MOLECULAR AND CELLULAR BIOLOGY, July 2010, p. 3582–3595
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 14
MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA
Damage Response and Double-Strand Break Repair?†
Girdhar G. Sharma,1§ Sairei So,2§ Arun Gupta,1,2§ Rakesh Kumar,1,2§ Christelle Cayrou,3‡
Nikita Avvakumov,3Utpal Bhadra,4Raj K. Pandita,1Matthew H. Porteus,2
David J. Chen,2Jacques Cote,3and Tej K. Pandita1,2*
Washington University School of Medicine, St. Louis, Missouri 631081; University of Texas, Southwestern Medical Center, Dallas,
Texas 753902; Laval University Cancer Research Center, Quebec City G1R 2J6, Canada3; and Centre for Cellular and
Molecular Biology, Hyderabad AP 500007, India4
Received 11 November 2009/Returned for modification 21 January 2010/Accepted 4 May 2010
The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here
we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and
double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered
expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by
blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase
catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-depen-
dent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate
that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous
recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and
colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac
(histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.
In eukaryotes, specifically in mammals, the mechanisms by
which the DNA damage response (DDR) components gain
access to broken DNA in compacted chromatin remain a mys-
tery. The DNA damage response occurs within the context of
chromatin, and its structure is altered post-DNA double-strand
break (DSB) induction. Major alterations include (i) chroma-
tin remodeling via ATP-dependent activities and covalent his-
tone modifications and (ii) incorporation of histone variants
into nucleosomes. Chromatin structure creates a natural bar-
rier to damaged DNA sites, which suggests that histone mod-
ifications will play a primary role in DDR by facilitating repair
protein access to DNA breaks (43, 58, 87, 88). While some
experimental evidence indicates that preexisting histone mod-
ifications may play an important role in DDR, the precise role
of chromatin status prior to DNA damage on DDR is yet to be
clearly established. For instance, biochemical and cell biology
studies indicate that repair proteins (53BP1, Schizosaccharo-
myces pombe Crb2 [SpCrb2], and Saccharomyces cerevisiae
Rad9 [ScRad9]) require methylated Lys79 of histone H3 (H3-
K79) (29) or methylated Lys20 of histone H4 (H4-K20) and/or
CBP/p300-mediated acetylation of histone H3 on lysine 56 (9,
15, 29, 66, 93) for focus formation at DNA-damaged sites.
These modifications are normally present on chromatin, and
none has been reported to change in response to ionizing
radiation (IR)-induced DNA damage. However, it is yet to be
established whether preexisting acetylation of specific histone
residues at the time of cellular exposure to IR plays any critical
role in DDR. While recent studies demonstrate that in human
cells, histone H3 acetylated at K9 (H3K9ac) and H3K56ac are
rapidly and reversibly reduced in response to DNA damage,
most histone acetylation modifications do not change appre-
ciably after genotoxic stress (80).
The amino-terminal tail of histone H4 is a well-described
target for posttranslational modification, including acetylation
(4, 19, 82). Reversible acetylation occurs at four lysines (posi-
tions 5, 8, 12, and 16) in vivo in most eukaryotes (4), and their
hyperacetylation could lead to unfolding of the nucleosomal
fiber (82). Acetylation of K16 is prevalent in Drosophila on the
hyperactive male Drosophila polytene X chromosomes (83),
where it contributes to transcriptional upregulation (22). In
yeast, H4K16ac does not correlate with active genes (37), while
all other known acetylation marks on histone H4 are linked to
enhanced transcription (16). The H4K16ac modification poses
a structural constraint on formation of higher-order chromatin.
It is therefore possible that this posttranslational modifica-
tion could contribute to DDR by forcing chromatin to keep
a more open configuration. In this role, H4K16ac would
potentially serve as a platform structure to generate proper
signaling for DDR.
The histone acetyltransferase (HAT) responsible for the ma-
jority of H4K16 acetylation in the cell is MOF (2, 24, 25, 46, 75,
79). A single histone H4K16ac modification modulates both
higher-order chromatin structure and functional interactions
between a nonhistone protein and the chromatin fiber (74).
The yeast histone acetyletransferase Esa1 (essential SAS2-re-
lated acetyltransferase), can acetylate lysine 16 of histone H4
* Corresponding author. Mailing address: Department of Radiation
Oncology, UT Southwestern Medical Center, 2201 Inwood Road,
NC7.116, Dallas, TX 75390. Phone: (214) 648-1918. Fax: (214) 648-
5995. E-mail: firstname.lastname@example.org.
‡ Present address: Institute of Human Genetics, Montpellier,
§ These authors contributed equally.
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 17 May 2010.
and is required for DNA repair in yeast (8). We have previ-
ously reported that cells expressing a HAT-dead human MOF
(hMOF) had a higher frequency of residual DNA DSBs and
chromosome aberrations after cellular exposure to IR; how-
ever, the reasons for the increased aberrations are not known
(25). While histone lysine modifications have been linked to
the recruitment of DNA repair factor in mammalian cells, it is
unknown whether reduction of H4K16ac will influence DDR.
Here we demonstrate that decreased levels of H4K16ac, due to
hMOF depletion, can alter DDR at several stages of DNA
DSB repair and abrogate both the non-homologous end-join-
ing (NHEJ) and homologous recombination (HR) pathways of
MATERIALS AND METHODS
Cell culture and derivation of cell lines. HEK293, MCF7, HCT116, GM5849,
and HL60 cells were maintained and transfected with plasmids as described
previously (25). A cDNA fragment encoding wild-type hMOF was cloned into
the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA) as de-
scribed previously (24, 25). Wild-type hMOF was created by using a PCR ap-
proach with appropriate primer pair combinations.
RNAi. Small interfering RNAs (siRNAs) for MOF, Tip60, and control lucif-
erase (Luc) were obtained from Dharmacon Research (Lafayette, CO). siRNA
was labeled with Cy3 by using the Label IT siRNA Tracker intracellular local-
ization kit (Mirus) following the manufacturer’s prescribed procedure. RNA
interference (RNAi) treatment of 293 and MCF7 cells, as well as cells from other
cell lines, was performed as described previously (25, 26, 54). Cells were used
72 h after transfection for all experimental purposes. In some of our experiments,
the siRNA sequence of CUCCCAGCCUGUAAAUAUGUU specific for the
untranscribed region (UTR) of hMOF from Dharmacon was used to knock down
Expression profiling. We have used the previously described modified ap-
proach for microarray analysis of gene expression (85). Total RNA was isolated
from cells using the RNeasy kit (Qiagen, Inc., Valencia, CA), and expression
analyses were performed using Affymetrix’s commercial gene expression arrays.
