Purkinje cell-specific males absent on the first (mMof) gene deletion results in an ataxia-telangiectasia-like neurological phenotype and backward walking in mice.
ABSTRACT The brains of ataxia telangiectasia (AT) patients display an aberrant loss of Purkinje cells (PCs) that is postulated to contribute to the observed deficits in motor coordination as well as in learning and cognitive function. AT patients have mutations in the ataxia telangiectasia mutated (ATM) gene [Savitsky et al. (1995) Science 268:1749-1753]. However, in Atm-deficient mice, the neurological defects are limited, and the PCs are not deformed or lost as observed in AT patients [Barlow et al. (1996) Cell 86:159-171]. Here we report that PC-specific deletion of the mouse males absent on the first (mMof) gene (Cre(-)), which encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac) and influences ATM function, is critical for PC longevity. Mice deficient for PC-specific Mof display impaired motor coordination, ataxia, a backward-walking phenotype, and a reduced life span. Treatment of Mof(F/F)/Pcp2-Cre(+) mice with histone deacetylase inhibitors modestly prolongs PC survival and delays death. Therefore, Mof expression and H4K16 acetylation are essential for PC survival and function, and their absence leads to PC loss and cerebellar dysfunction similar to that observed in AT patients.
- SourceAvailable from: Tej K Pandita[Show abstract] [Hide abstract]
ABSTRACT: Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.Cell Reports 06/2014; · 7.21 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and led to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Copyright © 2015, American Association for Cancer Research.Cancer research. 01/2015;
- [Show abstract] [Hide abstract]
ABSTRACT: Histone acetyl transferases (HATs) play distinct roles in many cellular processes and are frequently misregulated in cancers. Here, we study the regulatory potential of MYST1-(MOF)-containing MSL and NSL complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. We find that both complexes influence transcription by targeting promoters as well as TSS-distal enhancers. In contrast to flies, the MSL complex is not exclusively enriched on the X chromosome yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix, the major repressor of Xist lncRNA. MSL depletion leads to decreased Tsix expression, reduced REX1 recruitment, and consequently, enhanced accumulation of Xist and variable numbers of inactivated X chromosomes during early differentiation. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs.eLife Sciences 05/2014; · 8.52 Impact Factor
Purkinje cell-specific males absent on the first (mMof)
gene deletion results in an ataxia-telangiectasia-like
neurological phenotype and backward walking in mice
Rakesh Kumara,b, Clayton R. Hunta,b, Arun Guptaa,b, Suraj Nannepagac, Raj K. Panditab,d, Jerry W. Shayd,
Robert Bachooc, Thomas Ludwige, Dennis K. Burnsf, and Tej K. Panditaa,b,1
Departments ofaRadiation Oncology,cNeurology,dCell Biology, andfPathology, University of Texas Southwestern Medical Center, Dallas, TX 75390;
bDepartment of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108; andeInstitute for Cancer Genetics, Columbia University,
New York, NY 10032
Edited* by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved January 21, 2011 (received for review
November 4, 2010)
The brains of ataxia telangiectasia (AT) patients display an
aberrant loss of Purkinje cells (PCs) that is postulated to contribute
to the observed deficits in motor coordination as well as in
learning and cognitive function. AT patients have mutations in
the ataxia telangiectasia mutated (ATM) gene [Savitsky et al.
(1995) Science 268:1749–1753]. However, in Atm-deficient mice,
the neurological defects are limited, and the PCs are not deformed
or lost as observed in AT patients [Barlow et al. (1996) Cell 86:159–
171]. Here we report that PC-specific deletion of the mouse males
absent on the first (mMof) gene (Cre−), which encodes a protein
that specifically acetylates histone H4 at lysine 16 (H4K16ac) and
influences ATM function, is critical for PC longevity. Mice deficient
for PC-specific Mof display impaired motor coordination, ataxia,
a backward-walking phenotype, and a reduced life span. Treat-
ment of MofF/F/Pcp2-Cre+mice with histone deacetylase inhibitors
modestly prolongs PC survival and delays death. Therefore, Mof
expression and H4K16 acetylation are essential for PC survival and
function, and their absence leads to PC loss and cerebellar dysfunc-
tion similar to that observed in AT patients.
