Loss of the chromatin regulator MRG15 limits neural
stem/progenitor cell proliferation via increased
expression of the p21 Cdk inhibitor
Meizhen Chena, Olivia M. Pereira-Smitha,b, Kaoru Tominagaa,b,⁎
aBarshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio,
Texas 78245, USA
bDepartment of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio,
Texas 78245, USA
Received 22 October 2010; received in revised form 24 February 2011; accepted 15 April 2011
Available online 25 April 2011
and DNA damage repair. We have found that it is also important for neural stem/progenitor cell (NSC) function and
neurogenesis. Here, we demonstrate that expression of the cyclin-dependent kinase inhibitor p21 is specifically up-regulated in
Mrg15 deficient NSCs. Knockdown of p21 expression by p21 shRNA results in restoration of cell proliferation. This indicates that
p21 is directly involved in the growth defects observed in Mrg15 deficient NSCs. Activated p53 accumulates in Mrg15 deficient
NSCs and this most likely accounts for the up-regulation of p21 expression in the cells. We observed decreased p53 and p21
levels and a concomitant increase in the percentage of BrdU positive cells in Mrg15 null cultures following expression of p53
shRNA. DNA damage foci, as indicated by immunostaining for γH2AX and 53BP1, are detectable in a sub-population of Mrg15
deficient NSC cultures under normal growing conditions and the majority of p21-positive cells are also positive for 53BP1 foci.
Furthermore, Mrg15 deficient NSCs exhibit severe defects in DNA damage response following ionizing radiation. Our
observations highlight the importance of chromatin regulation and DNA damage response in NSC function and maintenance.
© 2011 Elsevier B.V. All rights reserved.
Chromatin regulation is crucial for many biological processes such as transcriptional regulation, DNA replication,
Establishment and maintenance of a functional central
nervous system depend on multipotent and self-renewable
neural stem/progenitor cells (NSCs) during development and
also in adult brain (Gage, 2000; Temple, 2001). The self-
renewal ability of NSCs is essential for maintaining the stem
cell pool for brain development and replacement of cells in
the brain during the lifespan of an organism (Shi et al., 2008;
Ma et al., 2009). NSCs also supply three major differentiated
cell types: neurons, astrocytes, and oligodendrocytes in the
brain for differentiation into restricted progenitor cells
(Reynolds and Weiss, 1992; Reynolds and Weiss, 1996).
⁎ Corresponding author at: Barshop 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. Fax: +1
210 562 5093.
E-mail address: firstname.lastname@example.org (K. Tominaga).
available at www.sciencedirect.com
Stem Cell Research (2011) 7, 75–88
1873-5061/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
Therefore, maintaining the appropriate balance between
self-renewal and multilineage differentiation in NSCs is
critical for development of functional brain networks in
adults as well as during embryonic development.
Chromatin structure can be dynamically changed by
covalent modifications of histone tails such as acetylation and
methylation. Chromatin regulation via these modifications is
involved in many important biological processes such as
transcription, DNA replication and DNA damage repair.
Chromatin regulation is also important for NSC function and
neurogenesis (Borrelli et al., 2008; Li and Zhao, 2008; Mehler,
The brain-specific knockout of Mll1 (mixed-lineage leukemia
1), a histone H3 lysine 4 (H3K4) methyltransferease in mice,
exhibits severely impaired neurogenesis (Lim et al., 2009).
Chromatin at the Dlx2 gene, which is a key downstream
regulator of neurogenesis, is bivalently marked by both
H3K4me3 and H3K27me3 and the Dlx2 gene fails to activate
(CREB binding protein) the histone acetyltransferease inhibits
differentiation of embryonic NSCs into all three lineages and
this most likely explains the cause of cognitive dysfunction in
Rubinstein–Taybi syndrome where one allele of CBP is deleted
or mutated (Wang et al., 2010). Interestingly, it has been
recently shown that deregulation of histone H4 lysine 12
(H4K12) acetylation is associated with age-dependent memory
impairment in the hippocampus in which NSCs are located
a hippocampal gene expression program associated with
memory consolidation in aged mice. HDAC2 but not HDAC1
negatively regulates memory formation and memory-related
gene expression through modulation of histone acetylations
Therefore, epigenetic mechanisms play crucial roles in NSC
function and neurogenesis by modulating important changes in
MRG15 was initially isolated as a member of gene family
that is related to cellular senescence and cell growth control
and is evolutionarily highly conserved (Bertram et al., 1999;
Marin and Baker, 2000; Bertram and Pereira-Smith, 2001).
MRG15 has a chromodomain in the N-terminus (Cavalli and
Paro, 1998; Jones et al., 2000; Brehm et al., 2004) and can
directly bind to di- or tri-methylated histone H3 at lysine 36
(H3K36me) through this domain (Joshi and Struhl, 2005;
Zhang et al., 2006; Sun et al., 2008). It has been shown that
MRG15 formscomplexes with both histone acetyltransferases
(HATs) and histone deacetylases (HDACs) (Ikura et al., 2000;
Cai et al., 2003; Doyon et al., 2004; Doyon and Cote, 2004;
Lee et al., 2009; Moshkin et al., 2009). Although a role of
MRG15-containing mSin3/HDAC complex in mammals still
remains unknown, similar complex in budding yeast, named
Rpd3S, is important for deacetylation of coding regions to
suppress spurious intragenic transcription through H3K36me
of a homologous complex in fission yeast causes increased
accessibility of DNA to genotoxic agents and widespread
abrogates global protective functions of chromatin (Nicolas
et al., 2007). MRG15 is also a stable component of Tip60 HAT
complex, which is implicated in transcriptional control as
well as DNA repair and apoptosis (Squatrito et al., 2006).