All experiments were performed at least in triplicate using enhanced green
fluorescent protein (EGPF) double-stranded RNA (dsRNA)-treated samples as
a control. We processed Raw Affymetrix CEL files by using MAS5.0 (92), and the
nonspecific filtering was applied to probe sets having interquantile range values
below 0.5. The remaining processed log2intensity values were analyzed using the
Bioconductor limma package (23) to detect differential gene expression. Statis-
tical significance of the results was determined using a moderated eBayes t test.
The resulting P values were adjusted using FDR (34). Probe sets were mapped
to genes using the annotation available in Ensembl (21).
Western blot analysis, IP, ChIP, and complex purification. Cell lysates for
Western blot analysis were prepared as previously described (54). Antibodies for
the different proteins analyzed have been described previously (24–26). Immu-
noblotting and detection of hMOF, H4K16 acetylation, Tip60, and the DNA-
dependent protein kinase catalytic subunit (DNA-PKcs) were done according to
a previously described procedure (24, 25). For immunoprecipitation (IP), cells
were broken in lysis buffer as described previously (25). Lysates were precleared
with purified immunoglobulin G (IgG) and protein A/G beads. Proteins were
immunoprecipitated with specific antibodies, and immunoprecipitates were
washed with lysis buffer as described previously (25). For chromatin immuno-
precipitation (ChIP), nuclear extracts were prepared as follows: 5 ? 108cells
were pelleted, washed in phosphate-buffered saline (PBS), swollen in hypotonic
buffer on ice (20 mM HEPES, pH 7.2, 0.3 M sucrose, 3 mM MgCl2, 3 mM
?-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride), and homogenized
with 20 strokes of a loose-fitting Dounce pestle. Cellular homogenates were
layered on a 0.65 M sucrose cushion and pelleted at 1,000 ? g for 5 min, and
nuclei were resuspended in DNase I buffer and digested by the procedure
previously described (47). Nuclear extracts in DNase I buffer were added to
protein A-agarose beads that were prebound with antibody. Immunoprecipitates
were washed with a buffer composed of 20 mM HEPES, pH 7.2, 10% glycerol,
0.35 M NaCl, 1 mM MgCl2, and 0.5 mM phenylmethylsulfonyl fluoride. For
tandem affinity purification (TAP) of native complexes with nuclear extracts,
fractionation on immunoglobulin G (IgG)-Sepharose and calmodulin resin was
performed as described previously (18). For the identification of the stable
components of purified complexes, we performed silver staining, tandem mass
spectrometry, and Western blotting as previously described (75). Chromatin
immunoprecipitation after formaldehyde-mediated in vivo cross-linking of DNA
with hMOF was performed with an hMOF-specific antibody as described previ-
ously (25, 69–71). Immunoprecipitated DNA from an equal number of control
and irradiated cells was purified by the standard procedure. The yield of DNA
was measured spectroscopically.
Immunofluorescence. Cell culture in chamber slides, fixation, and immuno-
staining were done as previously described (1, 28, 59). Fluorescent images of foci
were captured as described previously (26). Sections through nuclei were cap-
tured, and the images were obtained by projection of the individual sections as
recently described (48). The results shown are from three to four independent
experiments. Cells with a bubble-like appearance or micronuclei were not con-
sidered for IR-induced focus analysis. For immunostaining of meiocytes, 8 to 12
weeks old male mice maintained at the Washington University School of Med-
icine, St. Louis, MO, were sacrificed by cervical dislocation. Freshly obtained
testes were dissected in ice-cold minimal essential medium (MEM). Structurally
preserved nuclei for immunostaining were prepared by mincing fresh tissue in
MEM (Life Technologies). After removal of tissue pieces, the suspension was
immediately mixed with fixative (3.7% formaldehyde, 0.1 M sucrose) and placed
on a drop of hypotonic solution placed on precleaned slides. After air drying,
slides were stored at ?20°C until further use. Immunofluorescent staining was
done as described previously (24, 67).
Microirradiation. Cells were grown on coverslips, microirradiated with a laser
at 365 nm according to the described procedure (81) or with a laser at 405 nm in
combination with Hoechst 33342, and then incubated at 37°C for 10 min. Pre-
extraction was done with a buffer containing 0.5% Triton X-100 on ice for 20
min. Cells with and without preextraction were fixed with 4% paraformaldehyde,
incubated with 0.5% of Triton X-100, blocked with bovine serum albumin (BSA),
and incubated with specific antibody. Cells were washed with phosphate-buffered
saline (PBS), incubated with anti-rabbit IgG-Texas Red, washed with PBS, and
mounted with DAPI (4?,6-diamidino-2-phenylindole). For live cell imaging, cells
were grown on glass coverslip-bottomed chamber slides and maintained at 37°C
and 5% CO2during experiments.
hMOF retention assay. To determine the retention of MOF with DNA, 107
cells growing in exponential phase were irradiated and fixed with 4% formalde-
hyde at different times postirradiation. Total DNA from 107cells, coimmuno-
precipitated with MOF or retinoblastoma (Rb) antibody after in vivo cross-
linking, was performed by the standard procedure described previously (69, 71).
Immunoprecipitated DNA was purified by the standard phenol chloroform pro-
cedure (57), and DNA was quantified with a NanoDrop 2000 spectrometer
(Thermo Scientific, Inc.). The amount of DNA retained by MOF is presented in
arbitrary units of retention (MOF retention).
To determine how tightly hMOF is bound with DNA, cells with and without
exposure to IR were washed with ice-cold PBS, and cell fractionation was carried
out by four consecutive extractions with increasing detergent concentrations as
described previously (5). The supernatants were collected at each step after
extraction buffer treatment, and supernatants were cleared by centrifugation.