ataxia telangiectasia phenotype|chromatin modification|DNA damage
bellar ataxia (1). Postmortem examination of brains from AT
patients demonstrates significant loss and deformities in Purkinje
cells (PCs) (2). Although a cause-and-effect relationship has not
been proven, PC loss has a strong correlative association with
AT. Mice deficient in ataxia telangiectasia mutated (Atm) display
no gross ataxia or neuronal degeneration, but motor function is
stress in PCs of Atm−/−mice is increased (5), probably resulting
in increased DNA damage, the sensing and repair of which is
ATM dependent (6–8). The mammalian males absent on the first
(MOF) is an H4 histone acetylase that interacts with and influ-
ences downstream ATM functions in response to DNA damage
(9). Through histone H4 acetylated at lysine 16 (H4K16ac), MOF
has a critical role at multiple points in the cellular DNA damage
response and repair of DNA double-strand breaks (10). Acety-
lation of histone H4 at K16 by MOF also is an epigenetic signa-
ture of cellular proliferation during both embryogenesis and
oncogenesis (11), but whether MOF has a role in postmitotic cells
is not known. In this study, we determined that MOF is required
for postmitotic PC survival and that loss of MOF and H4K16
acetylation in PCs results in mice displaying neurological abnor-
malities similar to those encountered in AT patients.
he most striking and obvious neurological manifestation seen
in ataxia telangiectasia (AT) patients is a progressive cere-
Results and Discussion
Immunostaining of mouse cerebellum tissue sections confirmed
endogenous MOF expression in PCs and the presence of acet-
ylated H4K16 (Fig. S1). To deplete MOF specifically from PCs,
mice containing a conditional floxed mMof gene (MofF/F) in-
troduced by homologous recombination were crossed with mice
expressing Cre recombinase from the PC-specific Pcp2 gene
promoter (12) to obtain MofF/F/Pcp2-Cre−and MofF/F/Pcp2-Cre+
mice. The tissue-specific Pcp2 promoter becomes maximally ac-
tive 2–3 wk after birth, thus circumventing the embryonic-lethal
effect of homozygous mMof deletion (11). In cerebellum col-
lected from MofF/F/Pcp2-Cre−and MofF/F/Pcp2-Cre+mice at
various times (10–65 d) after birth, MOF expression was easily
detected in tissue from MofF/F/Pcp2-Cre−or MofF/F/Pcp2-Cre+
mouse PCs at postnatal day 15, but expression was lost by
postnatal day 25 in tissue from MofF/F/Pcp2-Cre+mice. As
expected, MOF expression in PC-adjacent cell types was un-
altered during the 65-d period examined (Fig. S2). Similarly,
H4K16ac was easily detected in PCs from MofF/F/Pcp2-Cre+mice
at postnatal day 15, but expression was gradually lost by post-
natal day 35 (Fig. S2). Calbindin (a PC-specific cell marker)
histochemical staining of cerebellum from age-matched MofF/F/
Pcp2-Cre−and MofF/F/Pcp2-Cre+mice indicated that detectable
PC loss in MofF/F/Pcp2-Cre+mice was first evident by postnatal
day 25, increased to 40% of PCs by postnatal day 45, and finally
reached 80% loss by postnatal day 65 (Fig. 1 and Figs. S3 and
S4). A significant decrease (P < 0.001; Student’s t test) in the
number of PCs in MofF/F/Pcp2-Cre+mice was observed from
about postnatal day 35 onwards, but there was no corresponding
decrease in PC numbers in MofF/F/Pcp2-Cre−mouse brains (Fig.
1). Along with the loss of PC observed in brains MofF/F/Pcp2-
Cre+mice, granular cells were visibly reduced (Fig. 1A i and ii).
Numerous PCs in tissue from MofF/F/Pcp2-Cre+mice contained
pyknotic nuclei, perhaps reflecting an altered chromatin state
caused by decreasing H4K16ac (Fig. S2), a chromatin modifi-
cation essential for protein–protein interactions (13). After the
initial appearance of pyknotic-like PCs in tissue from MofF/F/
Pcp2-Cre+mice, abnormal, coarse PC dendrites were detected
with calbindin staining (Fig. 2 and Fig. S4).
