Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit
DNA-repair defects post exposure to gamma ionizing radiation
Sandra N. Garciaa,1,2, Bhakti M. Kirtaneb,1, Andrej J. Podlutskya,
Olivia M. Pereira-Smitha, Kaoru Tominagaa,*
aBarshop Institute for Longevity and Aging Studies, Department of Cellular and Structural Biology, University of Texas Health Science Center
at San Antonio, 15535 Lambda Drive, STCBM #3.100, San Antonio, TX 78245, USA
bNational Institute for Research in Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai 400 012, India
Received 30 July 2007; revised 8 October 2007; accepted 11 October 2007
Available online 18 October 2007
Edited by Angel Nebreda
a core component of the NuA4/Tip60 histone acetyltransferase
complex that modifies chromatin structure. We here demonstrate
that Mrg15 null and heterozygous mouse embryonic fibroblasts
exhibit an impaired DNA-damage response post gamma irradia-
tion, when compared to wild-type cells. Defects in DNA-repair
and cell growth, and delayed recruitment of repair proteins to
sites of damage were observed. Formation of phosphorylated
H2AX and 53BP1 foci was delayed in Mrg15 mutant versus
wild-type cells following irradiation. These data implicate a no-
vel role for MRG15 in DNA-damage repair in mammalian cells.
? ? 2007 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
MORF4-related gene on chromosome 15 (MRG15) is
Keywords: MORF4; NuA4; Sin3-HDAC; ATM; 53BP1
Normal cells have a finite ability to divide in culture, a phe-
nomenon known as replicative senescence. Cell fusion of nor-
mal with immortal tumor cells demonstrated that senescence is
a dominant phenotype and provided the first evidence that
senescence is a mechanism of tumor suppression . These
studies resulted in the isolation of mortality factor on chromo-
some 4 (MORF4) as a senescence inducing gene. MORF4 is a
member of a family of transcription factors including the
MORF4-related gene on human chromosome 15 (MRG15)
MRG15 has a 96% similarity to MORF4 in amino acid se-
quence but fails to induce senescence upon introduction into
immortal cells. The most striking structural difference between
the two proteins is the presence of an N-terminal extension in
MRG15, which includes a chromodomain. Proteins containing
a chromodomain, characterized to date, have been found to be
chromatin remodeling factors involved in causing conforma-
tional changes in chromatin by ATP-dependent movement of
nucleosomes and modification of histones [4–6]. Histone mod-
ifying enzymes, the histone acetyltransferases and deacetylases
(HATs, HDACs), are present in complexes involved in tran-
scription and, recently, HAT complexes have been implicated
in DNA-damage detection and repair . MRG15 is present
in both the NuA4/Tip60-HAT and Sin3-HDAC chromatin
modifying complexes .
We have shown that MRG15 is important for cell prolifera-
tion in primary mouse embryonic fibroblasts (MEFs), and that
deletion of the gene in mice results in gross developmental de-
fects leading to embryonic lethality . We here demonstrate
that MRG15 is required for effective DNA-damage repair post
exposure to ionizing radiation (IR) in MEFs and is important
for efficient recruitment of DNA-repair proteins at sites of
damage and acetylation of H2A and H2AX. Loss of a single
copy of MRG15 in MEFs delays repair of DNA-damage post
irradiation, indicating that even a modest decrease in MRG15
levels affects the function of associated complexes. This sug-
gests a novel and critical role for MRG15 in DNA-repair in
2.1. Cell culture and gamma irradiation conditions
Generation of Mrg15 null and heterozygous (het) MEFs and condi-
tions for cell culture have been described previously . To determine
the optimal dose of IR, MEFs were exposed to 0, 2, 3, 5 and 10 Gy
from a137Cs source and seeded at 2500 cells per 60-mm tissue culture
dish in triplicate. Cells were incubated for 10 days, fixed and stained
and total colony number and cell numbers per colony scored . Clon-
ing efficiency was equivalent in cells exposed to 3–5 Gy and doses in
this range were used in all experiments, except for detection of
H2AX and 53BP1.