Purification of the Drosophila Tip60 complex demonstrated
that the components of the fly complex were very similar to
those in the mammalian complex (Kusch et al., 2004).
Moreover, it is evident that Tip60 and MRG15 are essential
components of this complex, as knockdown or deletion of
either gene results in an inability to repair DNA double strand
breaks (DSBs) as well as embryonic lethality. We, and others,
DSB repair (Murr et al., 2006; Garcia et al., 2007; Ikura et al.,
We have previously published that embryonic Mrg15 null
NSCs exhibit defects in growth as well as neuronal lineage
differentiation (Chen et al., 2009). In this study, we have
examined the molecular mechanism(s) of the growth defect
underlying Mrg15 deficiency in mouse embryonic NSCs by
comparing neurosphere cultures obtained from Mrg15 null
and wild-type embryonic brains. We have found increased
expression of p21Sdi1/Cip1/Waf1 (p21) in Mrg15 null NSC
cultures that most likely leads to the growth defect observed
in Mrg15 deficient cells, and provide evidence that ineffi-
cient DNA damage repair in Mrg15 null NSCs may contribute
to this growth defect.
p21 expression is upregulated in Mrg15 deficient
We have previously reported that Mrg15 deficient NSCs from
embryos show severe growth defects in vitro and in vivo (Chen
et al., 2009). Because BrdU incorporation of Mrg15 deficient
NSCsinculturewassignificantly reduced compared withthose
from wild-type, we speculated cell cycle control is dysregu-
lated in Mrg15 deficient NSCs. To further elucidate the
a cyclin-dependent kinase inhibitor, as a gene that was up-
regulated 3.3-fold in Mrg15 deficient NSCs compared with
wild-type cells. We further confirmed that p21 was upregu-
independent isolated clones of Mrg15 deficient NSCs by
quantitative RT-PCR (qRT-PCR) (Fig. 1A). We performed
statistical analysis using the unpaired t test to determine if
the difference in p21 expression levels in wild-type versus
Mrg15 deficient NSCs was significantly different or not. The p
value was 0.0788 when we compared the three wild-type with
Mrg15 null clones, and close to significance. This was because
one of the Mrg15 deficient NSC clones had relatively lower
from the analysis, the p value was highly significant 0.0005. It
appears that about 10% of the NSC clones do not show p21
upregulation though they still exhibit a growth defect,
suggesting the existence of a p21 independent mechanism
for growth defect observed.
We then determined the expression levels of p16INK4a
(Fig. 1B) and p19ARF (Fig. 1C) by qRT-PCR, since these other
cell cycle regulators have been shown to be important in
stem cell self-renewal (Jacobs et al., 1999; Molofsky et al.,
2006; Nishino et al., 2008). We did not find any correlation
between their expression and the cell growth defect of
76M. Chen et al.
Mrg15 deficient NSC cultures. p16 and p19 expression levels
were not statistically significantly different between wild-
type and Mrg15 deficient NSCs.
We also determined p21 protein levels in wild-type and
Mrg15 deficient neurosphere cultures by Western blot. As
expected, p21 protein expression was upregulated in early
passage of Mrg15 deficient but not wild-type neurosphere
cultures (Fig. 2 upper panel) and the difference was
statistically different (p value 0.0085, unpaired t test). This
suggests that p21 upregulation in Mrg15 deficient NSCs is
p21 expression was also upregulated in later passage (6th)
wild-type neurosphere cultures (Fig. 2 lower panel), at which
time these cultures also exhibited slow growth.
Downregulation of p21 expression improves cell
growth in Mrg15 deficient NSCs
To determine whether p21 is an active regulatory gene in the
growth defect of Mrg15 deficient NSCs, we performed p21
knockdown in these cells and examined the rate of BrdU
incorporation. Because p21 expression was also upregulated
in late passage of wild-type NSC cultures which showed slow
growth, we compared the effect of p21 knockdown in first
passage cultures of Mrg15 deficient NSCs with that of late
passage (6th) cultures of wild-type cells. We used a lentivirus
shRNA expression system for this purpose because p21
knockdown occurred efficiently (Fasano et al., 2007; Phoenix
and Temple, 2010) and siRNA transfection using liposomes
was highly toxic to the NSCs. Knockdown of p21 following
infection with a lentivirus expressing p21 shRNA occurred
efficiently (N80%) in both Mrg15 deficient and wild-type
NSCs (Fig. 3A) as described previously (Fasano et al., 2007).
Infection efficiency of lentivirus, as indicated by GFP
expression, was not different between Mrg15 deficient and
wild-type NSCs or empty vector and p21 shRNA constructs.
Knockdown of p21 in Mrg15 deficient NSCs significantly
increased BrdU incorporation (from 24% to 36%) (Fig. 3B).
This result indicates that upregulation of p21 expression
contributes to the growth defect in Mrg15 deficient NSCs.
