OS cells were transfected with a
found that IR rescued COP1-induced downregulation of MTA1
and p53, accompanied with a marked downregulation of COP1
(Fig. 4D), suggesting that IR stabilizes MTA1 by, at least in part,
inhibiting the COP1-mediated proteolysis. Because IR induces
rapid autodegradation of COP1 in an ATM-dependent manner
(22), we next determined whether the noted stabilization of
MTA1 in response to IR requires ATM protein using an A-T cell
line (AT22IJE-T) that was stably transfected with plasmids
encoding either wild-type ATM (ATM?/?/ATM) or an empty
expression vector (ATM?/?/vector) (32). We found that MTA1
was upregulated by IR in the ATM?/?/ATM but not in the
stabilization of MTA1 in response to IR. Collectively, these data
indicated a mandatory requirement of the ATM-dependent
degradation of COP1 for an increased stability of MTA1 by IR.
Modulation of the Coregulator Function of MTA1 by IR. As a tran-
scriptional coregulator, MTA1 exists in both corepressor or
coactivator complexes and can either repress or stimulate the
expression of cellular genes (2). Because genotoxic stress in-
creases the p300/HAT activity and MTA1-acetylation on K626
by p300 regulates its coactivator activity upon the BCAS3
chromatin (8), we next determined the effect of IR upon the
coregulatory activity of MTA1. For this purpose, U2OS cells
were left untreated or treated with 10 Gy of IR and subjected to
U2OS cells were transfected with the indicated expression vectors, immuno-
ted with the indicated antibodies. (D) U2OS cells were transfected with the
indicated expression plasmids, treated with cycloheximide, and collected at
different time points for Western blot analysis as described above. Western
blots were subjected to densitometric analysis, and results were normalized
based on actin expression levels, and reported in graphic form (right panel).
MTA1 destabilizes COP1 by promoting its autoubiquitination. (A)
tion in an ATM-dependent manner. (A and B) Whole-body mice (A) or U2OS
harvested at the indicated time points for Western blot analysis with the
indicated antibodies. (C) U2OS cells were treated or untreated with 10 Gy of
IR. After 1 h of IR treatment, cells were treated with cycloheximide and
collected at different time points for Western blotting analysis as described
above. (D) U2OS cells were transfected with a Flag-COP1 expression vector or
empty vector. After 36 h of transfection, cells were treated or untreated with
IR and harvested for Western blot analysis with the indicated antibodies. (E)
ATM?/?/vector or ATM?/?/ATM fibroblasts were treated with 10 Gy of IR and
harvested at the indicated time points for Western blot analysis using the
Disruption of the COP1-mediated proteolysis led to MTA1 stabiliza-
www.pnas.org?cgi?doi?10.1073?pnas.0908027106Li et al.
the sequential immunoprecipitation followed by Western blot
analyses. We found that IR-mediated increase in stability of
MTA1 is also accompanied by a corresponding increase in the
levels of K626 acetylated MTA1 (Fig. 5A) as well as an increased
interaction between the MTA1 and Pol II (Fig. 5B). In contrast,
IR exposure does not affect the interaction between MTA1 and
HDAC2 in the U2OS cells (Fig. 5B), suggesting that IR affects
the MTA1 coactivator but not corepressor function. In support
of this notion, we showed that indeed, IR promotes the recruit-
ment of MTA1/Pol II complex to the BCAS3 gene chromatin
revealed by a sequential double-ChIP (chromatin immunopre-
cipitation) assay (Fig. 5C). Moreover, IR stimulates BCAS3-
promoter activity in the wild-type MTA1 (MTA1?/?) MEFs and
that this effect was compromised in its knockout (MTA1?/?)
counterparts (Fig. 5D). Consistent with these findings, we found
MTA1?/?MEFs (Fig. 5E). Collectively, these findings reveal that
IR may affect the functionality of MTA1 in mammalian cells.
Revelation of a Novel Function of MTA1 in DNA Repair. Because
MTA1 is a DNA damage responsive protein (Fig. 4), we next
investigated the possible role of MTA1 in the DNA double-
strand break (DSB) repair following IR treatement. One of the
hallmarks of defective DNA repair is increased radiation sensi-
tivity. We first examined the effect of MTA1 deficiency
(MTA1?/?) on cell survival in response to IR by clonogenic
survival assay (33). We found that MTA1?/?MEFs were hy-
persensitive to IR exposure and exhibited a decreased clono-
genic survival compared to its wild-type controls (Fig. 6A),
suggesting a defect in DSB repair in MTA1?/?MEFs. Interest-
ingly, the noted hypersensitivity of MTA1?/?MEFs to IR
treatment was efficiently rescued by stable reintroduction of
MTA1 in the MTA1?/?MEFs (Fig. 6A and Fig. S7), indicating
that MTA1 is critical for efficient DSB repair.
We next examined the effect of MTA1 deficiency on the levels
of phosphorylated H2AX (?-H2AX), an established surrogate
marker for DSB signaling and the assembly of DNA repair
complexes at the site or in the vicinity of DSBs (34–36). Western
blot analysis using an anti–phospho-H2AX (Ser-139) antibody
revealed that in response to IR the levels of ?-H2AX were
greatly delayed in the MTA1?/?MEFs, whereas MTA1?/?
controls exhibited the typical kinetics of ?-H2AX, with its level
maximized at 30 min and declining afterward, reflecting DSB
generation and repair (Fig. 6B). Interestingly, reintroduction of
MTA1 in the MTA1?/?MEFs (MTA1?/?/MTA1) effectively
restored the delayed responsiveness of ?-H2AX to IR (Fig. 6B).
Moreover, there was no change in total H2AX protein levels
between MTA1?/?and MTA1?/?MEFs with or without IR
treatment, indicating that MTA1 is critical for the efficient
induction of H2AX phosphorylation in response to IR.