2.2. Colony formation and growth assays
Long-term (10 days) colony formation assays were performed as de-
scribed above, following 3 Gy exposure. For cell attachment/short-
term cloning efficiency and cell growth assays, c-irradiated (3 Gy) or
untreated MEFs were seeded at 100 cells per 35-mm tissue culture dish
in triplicate or 3 · 104cells per well in 24 well plates, respectively. Mass
cell growth was measured by the MTT assay  and cell number
determined from 1 to 5 days after irradiation, at 24 h intervals.
Abbreviations: HAT, histone acetyltransferase; HDAC, histone deace-
tylase; IR, ionizing radiation; MEFs, mouse embryonic fibroblasts;
MORF4, mortality factor on chromosome 4; MRG15, MORF4-
related gene on chromosome 15; TSA, trichostatin A; het, heterozy-
*Corresponding author. Fax: +1 210 562 5093.
E-mail address: email@example.com (K. Tominaga).
1These authors contributed equally to this work.
2Present address: Department of Biology, The University of Texas at
San Antonio, One UTSA Circle, BSB 2.03.42, San Antonio, TX 78249,
0014-5793/$32.00 ? 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 581 (2007) 5275–5281
2.3. Strand break detection using the alkaline comet assay
Strand break repair was analyzed by single-cell agarose gel electro-
phoresis under alkaline conditions as described previously . Cells
were irradiated (4 Gy), and harvested immediately or at 30 and
120 min post exposure to IR.
2.4. Immunoblot analysis for detection of histone acetyl-K5-H2A
(Ac-H2A) and phosphorylated H2AX (c-H2AX)
Histone proteins were acid extracted from trichostatin A (TSA)-trea-
ted cells (0.4 lM, 16 h) or irradiated cells (10 Gy) at 0, 30, 45, 60, 90
and 180 min post exposure according to manufacturer’s instructions
(Upstate Biotechnology, Charlottesville, VA). Acid extracted histones
from the same number of cells were loaded onto 15% SDS–polyacryl-
amide gels and Western blotted using anti-acetyl-K5-H2A (abcam,
ab1764), anti-phosphorylated H2AX (Ser139) (Upstate, #05-636), or
anti-histone H2A (Santa Cruz, sc-10807) antibodies, as described pre-
2.5. Indirect immunofluorescence to detect c-H2AX and 53BP1 foci
Cells were fixed at 0, 30 and 60 min post irradiation (10 Gy) with
cold 70% ethanol for 30 min at 4 ?C. Nonspecific binding was satu-
rated for 5 min at room temperature in block solution (1% bovine
serum albumin and 10% horse serum in PBS). After incubation with
anti-c-H2AX or 53BP1 antibodies, Fluorescein and Texas Red-conju-
gated secondary antibodies were added. Staining with 0.5 lg/ml DAPI
was done for 5 min. A Zeiss AxioVert 200M optical sectioning micro-
scope equipped with a Zeiss AxioCam B&W CCD camera was used to
collect digital images and three-dimensional deconvolution performed
with the Zeiss software to resolve foci.
3.1. Comet assays demonstrate Mrg15 null and heterozygous
(het) MEFs are defective in repair of IR induced DNA-
MRG15 is an essential component of the NuA4/Tip60-HAT
complex that has been shown to promote accessibility to chro-
matin and, thereby, facilitate recruitment of DNA-repair
machinery to sites of DNA-damage in Drosophila and mam-
malian cells . Post DNA-repair, other complexes, such as
the Sin3-HDAC complex, in which MRG15 is also a compo-
nent, have been postulated to restore condensed chromatin
at sites of damage to maintain genome integrity. In this study,
we analyzed Mrg15 null and het MEFs to determine if they
were defective in DNA-repair in response to IR. We initially
quantified DNA-damage using alkaline single-cell agarose gel
electrophoresis (comet assay).