Late passage wild-type NSC cultures also had a reduced rate
of BrdU incorporation (26%), which corresponded with slow
growth, and knockdown of p21 improved BrdU incorporation
(45%)(Fig. 3C). This suggests that p21 is a critical regulator
for passage-dependent growth regulation in NSCs. The effect
of p21 knockdown in Mrg15 deficient NSCs was less than that
in late passage wild-type NSCs indicating that other
molecular mechanisms may exist for the observed growth
defects in Mrg15 deficient NSCs, in addition to p21 up-
We further confirmed the effect of p21 knockdown on cell
cycle distribution in early passage Mrg15 deficient NSCs and
6th passage wild-type NSCs using FACS. Similar to the BrdU
incorporation results, the S phase population significantly
increased in both Mrg15 deficient and wild-type NSCs after
infection with lentivirus expressing p21 shRNA (Fig. 3E and G)
when compared with that of cells infected with lentivirus
containing the empty vector (Fig. 3D and F).
We have previously shown that nestin positive cell
populations in Mrg15 deficient NSC cultures were almost
expression, in early passage E10.5 Mrg15 deficient NSC cultures.
mRNA expression levels of p21 (A), p16INK4a (B), and 19ARF (C)
genes were measured by qRT-PCR from three wild-type and
three Mrg15 deficient NSC cultures. Data was normalized to
expression levels of the β-actin gene. Each data represents the
average of three independent assays (mean±SEM).
Increased expression of p21, but not p16/p19
representative Western blot of p21 is shown. Total cell lysates
were prepared from first or sixth passage of neurospheres from
three wild-type and three Mrg15 deficient embryos. Equal
amounts of protein were loaded, transferred to nitrocellulose
membrane, and probed with antibodies for p21 and β-actin
p21 protein accumulation in NSC cultures. A
77 Role of MRG15 in NSC proliferation
Cells expressing empty vector (EV) or p21 shRNA were analyzed by Western blot to p21, β-actin (loading control), GFP (indication of
infection efficiency), and MRG15. (B, C) Representative BrdU immunostaining in Mrg15 deficient cells (B) at first passage and wild-type
cells (C) at sixth passage. Single cells were plated on poly-L-lysine-coated coverslips, grown for 48 h, and incubated with 10 μM BrdU
for 1 h. Incorporated BrdU was detected by mouse anti-BrdU antibody followed by incubation with Alexa Fluor 594-conjugated
secondary antibody. The percentage of positive cells was determined by counting under a fluorescent microscope. Error bars
represent standard error of mean (SEM) of triplicate counts and p values determined using unpaired t test. (D, E, F, G) Cell cycle
distribution of GEF-positive Mrg15 deficient and wild-type NSCs after infection with a GFP-expressing lentivirus containing empty
vector or a p21 shRNA construct. (D) Mrg15 deficient NSCs with empty vector. (E) Mrg15 deficient NSCs with p21 shRNA construct. (F)
wild-type NSCs with empty vector. (G) wild-type NSCs with p21 shRNA construct.
p21 shRNA rescues cell proliferation in early passage (1st) Mrg15 deficient NSCs and late passage (6th) wild-type NSCs. (A)
78 M. Chen et al.
the same as that of wild-type cultures (Chen et al., 2009).
This suggests that the growth defect caused by p21
upregulation in Mrg15 deficient NSC cultures most likely
occurs in stem/progenitor populations and that these are not
increased by spontaneous differentiation in growth medium.
P53 is activated in Mrg15 deficient NSCs
It is well known that p21 expression is transcriptionally
regulated by the tumor suppressor gene p53. p53 is activated
by phosphorylation and acetylation in response to internal
and external stresses such as exposure to radiation, and
activated p53 upregulates expression of target genes,
wild-type NSCs for expression of the activated form of p53,
using an anti-phoshorylated p53Ser15 (Ser18 in mouse)
antibody, by Western blot (Fig. 4A). A higher amount of
activated p53 accumulated in Mrg15 deficient NSC cultures
compared with that in wild-type cultures, except in the case
of one clone of the 5 analyzed. When this clone was
eliminated from analysis the higher levels were significant,
p value 0.0132. Levels of p21 expression co-related with
accumulation of activated p53. We also confirmed complete
loss of Mrg15 expression in Mrg15 deficient NSCs.
We used a lentivirus encoding p53 shRNA (Ventura et al.,
2004) and an empty vector control to perform studies similar
to those done with p21, described above. Down-regulation of
p53 in Mrg15 deficient NSCs was confirmed by Western
analyses (Fig. 4B). BrdU incorporation indicated an increase
in positive cells following p53 knockdown in Mrg15 deficient
cells (Fig. 4C) and p21 positively stained cells were
decreased (Fig. 4D). This suggests that p53 is activated and
accumulates in Mrg15 deficient NSCs and this in turn
upregulates p21 expression.
DNA damage foci are detected in Mrg15 deficient
NSC cultures and are present in p21 positive cells
P53 is activated in response to intrinsic and extrinsic stimuli
such as DNA damage. Because activated p53 accumulates in
Mrg15 deficient NSC cultures, we examined whether there is
more DNA damage in Mrg15 deficient NSC cultures compared
using immunocytochemistry, because these two proteins form
foci at damage sites in DNA. Although we could not easily find
focus positive cells in wild-type NSC cultures (Fig. 5A, upper
panels), focus positive cells were easily detectable in Mrg15
panels). The focus number in Mrg15 deficient NSCs was usually
but the majority of these foci were γH2AX and 53BP1 double
positive. This result suggests that DNA damage accumulates in
Mrg15 deficient NSC cultures versus wild-type NSC cultures
under growing conditions.