The effect of PC-specific MOF loss on cerebellar cortical
cytoarchitecture was analyzed further by H&E staining of cere-
bellum sections. No differences in the granular, PC or molecular
layers were observed in tissue from 15-d-old MofF/F/Pcp2-Cre+or
MofF/F/Pcp2-Cre−mice. However, by postnatal day 25, occasional
Author contributions: R.K., R.B., T.L., D.K.B., and T.K.P. designed research; R.K., A.G., S.N.,
R.K.P., and T.L. performed research; T.L. and D.K.B. contributed new reagents/analytic
tools; R.K., C.R.H., J.W.S., R.B., D.K.B., and T.K.P. analyzed data; and C.R.H., J.W.S., D.K.B.,
and T.K.P. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 1, 2011
| vol. 108
| no. 9www.pnas.org/cgi/doi/10.1073/pnas.1016524108
pyknotic PC nuclei became apparent in tissue from MofF/F/Pcp2-
Cre+mice (Fig. 2 and Fig. S5). Most strikingly, by postnatal day
45 there was a substantial increase in the numbers of pyknotic
PCs and degenerating PCs, accompanied by areas of frank PC
loss and modestly increased numbers of Bergmann glia at the
granule cell–molecular layer interface in tissue from MofF/F/
Pcp2-Cre+mice (Fig. 2 and Figs. S6 and S7).
Cerebellum GFAP is necessary for the integrity of central
nervous system white-matter architecture and long-term main-
tenance of myelination (14). GFAP levels in immunostained
sections of cerebellum from 15-d-old MofF/F/Pcp2-Cre+and
MofF/F/Pcp2-Cre−mice were similar. However, at postnatal day
25 the intensity of GFAP staining was higher in tissue from MofF/F/
Pcp2-Cre+mice than in tissue from MofF/F/Pcp2-Cre−mice (Fig.
2 and Fig. S8), and this intensity increased further by day 45 (Fig.
2 and Fig. S9). The increased GFAP expression observed is
compatible with the Bergmann gliosis noted in areas of PC loss.
PCs send inhibitory projections to the deep cerebellar and
vestibular nuclei, which are the only source of output from the
cerebellar cortex (15). The most obvious clinical manifestation
seen in AT patients is progressive cerebellar ataxia (1) attribut-
able to the profound loss of PCs seen in postmortem brains (2).
Therefore, we investigated whether MOF depletion in mouse
PCs results in abnormal motor coordination. We observed that,
beginning around postnatal day 35, MofF/F/Pcp2-Cre+mice are
much less active than MofF/F/Pcp2-Cre−mice (Movie S1) and
display very slow forward walking (Movie S2). More in-
terestingly, by postnatal day 45 more than 65% of MofF/F/Pcp2-
Cre+mice display the curious phenotype of backward walking
(Fig. 3A and Movie S2), and there is a direct correlation between
the progression of PC loss and initiation of backward walking. By
postnatal day 55, MofF/F/Pcp2-Cre+mice exhibit only backward
walking in open field until they die (Fig. 4A and Movies S3 and
S4). A prominent visible ataxia of rapid head shaking was ob-
served only in MofF/F/Pcp2-Cre+mice after postnatal day 45
(Movie S5), a phenotype that has not been observed in Atm-null
mice (3, 4).
Results from sensorimotor testing indicated that MofF/F/Pcp2-
Cre+mice were impaired on the elevated platform test relative to
MofF/F/Pcp2-Cre−controls (Fig. 3B), as evidenced by the MofF/F/
Pcp2-Cre+mice spending significantly less time on the platform
[F(1,12) = 10.48; P = 0.007]. However, the groups did not differ
in other measures within the battery (e.g., ledge, pole, or 90° in-
clined or inverted screen), suggesting that the impaired perfor-
mance on the platform test represented a relatively specific
deficit, possibly involving disturbed balance. These tests were
performed at the age of 6–9 wk, when >40% of PCs were lost in
Rotarod testing indicated that the MofF/F/Pcp2-Cre+mice also
were impaired on tasks that required fine-motor coordination,
with deficits becoming more apparent with increasing task diffi-
culty. For example, the MofF/F/Pcp2-Cre+mice exhibited mild
performance deficits in their ability to remain on the stationary
rod (Fig. 3C) [main effect of Genotype (PC-specific mMof
knockout in mice vs. control): F(1,12) = 5.21; P = 0.042],
whereas their performance on the constant speed and acceler-
Calbindin staining of cerebellum. (i and ii) Immunofluorescence staining for
calbindin (red) and H4k16ac (green) of ventricle IV from cerebellum of MofF/F/
Pcp2-Cre−and MofF/F/Pcp2-Cre+mice at postnatal days 35 (i) and 65 (ii). Loss
of PCs is quite prominent in 65-d-old MofF/F/Pcp2-Cre+mice. (iii) PC iden-
tification by calbindin detection by HRP staining. Cerebellums of 65-d-old
MofF/F/Pcp2-Cre+mice show widespread loss of PCs and PC dendritic calbin-
din activity with some residual PC in the 10th lobe. (B) PC density at various
ages, demonstrating progressive PC loss in MofF/F/Pcp2-Cre+mice.