MEFs derived from E13.5 wild-type, Mrg15 null and het
embryos , were either mock treated (?IR) or exposed to
4 Gy IR (+IR) and harvested at various times post treatment.
DNA-damage in ?IR was low and no major differences were
observed between wild-type, Mrg15 null and het cells. At
10 min following exposure to IR, wild-type MEFs exhibited
a high percentage of DNA in the comet tail, representing dam-
aged DNA. However, by 120 min post exposure the cells had
efficiently repaired damaged DNA to levels comparable to
?IR controls (Fig. 1). In contrast, the Mrg15 null and het
MEFs had un-repaired DNA in the tail at 120 min. At least
two independent clones of MEF cell lines were analyzed for
each genetic background and decreased DNA-repair at
120 min was observed in the Mrg15 null and het MEF clones
tested. These results demonstrate that loss of even one copy of
MRG15 is sufficient to affect efficient repair of DNA-damage
3.2. Long- and short-term clonal and growth assays confirm that
Mrg15 null and het MEFs have impaired growth, not
increased apoptosis, following IR
Based on the results of the comet assay, we determined
whether cell growth of Mrg15 null and het cells was affected
Fig. 1. MRG15 is important for DNA-repair. Wild-type, Mrg15 het and null MEFs were untreated (?IR) or c-irradiated at 4 Gy (+IR) and
harvested at various times post exposure for comet analysis. Two clones of each genotype were tested. Distributions of percent cells with damaged
DNA in tails are shown.
S.N. Garcia et al. / FEBS Letters 581 (2007) 5275–5281
following DNA-damage using a clonogenic assay. Irradiated
Mrg15 null and het MEFs cloned poorly and the percentage
of large colonies were much fewer than in unirradiated con-
trols (Fig. 2A and B). Irradiated wild-type cells, in contrast,
cloned as well as unirradiated cells. This result was obvious
by viewing the dishes, and was confirmed when we counted
the number of colonies with over 100 cells in treated and un-
treated MEFs of all genotypes. In contrast to wild-type MEFs,
which had the same number of colonies in both conditions,
Mrg15 het and null MEFs showed reduced cell growth after
IR (Fig. 2C). Due to clonal heterogeneity, and to demonstrate
that the result was not unique to a single clonal isolate, we used
a minimum of two independently isolated wild-type, null and
het clones in these studies. The results demonstrate that loss
of one copy of MRG15 is sufficient to affect MEF cell growth
post exposure to IR.
We then determined the short-term survival of Mrg15 mu-
tant MEFs after IR using colony formation and cell growth as-
says. We counted attached cells (1 day) and number of cells per
colony (4 days) post-treatment. At day 1, cell numbers were no
different among genotypes or between untreated and treated
cells (data not shown). This indicates that apoptosis does not
contribute to the differential survival of Mrg15 mutant versus
wild-type MEFs observed in the long-term clonogenic assay,
after IR (Fig. 2). Data obtained at 4 days, confirmed this as
wild-type MEFs showed the same pattern of colony formation
in untreated and treated conditions (Fig. 3), whereas the
percentage of large clones in irradiated null and het MEFs
was decreased relative to untreated cells. Conversely, there
were more small clones following treatment in these cells. This
was further confirmed by mass cell culture growth measure-
ments done at 24 h intervals using the MTT assay, from days
1 to 5 after IR; wild-type cell growth unaffected by IR, mutant
MEF growth inhibited (data not shown).
These data suggest that reduced colony numbers after long-
term culture of Mrg15 mutant MEFs in response to IR, reflect
defects in growth and not increased apoptosis, and that wild-
type MEFs have efficiently repaired DNA-damage at this dose.
3.3. Histone acetylation is delayed in Mrg15 null and het MEFs
The NuA4/Tip60-HAT complex acetylates H2A at lysine 5
(K5) and TRRAP (another component of the Tip60 complex)
deficient MEFs exhibit impairment of acetylation of H2A post
IR that results in defective accessibility and recruitment of
additional repair proteins, such as 53BP1, to the damage sites
. In Drosophila melanogaster, dTip60 and dMRG15
proteins have been shown to be necessary for the exchange
of c-H2Av for unmodified H2Av at sites of repair . We
therefore determined whether MRG15 might be required for
proper acetylation of H2A in response to DNA-damage.