Western blot analysis of phosphorylated p53 at Ser15 is shown. Total cell lysates were prepared from early passage neurospheres from
three wild-type and five Mrg15 deficient embryos. Equal amounts of protein were loaded, transferred to nitrocellulose membrane,
and probed with antibodies for phospho-p53 (Ser15), p53, p21, MRG15, and β-actin (loading control). (B) Mrg15 deficient NSCs
expressing empty vector (EV) or p53 shRNA were analyzed by Western blot to p53 and β-actin (loading control). (C) Percentage of BrdU
positive cells in early passage of Mrg15 deficient NSCs after lentivirus infection expressing EV or p53 shRNA. The percentage of BrdU
positive cells by immunostaining was determined by counting under a fluorescent microscope. Error bars represent standard error of
mean (SEM) of counts from ten fields and p values determined using unpaired t test. (D) Percentage of p21 positive cells in early
passage of Mrg15 deficient NSCs after lentivirus infection expressing EV or p53 shRNA. The percentage of p21 positive cells by
immunostaining was determined by counting under a light microscope. Error bars represent standard error of mean (SEM) of counts
from ten fields and p values determined using unpaired t test.
Accumulation of activated p53 in Mrg15 deficient NSC cultures and effects of p53 shRNA knockdown. (A) A representative
79 Role of MRG15 in NSC proliferation
P21 expression is upregulated by activated p53 after DNA
damage and involved in cell cycle arrest to repair damaged
DNA in cells. To examine whether accumulation of p21 in
Mrg15 deficient NSCs is caused by DNA damage response, we
performed co-immunostaining with anti-p21 and anti-53BP1
antibodies (Fig. 5B). p21-positive staining was observed in
Mrg15 deficient NSC cultures but not in early passage wild-
type NSC cultures (Fig. 5C). The majority of p21 positive cells
in Mrg15 deficient NSC cultures were also 53BP1 focus
positive (62.5±7.6%, Mean±SEM, n=12 fields), whereas we
could not detect 53BP1 foci in early passage wild-type NSC
cultures. We did not detect 53BP1 foci in about 30–35% of
type (upper panel) and Mrg15 deficient (lower panel) NSCs were fixed and foci of γH2AX (red) and 53BP1 (green) in nuclei detected by
immunostaining. Nuclei were counterstained with DAPI. (B) Wild-type (upper panel) and Mrg15 deficient (lower panel) NSCs were
fixed, co-stained with mouse anti-p21 (red) and rabbit anti-53BP1 (green) antibodies, and counterstained with DAPI. Examples of p21/
53BP1 foci double positive cells in merged images are shown in separate pictures. (C) Percentage of p21 positive cells in early passage
NSC cultures. Error bars represent standard error of mean (SEM) from 8 field counts for wild-type and 12 field counts for Mrg15
deficient cells. p values were determined using the unpaired t test. Representative data from two experiments is shown.
Co-staining of p21 with DNA damage foci in Mrg15 deficient NSCs. (A) Detection of DNA damage foci in NSC cultures. Wild-
80 M. Chen et al.
p21 positive cells of in Mrg15 deficient NSCs. Thus, p21
accumulation in these cells may occur via DNA damage
Defects of DNA damage response in Mrg15 deficient
The results described above suggest that MRG15 is critical
for DNA damage repair in NSCs. We therefore examined the
DNA damage response of Mrg15 deficient NSCs compared
with that of wild-type NSCs. Mrg15 deficient and wild-type
NSCs were subjected to γ-irradiation at 10 Gy and focus
formation of 53BP1 and γH2AX at indicated time points was
measured by immunostaining. As expected, foci for γH2AX
and 53BP1 were easily detected at all time points except
for 0 time point after irradiation in wild-type NSCs
(Fig. 6A). In Mrg15 deficient NSCs, some cells already had
small but detectable foci for γH2AX and 53BP1 at the
0 time point (less than 10 foci per nucleus, corresponding
to baseline level). Although we could detect focus
formation of these in Mrg15 deficient NSCs in response to
γ-irradiation, focus formation for both γH2AX and 53BP1
was significantly delayed and foci of these were usually
smaller compared with those in wild-type NSCs (Fig. 6B, C
and D). This result showed that Mrg15 deficient NSCs have
defects in the DNA damage response and this could result in
inefficient DNA repair. This inefficient DNA damage
response may explain partly the growth defect of Mrg15
deficient NSCs in growing conditions.
It has been reported that MRG15 directly interacts with
PALB2 and is involved in DNA damage repair by homologous
recombination (HR) after IR (Sy et al., 2009; Hayakawa et al.,
2010). Next, we examined whether RAD51 focus formation
ing. RAD51 foci were negative in almost all cells in both
genotypes without IR, although a few cells had one or two
very small foci (less than 5 foci in the nucleus). About 30% of
wild-type NSCs had RAD51 foci at 3 h after IR (Fig. 7A and C).