The fractions were labeled as fractions I to IV. Pellets of about 107cells were first
resuspended in 150 ?l of fractionation buffer A for 5 min on ice. (Fractionation
buffer A contains 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, and
0.2% Nonidet P-40, supplemented with protease inhibitors [Sigma] and phos-
phatase inhibitors [10 mM NaF].) Fraction 1 from different samples was col-
lected following centrifugation at 1,000 ? g for 5 min, and pellets were washed
with the same buffer A. The supernatant was collected (fraction II); the nuclear
pellets were treated for 40 min on ice with 150 ?l of fractionation buffer B
containing 0.5% Nonidet P-40, which was subjected to centrifugation at 16,000 ?
g for 15 min; and supernatant was collected as fraction III. The remaining pellets
are fraction IV, which were finally lysed in SDS-PAGE buffer and boiled for 5
min. From each fraction, equal aliquots derived from equivalent cell numbers
were loaded on 10% SDS-PAGE gels and blotted as previously described (24, 25,
Proteins involved in IR-induced DDR are unaffected by
hMOF depletion. Our initial interest in hMOF was motivated
by its involvement in H4-Lys16 acetylation (H4K16ac) and its
ability to alter higher-order chromatin structure into a relaxed
conformation (72–74). This activity has been linked with tran-
scriptional upregulation in Drosophila (73), as is evidenced by
studies demonstrating that almost all active genes on the X
VOL. 30, 2010 MOF, H4K16ac, DDR, AND DSB REPAIR 3583
chromosome of Drosophila are associated with robust H4-
Lys16 acetylation (22). Our recent studies have revealed that
MOF inactivation or depletion in mammalian cells results in
loss of H4K16ac, which correlates with loss of cellular prolif-
eration (24). Since several studies have suggested that lack of
DDR components is likely to be responsible for loss of cellular
proliferation and that IR-induced DNA damage status corre-
lates with cell survival, we determined whether depletion of
hMOF downregulates any specific gene(s) involved in the
mammalian DDR. We compared the RNA expression profile,
using microarray analysis, in 293 cells with and without hMOF
depletion. A significant reduction in H4K16ac levels after
transfection with hMOF siRNA was observed after 72 h (see
Fig. S1A in the supplemental material), whereas no change in
H4K16ac levels was observed in cells depleted of another
histone acetyltransferase Tip60 (see Fig. S1A and B in the sup-
plemental material). While depletion of hMOF resulted in a
significant downregulation of 600 genes and upregulation of
431 genes, none of these gene-encoded proteins is known to be
involved in DDR, except XRCC4. (The complete microarray
data can be found at http://www.ncbi.nlm.nih.gov/geo/query
overexpression of hMOF in 293 cells significantly increased the
expression of 92 genes and downregulated 39 genes, but there
was no change in expression of genes encoding proteins in-
volved in DNA DSB repair (http://www.ncbi.nlm.nih.gov/geo
Consistent with the repair gene transcriptional profiles, we
found no significant reduction or increase in the level of known
DNA DSB repair proteins (ATM, Ku70, Ku80, ligase IV,
DNA-PKcs, Artemis, XLF1 [Cernunnos], NBS1, MRE11,
Rad50, Rad51, Rad52, Rad54, BRCA1, BRCA2, XRCC2,
XRCC3, Rad51B, Rad51C, Rad51D, XRCC1, 53BP-1, SMC1,
MDC1, FANCD2, CHK2, CHK1, Tip60, hSSB1, hSSB2,
H2AX, etc.) in hMOF-depleted cells (see Fig. S1C in the
supplemental material). These results suggest that hMOF de-
pletion does not affect the levels of the most commonly known
DNA DSB repair proteins at the transcriptional level. How-
ever, the effect of hMOF depletion on DDR could be due to
abrogation of posttranslational modifications of proteins in-
volved in DNA damage repair (24). Since the best-character-
ized posttranslational modification induced by DNA damage is
the phosphorylation of histone H2AX, which appears imme-
diately postirradiation as foci that colocalize with repairosomes
(58), we determined whether depletion of hMOF influences
the kinetics of appearance of ?-H2AX foci.
hMOF knockdown delays the appearance of postirradiation
?-H2AX foci. To test whether hMOF-dependent H4K16ac is
critical for DDR, IR-induced repair-associated focus forma-
tion was examined. Seventy-two hours posttransfection with
hMOF siRNA, 293 cells were irradiated with 1.5 Gy. Cells with
more than ?4 ?-H2AX foci were counted at different time
points postirradiation. We monitored simultaneously the trans-
fection of hMOF siRNA-Cy3, H4K16ac levels, and the fre-
quency of cells with ?-H2AX foci postirradiation (Fig. 1A; see
Fig. S2A and B in the supplemental material). Cells deficient in
hMOF contained reduced levels of H4K16ac (Fig. 1Aa) and
had a delayed appearance of IR-induced ?-H2AX foci (Fig.
1Ba) but did not show any delay in the disappearance of
?-H2AX foci once formed (Fig. 1Ab and Bb). Depletion of the
histone acetyltransferase Tip60 has been shown to abrogate
the disappearance of the damage-induced ?-H2AX variant in
Drosophila (38). In agreement with that observation, we found
that while depletion of Tip60 did not influence the levels of
H4K16ac (see Fig. S1A and B in the supplemental material)
and had minimal effect on the appearance of IR-induced
?-H2AX foci (Fig. 1Ac), it did induce a delay in the disappear-
ance of ?-H2AX foci (Fig. 1Ad and Bb).
To determine whether depletion of hMOF also influenced
the number of IR-induced ?-H2AX foci, the frequency of
?-H2AX foci per cell was quantified (see Fig. S2C and D in the
supplemental material). hMOF-depleted cells have delayed
induction of ?-H2AX focus formation (see Fig. S2C in the
supplemental material). These results were similar to mouse
FIG. 1. Correlation between status of H4K16ac levels and IR-in-
duced ?-H2AX focus formation in 293 cells. (A) Simultaneous detec-
tion of cells transfected with Cy3-labeled siRNA, H4K16ac status, and
?-H2AX focus appearance after exposure to 1.5 Gy of IR. (Aa and Ab)
Cells were transfected with hMOF siRNA and analyzed for ?-H2AX
foci after 5 min (Aa) and 300 min (Ab) of IR exposure. (Ac and Ad)
Cells were transfected with Tip60 siRNA and analyzed for ?-H2AX
foci after 5 min (Ac) and 300 min (Ad) of IR exposure. Panels Aa and
Ac are magnified cells as shown in Fig. S2A and B in the supplemental
material. (B) Frequency of cells with more than 2 ?-H2AX foci ob-
served at various time points postirradiation. For each time point, 100
cells were analyzed. Each experiment was repeated three times; the
mean numbers of cells with more than 2 foci are plotted against time.
(a) Appearance of ?-H2AX foci in cells with and without depletion of
either hMOF or Tip60. (b) Disappearance of ?-H2AX foci in cells with
and without depletion of either hMOF or Tip60.