Acid extracted histones were analyzed by Western blot to
assess whether there were any differences in the levels of
acetyl-H2A and c-H2AX in Mrg15 wild-type and null MEFs
following IR. To determine the specificity of the anti-Ac-K5-
H2A antibody, we prepared acid extracted histones from
wild-type and Mrg15 null MEFs treated with or without
trichostatin A (TSA), an inhibitor of class I HDACs, and per-
formed Western analyses using anti-Ac-K5-H2A or anti-H2A
antibodies (Fig. 4A). The major band recognized by the anti-
Ac-K5-H2A antibody was the size expected for H2A and the
intensity of this band increased significantly following TSA
treatment. We designated this band H2A. An additional pro-
tein, of a slightly higher molecular weight, also exhibited in-
creased acetylation following TSA treatment. This band
most likely is Ac-H2AX because the size was the same as that
of c-H2AX, as discussed below. There was no difference in the
response of wild-type or null cells following TSA treatment.
The total amount of H2A did not change after IR in both cell
types (Fig. 4B, upper panel). Acetylation of H2A in wild-type
MEFs increased significantly at 45 min after IR and these levels
were maintained at later time points. In contrast, acetylation of
H2A in Mrg15 null MEFs did not increase until 180 min after
IR (Fig. 4B, second panel). This impaired pattern of acetylation
of H2A was very similar to that observed in TRRAP deficient
MEFs following IR . When the blot was developed for a
longer time, an additional slightly higher molecular weight
band than Ac-H2A was observed (Fig. 4B third panel). The size
of this band was the same as that of c-H2AX. The anti-Ac-K5-
H2A antibody should recognize Ac-K5-H2AX as well as Ac-
H2A due to sequence similarity. We therefore conclude that
the upper band is indeed Ac-H2AX.
Acetylation of H2AX in wild-type MEFs increased after IR
and the kinetics were similar to that of H2A (Fig. 4B, third
Fig. 2. Mrg15 null and het MEFs are impaired in growth post
exposure to IR. (A) Representative wild-type and Mrg15 null MEF
clones exposed to 3 Gy and seeded at 2500 cells per dish. Cells were
grown for 10 days and stained with crystal violet. (B) Independent
clonal isolates of wild-type, Mrg15 null and het MEFs treated as in A.
(C) Quantitation of colony formation efficiency of Mrg15 mutant
MEFs (from 2B) after IR. Colonies with >100 cells were counted. The
percentage of untreated cells was set at 100%. (This is a representative
result for at least two independent clones for each genotype.) Error
bars represent S.E. for triplicate samples.
S.N. Garcia et al. / FEBS Letters 581 (2007) 5275–5281
panel). In contrast, acetylation of H2AX in Mrg15 null MEFs
did not increase until 180 min after IR, although basal levels
were higher than wild-type (Fig. 4B, third panel). Thus,
MRG15 is important for efficient acetylation of both H2A
and H2AX in mammalian cells, presumably through its asso-
ciation with the NuA4/Tip60-HAT complex.
The phosphorylation of H2AX (c-H2AX), a mammalian his-
tone variant, is another modification that occurs on chromatin
flanking strand breaks and is required for proper recruitment of
DNA-repair proteins to sites of damage . However, Mrg15
null MEFs did not exhibit defects in this phosphorylation pro-
cess post exposure to IR (Fig. 4B, bottom panel).
We further analyzed the formation of c-H2AX foci at differ-
ent times following IR in various Mrg15 MEF clones by
microscopy, using deconvolution algorithms. Western analysis
indicated phosphorylation levels of H2AX were increased
upon exposure to damage in Mrg15 null MEFs similar to what
was observed in wild-type MEFs. Immunostaining of wild-
type MEFs showed maximal foci formation as early as
30 min post exposure to IR (Fig. 4C and D). In null as well
as het cells some c-H2AX foci were observed at 30 min but
did not reach maximum levels until 60 min post IR (Fig. 4C
and D). These data suggest that even hemizygous expression
of MRG15 influences efficient formation of c-H2AX foci at
sites of damage in mammalian cells.