However, only 7% of Mrg15 deficient NSCs had RAD51 foci at
30, 90, and 180 min post exposure to 10 Gy, co-stained with mouse anti-γH2AX (red) and rabbit anti-53BP1 (green) antibodies, and
counterstained with DAPI to visualize nuclei. Examples of co-localization of γH2AX and 53PB1 foci in merged images are shown with
separated pictures. Representative cells from two experiments are shown. (C) Quantification of cells with γH2AX foci at 15, 30, 90,
and 180 min after IR (mean±SEM, n=5). (D) Quantification of cells with 53BP1 foci at 15, 30, 90, and 180 min after IR (mean±SEM,
n=5). p values determined using one-way ANOVA. Asterisk indicates, pb0.05; double asterisk indicates, pb0.01; triple asterisk
Defects in DNA damage response in Mrg15 deficient NSCs. Wild-type (A) and Mrg15 deficient (B) NSCs were fixed at 0, 15,
81 Role of MRG15 in NSC proliferation
the same time point after IR (Fig. 7B and C). Western analysis
revealed no significant difference in total protein levels of
RAD51 in wild-type versus Mrg15 deficient cells (Fig. 7D).
However, it is focus formation that is the critical response
suggesting that MRG15 is also important for RAD51 recruit-
ment at the damage sites after DNA damage and may affect
homologous recombination dependent DNA damage repair as
well as DNA damage response in NSCs.
Chromatin regulation is involved in DNA replication,
transcriptional regulation and DNA damage repair and is
a crucial step for stem cell self-renewal and function. We
had previously shown that inactivation of the chromatin
regulator MRG15 impairs proliferation of embryonic NSCs.
In this report, we demonstrate that this occurs through
activation of p53 and resulting increased expression of the
cdk inhibitor p21. We have found increased expression of
p21 and activated p53 in primary cell cultures of Mrg15
null NSCs but not wild-type. Focus formation of 53BP1,
which indicates the presence of DNA damage, also co-
localizes with p21 in Mrg15 null NSCs in growing culture
condition, without any extrinsic insults. Focus formation of
53BP1 after γ-irradiation is delayed in Mrg15 null NSCs
compared with wild-type NSCs. Our observations suggest
that chromatin regulation and DNA damage repair through
were fixed with or without IR at 180 min post exposure, co-stained with rabbit anti-RAD51 (green) and mouse anti-γH2AX (red)
antibodies, and counterstained with DAPI to visualize nuclei. Representative pictures for colocalization of RAD51 and γH2AX in merged
images are shown with separated pictures. Representative cells from two experiments are shown. (C) Quantification of cells with the
RAD51 foci at 180 min after IR (mean±SEM, n=5). p values determined using unpaired t test. (D) Detection of RAD51 expression level
by Western blot. Total cell lysates were prepared from NSCs with or without IR at 180 min post exposure and levels of RAD51 and β-
actin (loading control) were detected by Western blot.
Defects of RAD51 focus formation in Mrg15 deficient NSCs after IR exposure. Wild-type (A) and Mrg15 deficient (B) NSCs
82M. Chen et al.
MRG15 complex(es) are essential to establish and maintain
a functional NSC pool in mouse brain during development.
function in various stem cells including NSCs (Nijnik et al.,
2007; Rossi et al., 2007; McKinnon, 2009). Perturbations in
genes involved in DNA damage response signaling pathways
and/or DNA repair are associated with neurological disorders
such as neurodegeneration, microcephaly and brain tumors,
suggesting that the inability to respond to DNA damage
interferes with normal tissue homeostasis (McKinnon, 2009;
Lee and Mckinnon, 2007; Frappart and McKinnon, 2008). DNA
damage response and repair are critical for stem/progenitor
nervous system. Mice deficient for anyone of the many genes
that play a role in the cellular response to DNA damage (Atm,
et al., 2005; Shull et al., 2009) or genes actively involved in
DNArepair(BRCA2and Lig4)(Leeetal.,2000;O'Driscoll etal.,
2001; Frappart et al., 2007) all share a phenotype of
neurological failure due to defective DNA damage repair.
These deletions affect NSC self-renewal as well as neuronal
MRG15 is involved in DNA damage repair (Kusch et al.,
2004; Garcia et al., 2007) in addition to transcriptional
regulation of cell proliferation (Tominaga et al., 2005). Thus
there are two possibilities to explain the molecular mech-
anism by which MRG15 could be involved in the proliferative
defects in Mrg15 null NSCs that we have observed. These are
via the Tip60 complex or the PALB2/BRCA2 interaction
involving MRG15. In Drosophila, the Tip60 complex acety-
lates nucleosomal phospho-H2Av, a Drosophila H2AX homo-
log, in response to ionizing radiation and exchanges it with an
unmodified H2Av (Kusch et al., 2004) and knockdown of
either dTip60 or dMrg15 in Drosophila cells impaired this
acetylation and exchange of H2Av following irradiation. In
mammalian cells, depletion of either Tip60 or TRRAP, other
components of the Tip60 complex, results in impairment of
recruitment of DNA-repair proteins such as 53BP1 to damage
sites (Murr et al., 2006). The Tip60/TRRAP complex
acetylates histone H2A and H2AX at DNA damage sites and
thereby maintains open chromatin and facilitates access of
DNA repair machinery to DNA strand break sites. Ikura et al.
showed that H2AX acetylated by Tip60 after ionizing
radiation leads to ubiquitination by DNA damage induced
UBC13 (Ikura et al., 2007). Tip60 promotes the acetylation-
dependent ubiquitination of H2AX by UBC13, causing H2AX
release from chromatin and thereby facilitates chromatin
reorganization following DNA damage. We have also shown
that acetylation of histone H2A, in response to ionizing
radiation (IR), is impaired and recruitment of DNA repair
proteins delayed in Mrg15 null MEFs (Garcia et al., 2007).