3584 SHARMA ET AL.MOL. CELL. BIOL.
embryonic cells heterozygous for mouse Mof (mMof) (26),
which had reduced induction of ?-H2AX foci. In contrast,
Tip60 depletion had a minimal effect on initial IR-induced
?-H2AX foci, although such cells had a higher frequency of
residual ?-H2AX foci per cell (see Fig. S2D in the supplemen-
tal material). The kinetics of appearance and disappearance of
IR-induced ?-H2AX foci were similar with (Fig. 1B) and with-
out (see Fig. S2C and D in the supplemental material) Cy3
labeling of hMOF siRNA.
hMOF and Tip60 have important but independent roles in
the appearance and disappearance of ?-H2AX foci. Since
hMOF depletion delayed the appearance of IR-induced
?-H2AX foci while Tip60 depletion delayed their disappear-
ance, we determined how simultaneous depletion of hMOF
and Tip60 altered ?-H2AX focus dynamics. Cells doubly de-
pleted of hMOF and Tip60 showed similar kinetics of ?-H2AX
focus appearance, as observed in cells only depleted of hMOF
(see Fig. S3A in the supplemental material), and disappear-
ance, as was observed in cells only depleted of Tip60 (see Fig.
S3B in the supplemental material). These results suggest that
hMOF and Tip60 have different nonoverlapping roles during
IR-induced ?-H2AX focus appearance and disappearance.
Expression of a HAT-dead mutant hMOF (?hMOF) de-
creases IR-induced ?-H2AX focus formation. We have previ-
ously reported that overexpression of hMOF results in in-
creased H4K16ac levels (24, 25) and that cells overexpressing
hMOF show a slightly enhanced appearance of IR-induced
?-H2AX foci compared to control cells (see Fig. S4A in the
supplemental material). These results suggest that any increase
in the physiologically relevant levels of H4K16ac has a minimal
effect on IR-induced ?-H2AX focus formation. To further
investigate whether hMOF HAT activity is critical for IR-
induced ?-H2AX focus formation, we determined whether ec-
topic expression of previously described construct of hMOF
HAT-dead deletion mutant (?hMOF) (24, 25) abrogates IR-
induced ?-H2AX focus formation. Endogenous expression of
wild-type hMOF in cells expressing hemagglutinin (HA)-
tagged ?hMOF was knocked down by using a 3?UTR-specific
hMOF siRNA. Cells expressing ?hMOF had reduced levels of
IR-induced ?-H2AX focus formation. Furthermore, ?hMOF
failed to rescue the defect in ?-H2AX focus formation brought
on by depletion of endogenous wild-type hMOF (see Fig. S4B
in the supplemental material), further supporting a role for
H4K16ac status in IR-induced ?-H2AX focus formation.
Inhibition of histone deacetylase rescues IR-induced
?-H2AX focus appearance in hMOF-depleted cells. The above
results support the argument that H4K16ac levels are critical
for a timely DDR. To further confirm whether H4K16ac levels
correlate with IR-induced ?-H2AX focus appearance, we used
two different approaches. First we treated hMOF siRNA-
transfected and control 293 cells with the histone deacetylase
inhibitor trichostatin A (TSA) and determined the levels of
H4K16ac. Cells treated with TSA had increased levels of
H4K16ac without any change in hMOF levels (Fig. 2A). Cells
with and without MOF depletion had higher levels of H4K16ac
post-TSA treatment. Cells depleted of hMOF and treated with
TSA had faster kinetics of IR-induced ?-H2AX focus appear-
ance than untreated hMOF-depleted cells (Fig. 2B). This is
consistent with the observation that TSA-treated hMOF-de-
pleted cells also have enhanced growth rates (79). As tricho-
statin A is known to inhibit several histone deacetylases, we
wanted to determine whether the inactivation of SirT2, the
major histone deacetylase (HDAC) (90), which deacetylates
H4K16ac as well as H3K56ac (15), influences IR-induced
?-H2AX focus formation. SirT2-deficient cells had higher lev-
els of H4K14ac than control cells expressing SirT2 (Fig. 2C),
without showing any difference in the levels of MOF protein.
Cells deficient for SirT2 had a modestly higher frequency of
IR-induced ?-H2AX foci than cells with SirT2 (Fig. 2D). De-
pletion of MOF with siRNA in cells with and without SirT2 led
to decreased MOF as well as H4K16ac levels (Fig. 2C). Inter-
estingly, SirT2-deficient cells with knockdown of MOF
showed relatively higher levels of H4K16ac compared to
cells with SirT2 and depleted of MOF (Fig. 2D). Consistent
with the levels of H4K16ac, cells with SirT2 and depleted of
FIG. 2. Effect of H4K16ac levels on the appearance of IR-induced
?-H2AX foci. (A) 293 cells with and without knockdown of MOF were
treated with TSA and examined for H4K16ac levels. Lane C, control.
(B) Cells knocked down for hMOF and treated with TSA were irra-
diated and examined for appearance of ?-H2AX foci. (C) SirT2?/?
and SirT2?/?mouse embryonic fibroblasts with knockdown of MOF,
showing MOF and H4K16ac levels and (D) frequency of IR-induced
?-H2AX foci cells. (E) HL60 cells treated with DMSO for different
time periods showing MOF, H4K16ac, and histone H4. (F) HL60 cells
treated with DMSO or MOF knockdown and irradiated with 1.5 Gy,
showing frequency of cells with ?-H2AX postirradiation.
VOL. 30, 2010MOF, H4K16ac, DDR, AND DSB REPAIR 3585
MOF showed a longer delay in appearance of ?-H2AX foci
compared to SirT2-deficient cells with MOF knockdown,
which exhibited a minimal effect on IR-induced ?-H2AX
focus formation (Fig. 2D), further supporting the role of
H4K16ac in DDR.
We next determined the relationship between H4K16ac lev-
els and cell proliferation in HL60 cells. Treatment of HL60
cells with dimethyl sulfoxide (DMSO) induces their differen-
tiation and also reduces the levels of H4K16ac. A decrease in
the levels of hMOF was also observed in cells treated with
DMSO, whereas the levels of histone H4 did not change (Fig.
2E). After treatment with DMSO for 124 h, differentiated
HL60 cells displayed a decreased frequency of ?-H2AX foci
per cell following irradiation (Fig. 2F) as well as reduced
telomerase activity (see Fig. S5 in the supplemental material),
supporting the argument that decreased levels of H4K16ac
correlate with defective DDR.
Inactivation of ATM in hMOF-depleted cells decreases IR-
induced ?-H2AX focus formation. Ataxia-telangiectasia mu-
tated (ATM) protein is an early responder to DNA damage
with a key role in IR-induced phosphorylation of H2AX, and
in the absence of ATM, H2AX is either phosphorylated by
ATR or DNA-PKcs (10). Since, inactivation of ATM signifi-
cantly delays IR-induced H2AX phosphorylation, we tested
whether the formation of IR-induced ?-H2AX foci was af-
fected by inactivating ATM in hMOF-depleted cells. Cells
depleted of hMOF were treated with the ATM inhibitor, KU-
55933, for 24 h and then examined for IR-induced ?-H2AX
focus formation. We found that cells depleted with hMOF and
treated with KU-55933 had a prolonged delay in the appear-
ance of ?-H2AX foci when compared to cells only depleted of
hMOF or cells only treated with KU-55933 (see Fig. S6A in the
supplemental material). However, cells depleted of hMOF and
treated with KU-55933 had a higher number of residual
?-H2AX foci than cells with only hMOF depletion (see Fig.