PCs in cerebellum of MofF/F/Pcp2-Cre−and MofF/F/Pcp2-Cre+mice. (A)
Pcp2-Cre+and MofF/F/Pcp2-Cre−mice. (A) (Top) H&E staining reveals minimal
pyknotic PCs in 25-d-old MofF/F/Pcp2-Cre+mice, but by postnatal day 45 the
frequency of degenerating PCs with pyknotic nuclei and eosinophilic cyto-
plasm increases substantially. (Middle) GFAP staining reveals that levels are
similar in cerebellum of 25-d-old MofF/F/Pcp2-Cre+and MofF/F/Pcp2-Cre+mice;
however, GFAP levels are increased only in cerebellum of 45-d-old MofF/F/
Pcp2-Cre+mice, most conspicuously in areas of PC loss. (Bottom) Calbindin
staining shows similar frequency of PC cells in cerebellum of 25-d-old MofF/F/
Pcp2-Cre+and MofF/F/Pcp2-Cre+mice; however, PC degeneration along with
dendrite loss is quite visible in cerebellum of 45-d-old MofF/F/Pcp2-Cre+mice.
(B) Comparison of calbindin-stained PC from MofF/F/Pcp2-Cre−mice (Left)
and MofF/F/Pcp2-Cre+mice (Right) at higher magnification. DT, dendrite.
(i) Twenty-five days. (ii) Forty-five days.
Histologically stained sagittal sections of cerebellum from MofF/F/
Kumar et al.PNAS
| March 1, 2011
| vol. 108
| no. 9
ating Rotarod components was impaired (Fig. 3 D and E). Spe-
cifically, an analysis of the constant speed Rotarod data revealed
that, on average across test sessions, the MofF/F/Pcp2-Cre+mice
remained on the rotating rod for a significantly shorter time than
the MofF/F/Pcp2-Cre−mice, although performance generally im-
proved across test sessions [significant effects of Genotype,
F(1,12) = 11.98, P = 0.005, and Test Session, F(2,24) = 4.73,
P = 0.022]. Even greater performance impairments were ob-
served in the ability of MofF/F/Pcp2-Cre+mice to stay on the
accelerating Rotarod [significant effects of Genotype, F(1,12) =
22.63, P = 0.0005, and Test Session, F(2,24) = 11.60, P =
0.0003]. In summary, the Rotarod data suggest that MofF/F/Pcp2-
Cre+mice are impaired on tasks requiring fine-motor co-
ordination (constant speed Rotarod and accelerating Rotarod
tests) (Fig. 3 D and E). These behavioral analyses indicate that
MofF/F/Pcp2-Cre+mice have a behavioral phenotype of severe
movies. Significant backward walking of MofF/F/Pcp2-Cre+mice was observed after postnatal day 45. (B) Results from the elevated platform test showed that
MofF/F/Pcp2-Cre+mice were able to remain on a small, circular, elevated platform for a significantly shorter time than MofF/F/Pcp2-Cre−mice (*P = 0.007),
suggesting impaired balance in MofF/F/Pcp2-Cre+mice. (C–E) Results from the Rotarod test suggest that the MofF/F/Pcp2-Cre+mice had mild balance deficits
and severe impairments in fine-motor coordination. (C) On average across the test sessions, MofF/F/Pcp2-Cre+mice spent significantly less time than MofF/F/
Pcp2-Cre−mice on the stationary rod of the Rotarod test (**P = 0.042). (D) On the constant-speed Rotarod, MofF/F/Pcp2-Cre+mice spent a significantly shorter
amount of time than MofF/F/Pcp2-Cre−mice on the rotating rod (**P = 0.005) across the test sessions. Differences were significantly different between groups
for session 2 of trial 1 (*P = 0.008), and large differences also were observed for session 1 of trial 2, session 2 of trial 2, and session 3 of trial 1 (†P < 0.049). (E)
MofF/F/Pcp2-Cre+mice spent a significantly shorter period than MofF/F/Pcp2-Cre−mice on the accelerating rotating rod (**P = 0.0005) across the test sessions.