Because the Tip60 complex is important for self-renewal of
embryonic stem (ES) cells (Fazzio et al., 2008a, 2008b), the
role of MRG15 in proliferation defects of NSC may also occur
via the Tip60 complex.
Another possible connection between MRG15 and DNA
damage is PALB2. PALB2 was originally identified as an
interacting partner of BRCA2 which is a tumor suppressor for
breast and ovarian cancers and is required for the loading of
the BRCA2–RAD51 repair complex onto DNA. More recently,
it was shown that PALB2 can also bind to BRCA1 and that it is
an integral component of the BRCA1–BRCA2–RAD51 axis,
which is critical for the maintenance of genomic stability via
recombinational repair. Two groups have shown that MRG15
can bind directly to PALB2 and that knockdown of MRG15
affects homology-directed DNA repair (Sy et al., 2009;
Hayakawa et al., 2010), although results from these reports
are contradictory. Sy et al. showed that PALB2-deficient
EUF1341F cells reconstituted with MRG15-binding defective
PALB2 mutant exhibited increased gene conversion rates
although damage-induced RAD51 foci formation and mito-
mycin C sensitivity returned to normal in these cells. This
suggests that MRG15 inhibits homologous recombination
through PALB2 interaction. On the other hand, Hayakawa
et al. demonstrated that MRG15 deficient cells showed
reduced efficiency for homology-directed DNA repair and
hypersensitivity to DNA interstrand cross-linking agents
similar to PALB2 or BRCA2 deficient cells. They also showed
that MRG15 knockdown diminished the recruitment of
PALB2, BRCA2, and RAD51 to DNA damage sites. Although
we do understand this discrepancy, our previous and current
findings support the fact that MRG15 is an essential factor for
DNA damage repair in somatic cells as well as stem cells.
Deletion of Brca2 in the entire nervous system in mice leads
to microcephaly and defects in neurogenesis (Frappart et al.,
2007). p53 contributes to these phenotypes because simul-
taneous inactivation of p53 improves Brca2 depletion
phenotypes in mouse brains. p53 is responsible for both
cell growth defect and apoptosis in Brca2-deficient NSCs.
MRG15 may therefore also function through a BRCA2 pathway
The tumor suppressor p53 is an important regulator of
cell cycle and apoptosis in both developing and adult brain
and plays an important role in maintaining a proper balance
of neural stem/progenitor pools. It is known that one of the
p53 downstream target genes, p21, is also important for
maintaining NSC self-renewal during the lifespan of an
organism (Kippin et al., 2005; Pechnick et al., 2008). In the
absence of p53, NSCs isolated from adult mice as well as
mouse embryos exhibit a higher proliferation rate in culture
(Gil-Perotin et al., 2006; Meletis et al., 2006; Armesilla-Diaz
et al., 2009). On the other hand, p44Tg mice, in which p53
is constitutively activated, exhibit premature aging without
increased tumor risk, indicating that constitutive activation
of p53 limits NSC self-renewal following constitutive
expression of p21 (Medrano and Scrable, 2005; Medrano et
al., 2009). Therefore, it is possible that p53 activation
following increased p21 expression may limit self-renewal
potential in Mrg15 deficient NSCs. This is supported by the
fact that knockdown of p53 levels results in decreased p21
expression and an increase in BrdU-positive cycling cells in
both wild-type and Mrg15 null cells, as shown in this study.
It is known that p53 also inhibits neuronal differentiation
because p53 deficient NSCs differentiate into neuronal
lineage in higher rate (Meletis et al., 2006; Ferron et al.,
2009). We have previously shown Mrg15 deficient NSCs had
a defect in neuronal differentiation. This defect may also be
explained by upregulation of p53 activity in Mrg15 deficient
Epigenetic mechanisms are essential for normal brain
development and function and dysregulation in chromatin
regulation results in neurodegenerative disorders, such as
Alzheimer disease (Graff and Mansuy, 2009). It has been
shown that expression of cell cycle-related proteins
83 Role of MRG15 in NSC proliferation
increases in the degenerating neurons in Alzheimer disease
and re-entry into the cell cycle in neurons occurs aberrantly
(McShea et al., 1999; Raina et al., 2001; Evans et al., 2007).