3.4. Recruitment of 53BP1 to nuclear foci is delayed in Mrg15
We assessed recruitment of the DNA-repair factor 53BP1 to
strand breaks in Mrg15 mutants by immunofluorescence. Con-
sistent with the histone H2A and H2AX acetylation data
(Fig. 4B), formation of nuclear 53BP1 foci in Mrg15 null and
het MEFs, at various times post exposure to IR, was markedly
delayed when compared with wild-type cells (Fig. 5A). Foci
were observed at 30 min post IR in wild-type MEFs and this
number increased at 60 min post treatment. Relatively few to
no 53BP1 foci were present at 30 min post exposure in het
and null MEFs and, though the number of foci increased at
60 min post exposure, this was consistently lower when com-
pared to wild-type (Fig. 5B). Thus, in Mrg15 null and het
MEFs recruitment of the initial DNA-repair machinery is less
robust and delayed when compared with wild-type cells.
In this study, we demonstrate a role for MRG15 in repair of
DNA-damage and cell growth in response to IR in mammalian
cells, even at hemizygous levels of expression. This sensitivity
to MRG15 depletion most likely reflects its participation as
a cofactor in multiple chromatin modifying complexes, includ-
ing the NuA4/Tip60-HAT and Sin3-HDAC1 complexes, that
have been implicated in DNA-repair. In fact, deletion of other
components of the NuA4/Tip60-HAT complex has been
shown to result in defects in histone H4 and H2A acetylation
and, as a consequence, impaired recruitment of DNA-repair
machinery to sites of damage .
It has been shown that, in D. melanogaster, the NuA4/Tip60-
HAT complex is important for the exchange of c-H2Av for
unmodified H2Av at sites of DNA-repair . We have found
that Mrg15 null MEFs are delayed in H2A and H2AX acety-
lation and c-H2AX foci formation after IR. The MRG15 pro-
tein does not itself have HAT activity and, therefore, Mrg15
null cell defects in H2A and H2AX acetylation are most likely
due to improper targeting of the NuA4/Tip60-HAT complex
Fig. 3. Colony size distribution assay of MEFs. Untreated and c-irradiated MEFs (100 cells) were plated in 60-mm dishes and incubated for 4 days.
The cell number in each colony was determined.
S.N. Garcia et al. / FEBS Letters 581 (2007) 5275–5281
to sites of damage in the absence of this protein. Another pos-
sibility is that deletion of MRG15 disrupts the integrity of the
NuA4/Tip60-HAT complex in mammalian cells. The fact that
the yeast NuA4 complex is intact in cells deleted for the yeast
MRG15 homolog, EAF3 , does not eliminate the possibil-
ity that this process might differ across species. In support of
this, deletion of the EAF3 gene in yeast does not affect cell via-
bility, whereas deletion of MRG15 has many detrimental ef-
fects on mouse development . We observed delayed
acetylation in Mrg15 mutant MEFs and subsequently delayed
53BP1 foci formation in response to IR. MRG15 appears to be
more important for c-H2AX foci formation in contrast to
observations made in Drosophila . These results further
support the idea that the function of MRG15 varies with cell
type and origin, despite the high degree of conservation of
the protein across species.
ATM kinase is a key player in the activation of cell cycle
checkpoints in response to radiation-induced DNA-damage
 and Tip60 acetylates and activates ATM after DNA-dam-
age . MRG15, which associates with the Tip60 complex,
when decreased could potentially modify the action of this
complex and thereby affect down stream pathway(s) of
ATM. UV irradiation of cells also results in rapid phosphory-
lation of H2AX, but different kinases and pathways than those
involved in DNA double-strand break repair are activated .
Thus, Mrg15 null and het cells may incorrectly activate such
kinases or pathways as a compensatory mechanism for the de-
creased levels of MRG15 and thereby decreased activity of the
Tip60 complex. We have observed that the kinetics of c-H2AX
levels in Mrg15 null MEFs following IR were similar to those
of wild-type MEFs. However, maximal foci formation was de-
layed in Mrg15 null and het MEFs. This may result from (i)
activation of phosphatases which dephosphorylate c-H2AX
outside foci after initiation of c-H2AX foci formation and
(ii) redistribution of c-H2AX to DNA-damage sites through
unknown mechanisms. Our preliminary proteomic analysis
of MRG15 indicates that MRG15 interacts with many other
proteins and is associated with multiple protein complexes.