S6B in the supplemental material).
hMOF and H4K16ac are important for the recruitment of
key repair components to DNA damage sites. Chromatin
marked by the phosphorylated form of H2AX becomes occu-
pied by MDC1 (mediator of DNA damage-checkpoint protein
1) and 53BP1 (7). Therefore, we determined whether deple-
tion of hMOF influences the frequency of focus formation of
critical DDR components. First, we examined IR-induced
MDC1 focus formation in cells with and without hMOF de-
pletion. MDC1 is known to localize to sites of DNA DSB and
is critical for DDR (40). Cells were treated with 5 Gy of IR and
examined for MDC1 foci at increasing time intervals postirra-
diation. Cells depleted of hMOF had a reduced frequency of
MDC1 foci and higher level of residual MDC1 foci postirra-
diation compared to control cells (Fig. 3A). A similar effect of
FIG. 3. hMOF depletion influences IR-induced repair focus-associated proteins and hMOF interaction with chromatin. 293 cells with and
without depletion of hMOF with reduced levels of H4K16ac were irradiated with 6 Gy and quantified for foci at different time points
postirradiation. (A) For MDC1, cells with more than 5 foci were counted. (B) For 53BP1, cells with more than 3 foci were counted. (C) For
induction of hSSB1 foci, cells were irradiated with 2 Gy and cells with more than 2 foci were counted after 1 h postirradiation. (D) RFP-Rad52-
expressing 293 cells knocked down for MOF were grown in the presence of bromodeoxyuridine (BrdU) and incubated in Hoechst 33342.
Subnuclear DNA damage was induced in the designated rectangular boxes using a 405-nm laser on an LSM510 Zeiss confocal live-imaging system.
(E) Association of hMOF with chromatin was determined by ChIP analysis. Cells were irradiated with 5 Gy and fixed, chromatin immunopre-
cipitation was performed with an hMOF or Rb antibody, and DNA was quantified by absorbance at 260 nm by NanoDrop 2000. (F) Cells with
and without exposure to IR were analyzed for the retention of hMOF by Western blotting. hMOF was detected with a specific hMOF antibody.
3586 SHARMA ET AL.MOL. CELL. BIOL.
hMOF depletion was found for IR-induced 53BP1 focus ap-
pearance and disappearance (Fig. 3B).
We also tested whether hMOF influences IR-induced hu-
man single-stranded DNA binding protein (hSSB1) focus for-
mation (62). hSSB1 accumulates in the nucleus and colocalizes
with other known repair proteins but does not localize to
replication foci in S-phase cells, and its deficiency does not
influence S-phase progression (62). Since hSSB1 depletion
abrogates the cellular response to DSBs (62), we determined
whether hMOF depletion influences hSSB1 focus formation
postirradiation. Cells irradiated with 2 Gy were examined for
the frequency of hSSB1 foci 60 min postirradiation. The fre-
quency of cells with hSSB1 foci was significantly lower in
hMOF-depleted cells, supporting the role of hMOF in IR-
induced DDR-associated focus formation (Fig. 3C). Further-
more, we determined in live cells whether depletion of MOF
influences the recruitment of repair-associated Rad52 protein
to damaged sites. Recruitment of red fluorescent protein
(RFP)-Rad52 at a UV-laser-induced DNA damage site in cells
with and without MOF depletion was analyzed at 10-min in-
tervals up to 4 h postirradiation. Rad52 was observed to effi-
ciently label the entire laser track, but in cells with MOF
depletion and lower H4K16ac levels, labeling of the laser track
was not detected (Fig. 3D). Such failure of Rad52 accumula-
tion at damaged sites in MOF-depleted sites suggests that
MOF facilitates the recruitment of Rad52 at damaged sites.
DNA damage stimulates hMOF association with chromatin.
Our observations suggested that the delay in formation of
IR-induced DNA DSB repair-associated foci seen in hMOF-
depleted cells could be due to altered chromatin structure as a
result of reduced basal-level H4K16ac. Additionally, we
wanted to determine whether hMOF also contributes by alter-
ing chromatin structure during the process of DNA repair.
Therefore, we determined whether hMOF is retained on DNA
postdamage, as has been reported in the case of ATM (5).
Cells were exposed to 5 Gy of IR and collected at different
time points postirradiation, and chromatin immunoprecipita-
tion was performed using hMOF or Rb antibody. hMOF as-
sociation with DNA increased significantly at 2 h postirradia-
tion, whereas no such change in Rb association was observed
(Fig. 3E), suggesting that hMOF’s ability to alter chromatin
structure by acetylating H4K16 may be actively recruited and
necessary for proper DNA repair and resetting the chromatin
structure. To determine how tightly hMOF is associated with
DNA postirradiation, we carried out cellular fractionation with
successive detergent extractions to progressively remove
loosely bound proteins. Since the increased association of
hMOF with DNA was observed after 2 h of irradiation, we
chose to analyze cells for the retention of hMOF at 2, 4, and
6 h postirradiation. Four fractions were collected from cells
with and without irradiation. The first fraction (I) is the clar-
ified cell extract collected after cells were treated with 0.2%
Nonidet P-40-containing buffer. Fraction II is the extract from
the cell pellet of the first fraction, which was washed again in
0.2% Nonidet P-40-containing buffer. Fraction III contains a
longer extraction with 0.5% Nonidet P-40 which was carried
out on the remaining pellet from the second fraction. Fraction
IV contains the insoluble remains that were boiled in electro-
phoresis sample buffer. Consistent with the ChIP data, frac-
tions II to IV showed consistently increased retention of
hMOF in treated samples compared to untreated cells at 2 h
postirradiation (Fig. 3F).
Decreased MOF and H4K16ac abrogate ?-H2AX focus for-
mation at a defined DNA DSB site. To establish a direct link
between H4K16ac status and H2AX phosphorylation at the
site of DNA DSB, we used the system in which a single DSB is
induced at a defined genomic site in NIH 3T3-derived cell lines
(77). These cells (NIH2/4) were engineered to contain a single
copy of the 18-nucleotide I-SceI restriction site flanked on one
side by an array of 256 copies of the lac repressor binding site
and on the opposite side by 96 copies of the tetracycline re-
sponse element (77). The I-SceI restriction endonuclease
tagged with the ligand-binding domain of the glucocorticoid
receptor (I-SceI-GR) remained cytoplasmic in the absence of
triamcinolone acetonide (TA), and DNA DSBs were not de-
tected when noninduced cells were immunostained with anti-
body specific to ?-H2AX. Treatment with TA induced trans-
location of I-SceI to the nucleus and rapid induction of a single
DNA DSB, as visualized by detection of a single nuclear
?-H2AX focus by immunofluorescence (Fig. 4A). NIH2/4 cells
depleted of MOF by MOF siRNA (Fig. 4B) showed a delay in
the appearance of the TA-induced ?-H2AX focus (Fig. 4C).