Because MRG15 is involved in cell cycle regulation through
chromatin regulation, dysregulation of MRG15 may contrib-
ute to the initiation and/or progression of such neurodegen-
Our knowledge of the many functions of MRG15 continues
to expand. We have determined that some of the major
functions of MRG15 include transcriptional regulation via
complexes involving both HATs and HDACs. Although we here
demonstrate the importance of MRG15 in NSC proliferation
through a DNA damage response, we speculate that other
molecular mechanisms are also involved. MRG15 may
directly modulate expression of genes that are important
for cell cycle regulation. A small percentage of Mrg15
deficient NSCs did not show DNA damage foci, although p21
was overexpressed in these cells. MRG15 is a component of
mSin3/HDAC1/Pf1 complex and it has been shown that this
complex has a transcriptional repressor activity (Yochum and
Ayer, 2002; Hayakawa et al., 2007). Although this complex is
mainly recruited into coding regions of actively expressing
genes, which are marked by trimetylation of histone H3 at
lysine 36, to prevent uncontrolled chromatin relaxation
downstream of the transcriptional start sites (Jelinic et al.,
2011), it remains possible that this complex repressively
modulates the promoter activity of specific genes such as
p21. In fact, it has been shown that MRGX, MRG15 homolog,
also has a transcriptional repressor activity depending on the
cell-type analyzed (Tominaga et al., 2003). p21 expression
may be repressed by a MRG15-containing repressor complex
in wild-type NSCs under normal conditions and p21 accumu-
lation in Mrg15 deficient NSCs may occur through de-
repression of this promoter activity in a DNA damage
independent manner. This possibility is currently being
explored. It may also be possible that MRG15 negatively
controls p21 expression via the Tip60/p400 complex, of
which MRG15 is a component (Tyteca et al., 2006; Gevry
et al., 2007; Park et al., 2010). Additional studies are
required to determine the complexity of the molecular
Our study demonstrates a critical role of the chromatin
regulator MRG15 in proliferation of NSCs in vitro. Loss of
Mrg15 in NSCs shows increased 53BP1 focus formation,
activated p53, and a resulting increase in p21. This suggests
that maintenance of genomic integrity through chromatin
regulation is essential to establish a functional nervous
system. This study should provide insights into the molecular
mechanism(s) involved in chromatin regulation in NSC self-
renewal and function in the mouse brain.
Mrg15 heterozygous mice were maintained under pathogen-
free conditions with approval of the institutional animal care
committee and were treated in accordance with the NIH
Guide for the Care and Use of Laboratory Animals. We used a
mixed genetic background (C57BL/6J and 129SvEv) ofmice in
this study (Tominaga et al., 2005) as backcrossing to the
C57BL/6 background exacerbated the fetal phenotype.
Neural stem/progenitor cell (NSC) culture
Cerebral cortices of timed pregnant mouse embryos (E10.5)
were dissociated, single cells obtained by trypsin digestion
following trituration with a polished Pasteur pipette and
cultured in 35-mm tissue culture dishes (Corning Incorporat-
ed, Corning, NY, USA) in Neurobasal Medium (Gibco-BRL,
Gaithersburg, MD, USA) supplemented with 20 ng/ml FGF-2/
basic FGF (Upstate, Temecula, CA, USA), 20 ng/ml EGF (BD
Bioscience, Bedford, MA, USA), 2 mM L-glutamine, 1×B27,
and 50 U/ml penicillin- 50 μ/ml streptomycin (Gibco-BRL,
Gaithersburg, MD, USA). The cells were maintained in a
humidified incubator at 37 °C in 95% air/5% CO2for 1 week
and neurospheres were isolated. Isolated neurospheres were
trypsinized, cells were counted using a hemocytometer and
used for the next experiments.
PCR array and quantitative RT-PCR (qRT-PCR)
Total RNA was isolated from neurospheres lysed with Trizol
Reagent (Invitrogen) and purified with the RT2qPCR-Grade
RNA Isolation Kit (SABiosciences, PA-001). Elimination of
contaminating DNA and cDNA synthesis was performed with
RT2First Strand Kit (SABiosciences, C-03) and real-time PCR
performed by ABI Prism 7500 sequence detector (Applied
Biosystems) using the RT2 Profiler™ PCR Array Mouse Cell
Cycle (SABiosciences, PAMM-020A), according to the manu-
facturer's instructions. Data analyses were performed using
the PCR Array Data Analysis Web Portal.
For qRT-PCR, cDNAs were synthesized from neurosphere
derived total RNA (0.3 μg) by priming with 200 ng of random
hexamer, 0.5 mM dNTP and 100 units of SuperScript II Reverse
Transcriptase (Invitrogen) at 42 °C for 1 h. Real-time PCR was
carried out using SYBR® Green PCR Master Mix (Invitrogen).
Fluorescence was monitored by GeneAmp 7900HT Sequence
Detection system. Expression levels of target genes were
normalized to those of β-actin. The following primer sets
were used; p21-5′ (5′-GCAGATCCACAGCGATATCCAG-3′) and
p21-3′ (5′-CGAAGAGACAACGGCACACTTT-3′), p16-5′ (5′-
CGAACTCTTTCGGTCGTACCC-3′) and p16-3′ (5′-CGAATCTG-
CACCGTAGTTGAGC-3′), p19-5′ (5′-GTTCTTGGTCACTGTGAG-
GATTCAG-3′) and p19-3′ (5′-CCATCATCATCACCTGGTCCAG-3′),
β-actin-5′ (5′-ACCAGTTCGCCATGGATGAC-3′) and β-actin-3′
(5′-TGCCGGAGGCGTTGTC-3′). The unpaired t test was used
for statistical analyses.
The cells were washed with PBS and lysed with lysis buffer
(20 mM Tris–HCl [pH 7.5], 1% NP-40, 150 mM NaCl, 10%
glycerol, and protease inhibitor cocktail set I [Calbiochem]).