Pairwise comparisons showed significant differences between groups in session 1 of trial 2, and in session 3 of both trials (*P < 0.003); large differences also
were also observed in session 2 of trial 1 (††P = 0.009) and in the other remaining trials (†P < 0.035).
Behavioral studies of MofF/F/Pcp2-Cre−and MofF/F/Pcp2-Cre+mice. (A) MofF/F/Pcp2-Cre+mice showed prominent backward walking as shown in SI
| www.pnas.org/cgi/doi/10.1073/pnas.1016524108Kumar et al.
impairment in fine-motor coordination and possibly mild balance
disturbances. These results are consistent with previous findings
using the same tests to characterize functional deficits in other
strains of mutant mice that have synaptic defects in cerebellar
PCs (16), mice that have nonreceptor Abl/Arg tyrosine kinase-
related cerebellum defects (17), and Atm-deficient mice (3).
The physiological effect of PC loss induced by MOF depletion
was readily visible (Fig. 4). Although the total body weight of
MofF/F/Pcp2-Cre+and MofF/F/Pcp2-Cre−mice was similar at
postnatal day 15, the growth of MofF/F/Pcp2-Cre+mice was sig-
nificantly reduced subsequently, with maximal total body weight
reached 25–45 d after birth that was <50% of the weight of age-
matched MofF/F/Pcp2-Cre−mice. After age 45 d, the body weight
of MofF/F/Pcp2-Cre+mice began to decline (Fig. 4 A and B). The
loss of body weight in MofF/F/Pcp2-Cre+mice correlated with
a decrease in food intake compared with MofF/F/Pcp2-Cre−mice
(Fig. 4C). Overall survival was dramatically decreased in MofF/F/
Pcp2-Cre+mice, with the majority dying within 55–65 d after
birth and none surviving longer than 75 d (Fig. 4D).
The H4K16ac modification poses a structural constraint on
the formation of higher-order chromatin (13), perhaps inducing
an open chromatin configuration that is more readily accessible
to transcription as well as DNA repair. Acetylation of histone H4
at K16 also is critical for protein–protein interactions (18), and
the only mammalian protein known to perform this function is
MOF (9, 10, 19, 20), Recently, we reported that cells treated with
trichostatin A (TSA) had increased levels of H4K16ac but un-
altered MOF protein levels (10). We therefore investigated
postnatal day 45. (B) Body weight of MofF/F/Pcp2-Cre+mice decreased after postnatal day 30. (C) Comparison of food intake by MofF/F/Pcp2-Cre+and MofF/F/
Pcp2-Cre−mice. (D) Survival of MofF/F/Pcp2-Cre+mice was significantly decreased relative to MofF/+/Pcp2-Cre−mice. Mice with PC-specific knockout of the
mMof gene (MofF/F/Pcp2-Cre+) die at 61 ± 12 d after birth, whereas MofF/F/Pcp2-Cre−mice remain healthy. (E) TSA has a positive effect on the cumulative
survival of MofF/F/Pcp2-Cre+mice but not MofF/F/Pcp2-Cre−mice. (F) TSA affects the frequency of PCs in MofF/F/Pcp2-Cre+mice but not in MofF/F/Pcp2-Cre−mice.
Effect of MOF and HDACi treatment on body weight, survival, and PC numbers. (A) MofF/F/Pcp2-Cre+(Left) and MofF/F/Pcp2-Cre−(Right) mice at
Kumar et al. PNAS
| March 1, 2011
| vol. 108
| no. 9
whether inhibition of H4K16ac de-acetylation with two different
histone deacetylase inhibitors (HDACi), TSA and EX-527 (21),
would improve survival by delaying PC loss in MofF/F/Pcp2-Cre+
mice. Short-term drug treatment of MofF/F/Pcp2-Cre+mice ini-
tiated 30–35 d after birth delayed mouse death (Fig. 4E). A
further delay in initiating treatment to postnatal day 55 failed to
extend lifespan (Fig. 4 and Fig. S10). If drug treatment began at
postnatal day 35, a slight, but still significant, increase in PC
survival was observed in MofF/F/Pcp2-Cre+mice, and such mice
also had a 10–12% increase in lifespan.