These kinetics were similar to those seen in cells irradiated
with IR (Fig. 1).
NIH2/4 cells with and without MOF depletion were treated
with TA and analyzed for MOF as well as H4K16ac status.
Cells treated with TA were fixed at different time points after
TA treatment, and quantification of MOF- and H4K16ac-
bound DNA was done by fluorescent intensity (Fig. 4A; see
Fig. S7 in the supplemental material) and real-time PCR (Fig.
4D). The fluorescent intensity measurement of ?-H2AX,
MOF, and H4K16ac at and around the I-SceI DSB site re-
vealed that the accumulation of ?-H2AX peaked at the DSB
site, whereas MOF and H4K16ac levels did not change around
the DNA DSB (Fig. 4A). We found that cells depleted of
hMOF had reduced numbers of ?-H2AX foci, which corre-
lated with reduced global levels of H4K16ac (see Fig. S8 in the
supplemental material). Thus, the delayed appearance of
?-H2AX foci following knockdown of MOF appears to be
correlated with the globally decreased levels of H4K16ac, sug-
gesting that H4K16ac has a role in DNA DSB-induced
?-H2AX focus formation (see Fig. S8 in the supplemental
material). However, chromatin immunoprecipitation (62, 64)
revealed that the levels of MOF as well as H4K16ac present at
the I-SceI (bp 94 to 378) site did not change following induc-
tion of I-SceI-induced DSB (Fig. 4D). These results suggest
that DNA DSB does not induce changes of MOF and
H4K16ac levels at the site of damage.
hMOF depletion alters DNA DSB repair by NHEJ. We
determined whether depletion of hMOF and lack of H4K16ac
altered non-homologous end-joining-mediated DNA DSB re-
pair. In the first assay, we performed ligation-mediated (LM)-
PCR using the approach described in reference 77 and found
that NIH2/4 cells depleted of MOF displayed delayed DSB
repair, which is consistent with the observation of the delayed
appearance of ?-H2AX foci. We further supported these find-
ings by using an engineered cell line that can express the red
fluorescent protein (RFP) only upon repair of I-SceI-induced
DSBs by NHEJ (Fig. 4E). Depletion of MOF resulted in a
decreased number of cells with RFP (Fig. 4F). These data
VOL. 30, 2010MOF, H4K16ac, DDR, AND DSB REPAIR 3587
clearly indicate that the cell’s H4K16ac status plays a critical
role in the NHEJ pathway.
hMOF interacts with DNA-PKcs: hMOF depletion abro-
gates IR-induced ATM-dependent DNA-PKcs phosphoryla-
tion. The above results indicate that MOF is involved in the
NHEJ pathway. In order to determine whether MOF physi-
cally interacts with proteins directly involved in DNA DSB, we
performed a general search for hMOF-interacting proteins
using a tandem-affinity-purification (TAP)-tagged hMOF ex-
pressed in human cells. Proteins copurifying with MOF were
identified by mass spectrometry and confirmed by Western
blotting. hMOF is a component of both hMOF-host cell factor
(HCF)-hMSL1v-hNSL and hMOF-hMSL1/2/3 complexes,
while hMSL3 is only a component of hMOF-hMSL1/2/3 com-
plexes (11, 39, 42, 75). The Western blot analysis indicates that
DNA-PK is more associated with the hMOF-hMSL1v-HCF
FIG. 4. MOF depletion results in delayed appearance of ?-H2AX focus at a site-specific DNA DSB. (A) Mouse NIH2/4 cells with and without
treatment with TA were fixed and examined for the presence of a ?-H2AX focus. The fluorescence of ?-H2AX, MOF, and DNA was measured
across the nucleus and plotted on the top of the cell nucleus. Immunofluorescence was performed with anti-?-H2AX, anti-H4K16ac, and anti-MOF
antibody, and cells were counterstained with DAPI. Fluorescent intensities were measured using Isis or Zen software for each channel. (B) Western
blot detection of MOF 72 h posttransfection with siRNA in NIH2/4 cells. (C) NIH2/4 cells with and without MOF depletion were treated with TA
and fixed at different time points to detect a ?-H2AX focus by immunostaining. (D) ChIP analysis in I-SceI-induced cells with and without
knockdown of MOF examined for bound MOF, H4K16ac, and histone H4. No changes of MOF and H4K16ac levels at the site of damage were
detected. (E and F) Effect of MOF on NHEJ. (E) 293 engineered cell line that can become fluorescent (RFP) only upon repair of I-SceI-induced
DSBs by NHEJ. CMV, cytomegalovirus; CBA, chicken ?-actin promoter. (F) Depletion of MOF results decreased frequency of cells with RFP.
These data clearly indicate that the cell’s H4K16ac status plays a critical role in the NHEJ pathway.
3588 SHARMA ET AL.MOL. CELL. BIOL.
complex than with the hMSL complex (Fig. 5). In contrast to
the purified hMOF-hMSL complex obtained using TAP-
tagged hMSL3L1, the pattern of bands obtained with hMOF-
TAP was less clear, with hMOF-TAP being by far the most
prominent band (Fig. 5). This result indicates that hMOF can
associate with polypeptides other than the conserved MSL
proteins: for instance, with host cell factor 1 (HCF-1) (Fig. 5).
We confirmed that HCF-1 associates specifically with hMOF
by performing Western blot analysis with an anti-HCF anti-
body on hMOF TAP-, hMSL3L1 TAP-, and mock TAP-puri-
fied samples (Fig. 5B). These data clearly indicate that hMOF
associates with HCF-1 in a complex distinct from the hMSL
complex obtained with hMSL3L1.
Additionally hMOF was found to copurify with a significant
amount of DNA-dependent protein kinase catalytic unit
(DNA-PKcs) (Fig. 5A and B; see Fig. S9A in the supplemental
material). The activity of this serine/threonine protein kinase is
stimulated by its recruitment to the DNA DSB via the Ku
heterodimer, Ku80/Ku70. Since DNA-PKcs is critical for DNA
repair via the NHEJ pathway and also plays a role in the
signaling response to DNA damage (58, 68), we determined
whether depletion of hMOF influences IR-induced phosphor-
ylation of DNA-PKcs. Cells depleted of hMOF had decreased
ATM-dependent IR-induced DNA-PKcs phosphorylation at
the pT2609 site but not at the pS2056 DNA-PKcs autophos-
phorylation site (Fig. 5C). In contrast, Tip60 depletion had no
effect on IR-induced DNA-PKcs phosphorylation at T2609.