The genotype of Mrg15 het or null most likely affects different
processes than wild-type and results in the phenotypes we have
observed. Details regarding this remain to be determined.
An example of the complexity of MRG15 function(s) is the
fact that it interacts with a number of proteins that are not
associated with the NuA4/Tip60-HAT and Sin3-HDAC
Fig. 4. Mrg15 null MEFs show delayed H2A-K5 acetylation and c-H2AX foci formation in response to IR. (A) Histones were acid extracted from
wild-type and Mrg15 null MEFs treated without (?) or with (+) 0.4 lM trichostatin A for 16 h. Immunoblots were probed with anti-acetyl-K5-H2A
and anti-H2A antibodies. (B) Histones were acid extracted from wild-type and Mrg15 null MEFs at various time points post treatment with 10 Gy.
Immunoblots were probed with anti-acetyl-K5-H2A antibodies (second and third panels), stripped and probed with anti-c-Ser139-H2AX (fourth
panel) and, finally, stripped and probed with anti-H2A antibodies (top panel) as loading control. (C) Cells from wild-type, het, and null MEFs were
fixed and stained with anti-c-H2AX (FITC) antibodies at 0, 30 and 60 min post exposure to 10 Gy. Representative cells from three experiments are
shown. Scale bars, 10 lm. (D) The number of c-H2AX foci per cell after treatment with IR, from two independent experiments.
S.N. Garcia et al. / FEBS Letters 581 (2007) 5275–5281
complexes, such as the hMOF-HAT and the retinoblastoma
protein [22,23]. It has recently been shown that the hMOF-
HAT acetylates the ATM kinase thereby modulating phos-
phorylation of its downstream DNA-repair targets in response
to IR . Thus, MRG15 function(s) in DNA-repair could be
mediated through such protein associations, that occur inde-
pendent of NuA4 and HDAC interactions. Many of these
MRG15-interacting proteins are not conserved across species,
suggesting that MRG15 will likely function in DNA-repair
through a number of distinct mechanisms that are cell type
and species dependent.
In summary, we have demonstrated a requirement for
MRG15 in DNA-repair and cell growth in response to IR
and this is dependent on protein levels, as hemizygous expres-
sion of MRG15 results in the same effect on DNA-repair as in
null cells. Additionally, MRG15 is important for efficient acet-
ylation of both H2A and H2AX, formation of c-H2AX foci
and as a consequence the recruitment of DNA-repair machin-
ery to sites of damage. These results underscore the sensitivity
of precise amounts of functional DNA-repair proteins for
maintenance of an intact genome, and emphasize the impor-
tance of analyzing protein function in different species. Over-
or under-expression of key proteins can disrupt a delicate
balance resulting in a decrease in the efficacy of DNA-repair
and related processes including cell growth, apoptosis, cell
senescence and tumorigenicity.
Acknowledgements: We thank Christina Hawks for assistance with
immunofluorescence microscopy, Natalia Podlutskaja for comet anal-
ysis and Emiko Tominaga for MEF culture. We also thank James R.
Smith, Christi Walter and James Jackson for comments on the manu-
script. This work was supported by NIA Grants, T32AG021890
(S.N.G.) and KO7AG25063 (A.P.), the Ellison Medical Foundation
(O.M.P.-S.), and the American Federation for Aging Research (A.P.
Fig. 5. 53BP1 foci formation is delayed in Mrg15 null MEFs post exposure to IR. (A) Cells from wild-type, het, and null MEFs were fixed and
stained with anti-53BP1 (Texas red) antibodies at various times post exposure to 10 Gy. Representative cells from three experiments are shown. Scale
bars, 10 lm. (B) The number of 53BP1 foci per cell after treatment with IR, from two independent experiments.
S.N. Garcia et al. / FEBS Letters 581 (2007) 5275–5281
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