H2AX phosphorylation can be detected by immunofluores-
cence, resulting in individual foci within the cell nucleus that can
be counted and are a measure of DSBs (37). We next examined
whether MTA1 deficiency affects the formation of ?-H2AX foci
by immunofluorescent staining using a phospho-H2AX (Ser-
(A and B) Protein extracts from U2OS cells treated or untreated with IR were
subjected to IP analysis with the indicated antibodies, followed by Western
blotting analysis with the indicated antibodies. (C) Sequential double-ChIP
assay of recruitment of MTA1/Pol II complex to the BCAS3 promoter. U2OS
cells were treated with or without IR and harvested after 2 h of IR treatment
antibody were followed by the second ChIPs by an anti-Pol II antibody. (D)
MTA1?/?and MTA1?/?MEFs were transfected with BCAS3-luciferase pro-
moter expression vector and treated with or without IR. Cells were lysed after
analysis of BCAS3 protein expression in MTA1?/?and MTA1?/?MEFs treated
with or without IR.
assay of MTA1?/?, MTA1?/?, and MTA1?/?/MTA1 MEFs treated with or with-
out different doses of IR. Cells were counted, plated, and subjected to indi-
cated doses of radiation and colonies formed over 14 days. Surviving colonies
were plotted as a function of cells plated and normalized by the plating
efficiency for each condition. (B and C) MTA1?/?, MTA1?/?, and MTA1?/?/
MTA1 MEFs were treated with or without IR and harvested at the indicated
time points for Western blot analysis (B) or immunofluorescence staining (C)
is presented here.
MTA1 is required for optimal DNA repair. (A) Clonogenic survival
Li et al.PNAS ?
October 13, 2009 ?
vol. 106 ?
no. 41 ?
139) antibody. After exposure of MTA1?/?MEFs to IR, we
found a notable reduction in the number of ?-H2AX–containing
repair foci and in the maximum achievable levels of ?-H2AX
protein compared with values in MTA1?/?controls (Fig. 6C and
Fig. S8). Of interest, the noted delay in responsiveness of the
ability of MTA1?/?MEFs to form the ?-H2AX foci was
efficiently rescued by stable reintroduction of MTA1 in the
MTA1?/?MEFs (Fig. 6C and Fig. S8), suggesting that MTA1 is
critical for the formation of ?-H2AX foci after DNA damage. As
MTA1?/?MEFs still contain MTA2 and MTA3 (Fig. S9), these
findings suggest that MTA1 is required for efficient DSB repair
as well as cell survival after DNA damage.
Our collective findings provide evidence that MTA1, a tran-
scriptional coregulator, is ubiquitinated and targeted for pro-
teasomal degradation and that the process is mediated by a
RING finger ubiquitin ligase COP1. Interestingly, we found that
MTA1, in turn, destabilizes COP1 by promoting its autoubiqui-
tination, thereby creating an autoregulatory feedback loop be-
tween MTA1 and COP1 for controlling their protein stability
(Fig. 6D). Moreover, we show that MTA1 is a DNA damage
responsive protein; it is stabilized and activated in response to IR
through, at least in part, disruption of the COP1-mediated
proteolysis. Importantly, such posttranslational modification of
MTA1 affects the functionality of MTA1 as a coactivator on its
target chromatin. The biologic significance of these findings was
further revealed by the use of genetically engineered MTA1-
knockout MEFs. We found that MTA1 is required for optimal
DSB repair, and inactivation of MTA1 therefore increases the
cellular sensitivity to IR-induced DNA damage. DNA damaging
the mainstays of most current cancer treatment regimens. Given
the fact that MTA1 is widely upregulated in human cancers and
is closely associated with poor survival in patients with cancers
(5, 6), this study shows that MTA1 is a potential therapeutic target
that could be used to enhance the effectiveness of IR or DNA-
damaging chemotherapy by inhibiting the action MTA1. These
findings provide insights into the regulation of MTA1 protein and
define a novel function of MTA1 in DNA damage response.
Materials and Methods
Culture Collection (Manassas, VA). AT22IJE-T (A-T), a fibroblast cell line de-
rived from an ataxia-telangiectasia patient, and lines stably transfected with
either an empty expression vector (ATM?/?/vector) or full-length ATM cDNA
(ATM?/?/ATM) were gifts from Dr. Yosef Shiloh (Tel Aviv University, Tel Aviv,
embryos at day 9 of development by using a standard protocol. To establish
cell lines stably expressing MTA1, MTA1?/?MEFs were transfected with ex-
Transfection Reagent (Roche Applied Science, Indianapolis, IN) and then
subjected to selection after 24 h of transfection with 10 ?g/ml of blasticidin
(Invitrogen, Carlsbad, CA) for 2 weeks. The resulting colonies were isolated
and analyzed for V5-MTA1 expression by immunoblotting. All of the cell lines
were grown in the recommended media by the providers supplemented with
10% fetal bovine serum (FBS) and 1? antibiotic-antimycotic solution in a
from Invitrogen (Carlsbad, CA) if not otherwise stated.
The detailed Materials and Methods are provided in the SI Text. Primers
used for generating various mutants and quantitative real-time polymerase
chain reactions are provided in Table S1 and Table S2, respectively.
ACKNOWLEDGMENTS. We are especially grateful to Seetharaman Balasent-
hil, Amjad H. Talukder, and Shao-Hua Peng for technical assistance in the in
vivo ubiquitination assay, molecular cloning of COP1 expression vectors, and
providing HA-Ub expression vector and ATM?/?/vector and ATM?/?/ATM cell
lines, respectively. This study was supported by National Institutes of Health
grant CA98823 (to R.K.).
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