These results suggest that hMOF is critical for the activation of
We further determined the impact of hMOF on DNA-PKcs
accumulation at DSB sites by using live cell imaging combined
with microirradiation of cells expressing yellow fluorescent
protein (YFP)-tagged DNA-PKcs. We observed a modest in-
crease in the YFP-tagged DNA-PKcs accumulation at the site
of damage (Fig. 5D). To determine the influence of hMOF
depletion on DNA-PKcs accumulation at the site of damage,
cells were irradiated and time-lapse imaging to monitor YFP
intensity was performed (Fig. 5D). Cells depleted of hMOF
FIG. 5. Interaction of hMOF with DNA-PKcs. (A) hMOF-TAP is
purified with distinct sets of associated proteins. The calmodulin frac-
tion of hMOF-TAP was separated by SDS-PAGE and stained with
Sypro Ruby Red, and gel slices were subjected to in-gel digestion and
mass spectrometry. M, molecular mass marker. Peptides correspond-
ing to hMSLs and other proteins were identified (numbers of peptides
are shown in parentheses) (see Fig. S9 in the supplemental material).
DNA-PKcs copurifies preferentially with hMOF. hMOF TAP-,
hMSL3L1 TAP-, and mock TAP-purified fractions were assayed for
the presence of hMSL1, HCF-1, and DNA-PKcs by Western blotting
(B). (C) Cells with and without depletion of hMOF or Tip60 were
irradiated with 5 Gy and examined for DNA-PKcs phosphorylation
using antibodies specific for pS2056 and pT2609. Depletion of hMOF
resulted in loss of ATM-dependent phosphorylation of DNA-PKcs
(pT2609), and no such loss was observed in Tip60-depleted cells.
(D) Recruitment of DNA-PKcs to the laser-induced DNA DSBs. Cells
were transfected with YFP-DNA-PKcs and microirradiated. Time-
lapse imaging of YFP-DNA-PKcs-expressing U2OS cells was done
before and after microirradiation. (E) Cells stably expressing YFP-
DNA-PKcs were treated with control or hMOF siRNA for 72 h, and
microirradiated. (F) Initial accumulation kinetics (see Fig. S9B in the
supplemental material) and relative fluorescence for a 2-h time course
of treatment with YFP-DNA-PKcs at laser-generated DSBs. Error
bars represent the standard deviations (SD) from three different ex-
VOL. 30, 2010 MOF, H4K16ac, DDR, AND DSB REPAIR3589
showed slower kinetics of the appearance as well as disappear-
ance of fluorescent focus (Fig. 5E and F; see Fig. S9B in the
supplemental material). These results suggest that hMOF de-
pletion influences IR-induced ATM-dependent DNA-PKcs
phosphorylation as well as its accumulation at the sites of
damage (Fig. 5F).
hMOF depletion alters DNA DSB repair by the HR path-
way. DNA DSBs are repaired by either the NHEJ or HR
pathways (13, 27, 31, 65, 68, 76). Homologous recombination
requires a sister chromatid, homolog, or homologous sequence
on a heterolog to be used as a template for DNA DSB repair
and thus requires more significant chromatin remodeling and
greater DNA accessibility to facilitate DNA unwinding, strand
invasion, and DNA replication-based repair mechanisms. We
investigated the role of hMOF in HR repair by first examining
the effect of hMOF depletion on Rad51 focus formation.
The Rad51 protein is a central player in homolog-directed
recombinational repair (58, 63). It localizes to a homologous
sequence and forms initial joints, even on the surface of nu-
cleosomes (63). Similarly, Rad51 also colocalizes with ?-H2AX
and Dmc1 foci on synaptonemal complexes at the leptotene
and zygotene stages of meiosis, where recombination events
also take place (20). Cells with and without hMOF depletion
were irradiated with different doses of IR, and Rad51 foci were
examined 3 h after irradiation. Depletion of hMOF reduced
the frequency of cells with IR-induced Rad51 foci (Fig. 6A),
suggesting that hMOF has a role in the repair of DNA DSB
Since hMOF depletion decreases hSSBI foci (Fig. 3C) as
well as Rad51 focus formation (Fig. 6A), and both proteins
have a role in HR, we directly determined the influence of
hMOF on HR by two different approaches. In the first ap-
proach, cells depleted of hMOF were treated with mitomycin
C and examined for sister chromatid exchanges (SCEs) by the
previously described procedure (49, 51). Mitomycin C treat-
ment increased the frequency of SCEs, reflecting increased
FIG. 6. MOF plays a role in HR. (A) Cells with and without MOF or Tip60 knockdown were irradiated with different doses, and cells were
quantified for the presence of Rad51 foci after 4 h of irradiation. (B) Impairment of I-SceI-induced HR in hMOF-deficient cells was found. HR
frequencies are shown with (?) or without (?) I-SceI induction in untreated cells, in cells treated with control siRNA, and in cells treated with
hMOF- or BRCA1-specific siRNA (54). The results presented are the mean and standard error from three independent experiments. (C) Lo-
calization of MOF on mouse meiotic chromosomes during prophase 1. Spermatocyte spreads from male mice were immunostained with anti-MOF,
anti-TRF1, and anti-SCP. MOF is in red, synaptonemal complex protein SCP3 is green, and TRF1 is white on chromosomes from mouse
spermatocytes. The presence of MOF on synaptonemal complexes from leptotene to mid-pachytene suggests the role of MOF in meiotic
3590 SHARMA ET AL.MOL. CELL. BIOL.
recombination frequency. Cells depleted for hMOF showed a
decreased frequency of mitomycin C-induced SCEs (see Fig.
S10 in the supplemental material), suggesting defective recom-
bination. To further test whether HR is affected, we examined
the reconstitution frequency of a GFP reporter gene within a
chromosomally integrated plasmid substrate (61) in cells with
or without reduced levels of hMOF. Following I-SceI transfec-
tion, the population of cells containing the pDR-GFP substrate
demonstrated a ?80-fold increase in the number of GFP-
positive members compared with cells containing the control
vector, indicating that almost all of the GFP-positive cells
resulted from DSB-induced recombination. pDR-GFP cells
treated with hMOF siRNA had an ?3-fold reduction in the
number of GFP-positive cells in comparison to control siRNA-
transfected cells (t test; P ? 0.001) (Fig. 6B). These data along
with the observation of reduced frequency of Rad51 foci and
inefficient recruitment of Rad52 on the damaged chromatin
provide strong evidence that hMOF affects the HR pathway
for DNA DSB repair.