There was no difference in the number of PCs in MofF/F/Pcp2-
Cre+or MofF/F/Pcp2-Cre−mice after treatment with TSA during
postnatal days 10–15. This lack of effect probably reflects the
period before cellular MOF levels began decreasing because of
Cre recombinase expression. However, when mice were treated
with TSA beginning on postnatal day 20, PC loss was significantly
delayed in MofF/F/Pcp2-Cre+mice as compared with MofF/F/Pcp2-
Cre−mice (Fig. 4F). The delay in loss of PCs in MofF/F/Pcp2-Cre+
mice was most prominent when mice were treated during post-
natal days 25–45. However, TSA had a minimal effect on MofF/F/
Pcp2-Cre+mice when treatment was initiated at postnatal day 45.
We tested another HDACi, EX-527, which also is known to in-
crease H4K16ac levels (21, 22). MofF/F/Pcp2-Cre+mice adminis-
tered EX-527 also displayed a delay in PC loss (Fig. S10). HDACi
treatment in MofF/F/Pcp2-Cre+mice not only delayed the loss of
12% (Fig. 4 and Fig. S10). Because in vitro treatment with TSA as
well as EX-527 is known to increase H4K16ac levels (10, 21, 22),
the present studies support the argument that H4K16ac has
a critical role in maintaining PC functions.
We report here that PC-specific deletion of the Mof gene in
mice produces neurological abnormalities attributable to cere-
bellar dysfunction that are similar to those seen in AT patients,
including impaired fine-motor coordination, balance deficits,
ataxia, and a most unusual backward-walking phenotype. Exam-
ination of the corresponding mouse cerebellum indicates PC de-
pletion, which is also observed postmortem in AT patient brains.
Taken together, these results suggest that the PC-specific Mof-
deletion mouse mimics the physiological developments seen in
AT patients. MOF also interacts with and influences downstream
ATM functions in response to DNA damage (9, 10, 23), sug-
gesting defective communication along the MOF/ATM pathway
in PCs. Analysis of this pathway under conditions of oxidative
stress, as occurs in the absence of ATM, may offer explanations as
to the development of other AT-related symptoms. In addition,
these mice provide a valuable means for understanding the role
of chromatin-modifying factors, such as MOF, in the function and
survival of nonproliferating postmitotic cells, especially neuronal
cells. Further advances will require the development of specific
deacetylation inhibitors, such as ones targeting the MOF modi-
fication product histone H4K16ac, which could facilitate future
Materials and Methods
Generation of PC-Specific mMof-Deficient Mice. Targeting vectors for the
mMof locus were constructed to generate an in vivo deletion of the mMof
gene in mice. The details of generating a conditional mMof allele by
inserting a single loxP site together with a flippase recombination target
(FRT)-flanked hygromycin resistance gene cassette 1.5 kb upstream of the
mMof transcription initiation site and a second loxP site within intron 3 were
described recently (11). The construct was electroporated into W9.5 ES cells
to generate Mofflox/+cells. Details for generation of Mofflox/+/Rosa26creERT2/+
and MofDflox/flox/Rosa26creERT2/+ES cell clones have been described (11).
To generate Purkinje cell-specific Mof-deficient mice using the Cre/loxP
recombination system, Mofflox/floxmice, which contained two loxP sites
flanking exon 3 of the Mof gene, were crossed with pcp2-Cre transgenic
mice [B6.129-Tg(Pcp2-cre)2Mpin/J] (Jackson Laboratory) expressing Cre
recombinase under the control of a L7/Pcp2 promoter (24). All procedures
relating to the care and treatment of the animals were performed in ac-
cordance with the National Institutes of Health guidelines at Washington
University School of Medicine, St. Louis, Columbia University, New York, and
University of Texas Southwestern Medical Center, Dallas.
PCR Analysis of Genomic DNA. Genomic DNA was prepared from mouse tail
and several brain regions, including the olfactory bulb, cortex, and cere-
bellum, using the HotSHOT method (25). PCR was performed using a Robo-
Cycler (Stratagene) with Taq DNA polymerase (QiaTaq; Qiagen). The
procedures for primer selection and conditions used for PCR genotyping
were as described previously (11).
Histology and Immunohistochemistry. We analyzed the status of cerebellum
PCs using fresh-frozen sections as well as paraffin-embedded sections. Mice
were perfused with 4% paraformaldehyde, and dissected brains were
perfusion-fixed for 36 h before paraffin embedding.