The lysates were kept on ice for 30 min and centrifuged at
20,000×g for 15 min. Protein concentration of the superna-
tants was determined by the Bradford protein assay (Bio-Rad)
using BSA as a standard. The total proteins were separated on
84M. Chen et al.
12.5% or 8% SDS-PAGE and transferred to nitrocellulose
membrane. Membranes were blocked in 2% goat serum/0.5%
skimmed milk in PBST (0.05% Tween 20) for 1 h and then
probed with primary antibody overnight at 4 °C. Primary
antibodies used were as follows: rabbit anti-p21 (Santa Crzu,
H-164, 1:1000), mouse anti-p21 (BD Biosciences, SXM30,
1:400), rabbit anti-phospho-p53 at Ser15 (Cell Signaling,
9284, 1:1000), rabbit anti-p53 (Vector Laboratories, CM5,
1:1000), rabbit anti-MRG15 (1:1000) (Tominaga et al., 2005),
and mouse anti-β-actin (abcam, AC-15, 1:5000). Beta-actin
was used as a loading control. Horseradish peroxidase-
conjugated secondary antibodies (Santa Cruz) were used at
1:4000 for 1 h at room temperature and SuperSignal West
system was used for signal detection.
A shRNA construct for p21 and empty vector were obtained
from Drs. Phoenix and Temple (Fasano et al., 2007; Phoenix
and Temple, 2010). This shRNA-expressing lentiviral plasmid
was cotransfected with plasmids pMD2.G and psPAX2 into 293
T cells. Virus-containing medium was collected at 48 h and
72 h after transfection, filtered, and concentrated by ultra-
centrifugation. For viral transduction into NSCs, lentivirus-
from cultures 24 h after infection and cells were further
incubated for 48 h to ensure GFP expression. The GFP-positive
infected cells were sorted by FACS, counted, and seeded onto
pulsed with 10 μM BrdU for 1 h and positive cells detected
using the immunocytochemistry method using mouse anti-
BrdU (BD Biosciences, B44, 1:5) and Alexa Fluor 594-
conjugated secondary antibodies (Invitrogen, 1:1000 dilution)
described previously (Chen et al., 2009). The percentage of
BrdU-positive cells was determined by counting under a
fluorescent microscope, and at least 200 cells per sample
A similar approach was used with a lentivirus expressing
p53 shRNA (pSicoR p53) (Ventura et al., 2004) and empty
vector (pGIPZ), obtained from Dr. Iwakuma.
Cell cycle analysis
The GFP-positive cells (1×106) were fixed in 70% ethanol and
washed with PBS. The cells were incubated in PBS containing
0.1 mg/ml RNase (Sigma) at 37 °C for 30 min and 50 μg/ml
propidium iodide (Sigma) was added to each sample. Cells
were examined using a FACScalibur (Becton Dickinson) and
data were analyzed using CellQuest and ModFit LT softwares.
Cells were plated on poly-L-lysine-coated coverslips and
cultured for 2 days with Neurobasal Medium supplemented
with B27 and cell growth factors. For γ-irradiation experi-
ments, the cells were exposed to ionizing radiation (IR) at
10 Gy from a137Cs source and then incubated until indicated
time points. The cellswerefixed with4% paraformaldehydein
PBS for 10 min at room temperature. Fixed cells were washed
with PBS and permeabilized with PBST (0.1% Triton X-100) for
10 min at room temperature. Nonspecific binding was blocked
with 10% goat serum in PBST for 30 min at room temperature.
Cells were incubated with primary antibodies overnight at
4 °C. Primary antibodiesusedwereasfollows:mouseanti-p21
(BD Biosciences, SXM30, 1:250), mouse anti-γH2AX at Ser139
(Millipore, JBW301, 1:400), rabbit anti-53BP1 (Bethyl, BL182,
1:400), and rabbit anti-RAD51 (Santa Cruz, H-92, 1:200). Cells
were washed three times with PBST, then incubated with
Alexa Fluor 488 or 594-conjugated secondary antibodies
(Invitrogen, 1:1000 dilution) for 1 h at 37 °C. After washing
the cells were counterstained with 0.1 μg/ml DAPI (4′, 6-
diamidino-2-phenylindole, Sigma). Coverslips were mounted
onto Superfrost® Microscope Slides (Fisher Scientific) with
Fluoro-Gel (Electron Microscope Science) and fluorescent
images were acquired using a Zeiss Axiovert 200M microscope
with X-Cite™ 120 fluorescence illumination unit and AxioCam
MRm digital camera (Carl Zeiss Inc., Germany). All photo-
graphs were taken using X100 oil immersion objective.
MORF4 related factor on human chromosome 15
neural stem/progenitor cells
DNA double strand breaks
We thank Drs. Timothy N. Phoenix and Sally Temple and Dr.
Tomoo Iwakuma for providing lentivirus vectors for p21 shRNA
and p53 shRNA (pSicoR p53) and technical advice. We also
thank Dr. Benjamin J. Daniel for FACSanalyses and Mrs. Emiko
Tominaga for some experiments. This study was supported by
funding from NIH/NIA RO1AG032134 and SALSI to OMPS and
American Heart Association grant 0765084Y to KT.
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