Fixation and Sectioning. After harvest, mouse brains were removed and
perfusion-fixed with 4% paraformaldehyde. After fixation, hindbrains
(brainstem and cerebellum) were divided into four sections in the sagittal
plane. Tissues were dehydrated in xylene, embedded in paraffin, and sec-
tioned on a rotary microtome at 3-mm thicknesses. For routine histology,
paraffin sections were rehydrated, stained with H&E, and evaluated by
Immunohistochemistry. Immunostainingwas performed atroom temperature
on a BenchMarkXT automated immunostainer, using the UltraVIEW system
with HRP and diaminobenzidine (DAB) chromogen (Ventana Medical Sys-
tems). Optimum primary antibody dilutions were predetermined using
known positive-control tissues. Paraffin sections were cut at 3 μm on a rotary
microtome, mounted on positively charged glass slides, and air-dried over-
night. Sections then were placed onto the BenchMarkXT where the depar-
affinization and heat retrieval were performed. Sections then were
incubated for 1 h with primary mouse monoclonal antibody to calbindin
(1:50 dilution; Novocastra/Leica) or rabbit polyclonal antibody to glial
fibrillary acidic protein (1:400 dilution; Biocare) diluted in ChemMate buffer
(Ventana Medical Systems) or with buffer alone as a negative reagent
control. After washing in buffer, sections were incubated with a freshly
prepared mixture of DAB and H2O2in buffer for 8 min, followed by washing
in buffer and then in water. Sections were counterstained with hematoxylin,
dehydrated in a graded series of ethanols and xylene, and coverslipped.
Slides were reviewed by light microscopy. Positive reactions with DAB were
identified as dark brown reaction product.
PC Counting. The number of PCs was quantified in midsagittal, 100-mm-thick
cerebellar sections prepared from age-matched MofF/F/Pcp2-Cre−and MofF/F/
Pcp2-Cre+mice at different ages (n = 4 for each age group). Optical images
of PCs immunostained with anti-calbindin antibody were taken using 2×,
10×, and 20× objective lenses.
Reagents. TSA purchased from Sigma was dissolved in DMSO and adminis-
tered in vivo at 1 mg·kg of body weight−1·d−1for 10 d. EX527 was purchased
from Tocris and dissolved in 4% DMSO/10% cyclodextrin in PBS and ad-
ministered in vivo at 1 mg·kg of body weight−1·d−1for 10 d.
Quantitation of Food Intake. Standard procedures (26) for quantitating
mouse food intake were used. In brief, mice were acclimatized to individual
cages for 1 wk. Food intake was measured every week. The body weight of
individual mice was measured before and after each test series. Chow intake
was measured by weighing the cage tops containing food pellets. Cage
bottoms were covered with polyvinyl sheets when the cage tops were
weighed, and any food spilled on sheets was collected and weighed. Food
intake was corrected for spillage.
Behavioral Studies. To provide an initial characterization of the functional
deficits in MofF/F/Pcp2-Cre+mice, they and littermate MofF/F/Pcp2-Cre−con-
trols were evaluated on a battery of sensorimotor measures and the Rotarod
test at 6–9 wk of age. These behavioral analyses have been used previously
to assess ataxias, including ataxias of cerebellar origin, in several different
mutant mouse strains (16, 27, 28), and these methodologies are described
briefly in SI Materials and Methods. All behavioral tests were conducted by
individuals who were unaware of the genotypic status of the mice (SI
Materials and Methods).
| www.pnas.org/cgi/doi/10.1073/pnas.1016524108Kumar et al.
MarkGoldberg,andAnnStowefor helpwithanimalbehavior studies,suggestions,
and comments. We thank members of the T.K.P. laboratory for helpful discussions
This work was supported by National Institutes of Health/National Cancer Institute
Grants R01CA123232, R01CA129537, RO1CA154320, and R13CA130756.
1. Sedgwick RP, Boder E (1960) Progressive ataxia in childhood with particular reference
to ataxia-telangiectasia. Neurology 10:705–715.
2. Boder E, Sedgwick RP (1958) Ataxia-telangiectasia; a familial syndrome of progressive
cerebellar ataxia, oculocutaneous telangiectasia and frequent pulmonary infection.
3. Barlow C, et al. (1996) Atm-deficient mice: A paradigm of ataxia telangiectasia. Cell
4. Barlow C, et al. (2000) ATM is a cytoplasmic protein in mouse brain required to
prevent lysosomal accumulation. Proc Natl Acad Sci USA 97:871–876.
5. Huang C, Sloan EA, Boerkoel CF (2003) Chromatin remodeling and human disease.
Curr Opin Genet Dev 13:246–252.
6. Lavin MF (2008) Ataxia-telangiectasia: From a rare disorder to a paradigm for cell
signalling and cancer. Nat Rev Mol Cell Biol 9:759–769.
7. Pandita TK, Richardson C (2009) Chromatin remodeling finds its place in the DNA
double-strand break response. Nucleic Acids Res 37:1363–1377.
8. Pandita TK (2003) A multifaceted role for ATM in genome maintenance. Expert Rev
Mol Med 5:1–21.
9. Gupta A, et al. (2005) Involvement of human MOF in ATM function. Mol Cell Biol 25:
10. Sharma GG, et al. (2010) MOF and histone H4 acetylation at lysine 16 are critical for
DNA damage response and double-strand break repair. Mol Cell Biol 30:3582–3595.
11. Gupta A, et al. (2008) The mammalian ortholog of Drosophila MOF that acetylates
histone H4 lysine 16 is essential for embryogenesis and oncogenesis. Mol Cell Biol 28:
12. Zhang XM, et al. (2004) Highly restricted expression of Cre recombinase in cerebellar
Purkinje cells. Genesis 40:45–51.
13. Shogren-Knaak M, et al. (2006) Histone H4-K16 acetylation controls chromatin
structure and protein interactions. Science 311:844–847.
14. Gerbert J, Burns S, Liedtke LA (1996) Anesthesia blocks of the lower extremity.
Comparing the Biojector with needle and syringe. J Am Podiatr Med Assoc 86:
15. Ito M, Yoshida M, Obata K, Kawai N, Udo M (1970) Inhibitory control of
intracerebellar nuclei by the Purkinje cell axons. Exp Brain Res 10:64–80.
16. Grady RM, Wozniak DF, Ohlemiller KK, Sanes JR (2006) Cerebellar synaptic defects
and abnormal motor behavior in mice lacking alpha- and beta-dystrobrevin.
J Neurosci 26:2841–2851.
17. Qiu Z, Cang Y, Goff SP (2010) Abl family tyrosine kinases are essential for basement
membrane integrity and cortical lamination in the cerebellum. J Neurosci 30:
18. Neal KC, Pannuti A, Smith ER, Lucchesi JC (2000) A new human member of the MYST
family of histone acetyl transferases with high sequence similarity to Drosophila MOF.
Biochim Biophys Acta 1490:170–174.
19. Smith ER, et al. (2005) A human protein complex homologous to the Drosophila MSL
complex is responsible for the majority of histone H4 acetylation at lysine 16. Mol Cell
20. Taipale M, et al. (2005) hMOF histone acetyltransferase is required for histone H4
lysine 16 acetylation in mammalian cells. Mol Cell Biol 25:6798–6810.
21. Solomon JM, et al. (2006) Inhibition of SIRT1 catalytic activity increases p53
acetylation but does not alter cell survival following DNA damage. Mol Cell Biol 26:
22. Hajji N, et al. (2010) Opposing effects of hMOF and SIRT1 on H4K16 acetylation and
the sensitivity to the topoisomerase II inhibitor etoposide. Oncogene 29:2192–2204.
23. Li X, et al. (2010) MOF and H4 K16 acetylation play important roles in DNA damage
repair by modulating recruitment of DNA damage repair protein Mdc1. Mol Cell Biol
24. Barski JJ, Dethleffsen K, Meyer M (2000) Cre recombinase expression in cerebellar
Purkinje cells. Genesis 28:93–98.
25. Truett GE, et al. (2000) Preparation of PCR-quality mouse genomic DNA with hot
sodium hydroxide and Tris (HotSHOT). Biotechniques 29:52, 54.
26. Bachmanov AA, Reed DR, Tordoff MG, Price RA, Beauchamp GK (2001) Nutrient
preference and diet-induced adiposity in C57BL/6ByJ and 129P3/J mice. Physiol Behav
27. Wang Q, et al. (2002) Ataxia and paroxysmal dyskinesia in mice lacking axonally
transported FGF14. Neuron 35:25–38.
28. Griffey MA, et al. (2006) CNS-directed AAV2-mediated gene therapy ameliorates
functional deficits in a murine model of infantile neuronal ceroid lipofuscinosis.
Mol Ther 13:538–547.
Kumar et al. PNAS
| March 1, 2011
| vol. 108
| no. 9