Further validation for a role of MOF in DNA DSB repair by
HR was obtained by analysis of this HAT in meiotic cells,
which are programmed for homologous recombination. Since
meiotic recombination is initiated by DSBs that are introduced
into leptotene DNA by the conserved SPO11 transesterase (33,
41), we determined the localization of MOF during the lepto-
tene-to-pachytene stages of male meiosis in the testes of mice.
Interestingly, MOF was found associated with the synaptone-
mal complexes through the progression of pairing and synapsis
of the homologous chromosomes in the first meiotic division
from leptotene to late pachytene (Fig. 6C). This confined lo-
calization of MOF to the chromatin domains undergoing active
pairing and recombination further argues that MOF has a role
in DNA DSB meiotic recombination. Taken together, the
present data suggest that MOF plays a functional role in the
process of homologous recombination.
We sought to determine whether the ubiquitous, preexisting
basal levels of H4K16 acetylation have a role in DDR and what
impact H4K16ac’s absence may have on DNA DSB repair by
NHEJ or HR pathways. Our results provide strong evidence
that preexisting H4K16ac does facilitate DNA damage recog-
nition (Fig. 7). Based on the established role of this modifica-
tion in chromatin organization, our observations are consistent
with the model wherein basal levels of H4K16ac maintain a
chromatin structure conducive for efficient DNA damage re-
pair. This model (Fig. 7) is based on the fact that specifically
altering the levels of H4K16ac by various approaches directly
affected IR-induced ?-H2AX focus formation: (i) MOF deple-
tion reduced H4K16ac levels and delayed/abrogated IR-in-
duced focus formation of ?-H2AX, MDC1, 53BP1, Rad51, and
hSSB1; (ii) depletion of Tip60, another HAT, had no impact
on H4K16ac levels and no influence on ?-H2AX focus forma-
tion; (iii) depletion of Tip60 did not enhance the delay in the
IR-induced ?-H2AX focus formation in cells depleted for
hMOF; (iv) cells expressing HAT-dead MOF had a delay in
the appearance of IR-induced ?-H2AX focus formation; (v)
treatment of cells with TSA enhanced H4K16ac levels and
reversed the delay in ?-H2AX focus formation upon depletion
of hMOF; (vi) MOF depletion had a minimal effect on the
appearance of IR-induced ?-H2AX foci in SirT2?/?cells as
compared to SirT2?/?cells; and (vii) MOF depletion along
with ATM inactivation increased the delay in the formation of
IR-induced ?-H2AX foci.
Our comprehensive analysis of the correlation between the
chromatin modifier MOF, modified histone H4 residue K16ac,
and IR-induced DDR strongly supports the argument that
acetylation of histone H4K16 strongly influences DDR (Fig.
7). The role of H4K16ac in DDR is further supported by the
fact that the acetylation at the N-terminal tails of histones H3
and H4 correlates with the establishment of an open euchro-
matin conformation that is transcriptionally active. Conversely,
hypoacetylation of H3 and H4 tails is associated with hetero-
chromatin (36, 89). Our results argue that the two different
forms of chromatin could differentially influence generation of
DDR-associated signaling. Histone acetylation impacts chro-
matin structure through the neutralization of the positive
charges on lysine residues, altering intra- and internucleosomal
interactions of the chromatin fiber and thus facilitating decon-
densation and enhancing nucleosomal DNA accessibility (6,
35). It is well established that histone acetylation is recognized
by transcriptional factors or ATP-dependent remodeling activ-
ities (91), but since acetylated K16 (H4K16ac) is present in
?60% of the total H4 molecules of mammalian cells (84), it is
likely that it also has a specific structural role in chromatin-
based processes such as DDR. Shogren-Knaak and coworkers
have demonstrated the structural significance of histone H4 at
lysine 16 (H4K16ac) as its incorporation into nucleosomal ar-
rays abrogates the formation of compact 30-nm-like fibers (74).
The adaptor protein 14-3-3 binds the phosphorylated nucleo-
some and recruits MOF, which triggers the acetylation of his-
tone H4 at lysine 16 (94). Nucleosomes with H4K16ac in the
fiber have a decreased ability to form cross-fiber interactions
(74), and thus the chromatin remains in an open state, condu-
cive to DDR. This assumption is consistent with the fact that
H4K16ac levels peak in S phase (91), the cell cycle phase most
efficient for DNA DSB repair and which has less IR-induced
FIG. 7. MOF and H4K16ac influence DDR at multiple stages of
DNA DSB repair pathways. MOF is a major HAT for H4K16ac, and
its levels determine IR-induced repairosome formation. MOF inter-
acts with DNA-PKcs and also localizes on the synaptonemal complex
(SCP3) of meiocytes, thus linking to both the DNA DSB repair path-
VOL. 30, 2010 MOF, H4K16ac, DDR, AND DSB REPAIR3591
cell killing than during the G1and G2phases of the cell cycle
Histone acetyltransferases exist as components of multisub-
unit protein complexes involved in different processes such as
transcription activation, gene silencing, and cell cycle progres-
sion as well as DNA damage repair (12). Such complexes have
been reported to contain ATM-related proteins such as
TRRAP, which is found in human Tip60 and PCAF complexes
(3). ATM is implicated in mitogenic signal transduction, chro-
mosome condensation, meiotic recombination, cell cycle con-
trol, and telomere maintenance (50, 52, 53).
The effect of MOF on ATM activation is by an indirect
mechanism, and inactivation of ATM or p53 does not rescue
lethality due to MOF deletion (24). In addition, our current
studies reveal that depletion of MOF does not influence the
levels of proteins known to be involved in DDR, suggesting
that MOF-dependent H4K16ac primarily affects DDR through
a direct impact on chromatin structure (Fig. 7). Given the
significance of H4K16ac, its absence could result in reduced
transmission of signals generated from DNA damage sites, and
this would delay the timely course of repair. Furthermore,
analysis of H4K16ac levels at the site of I-SceI-induced DSB in
MOF-depleted cells revealed a direct correlation between loss
of H4K16ac and abrogation of ?-H2AX focus appearance.
In addition to histone kinases, several studies have revealed
the role of HAT complexes in DNA DSB repair. It has been
reported that HAT complexes act in concert with the ATP-
dependent SWI/SNF and RSC (which remodels the structure
of chromatin)-containing chromatin-remodeling complexes to
facilitate DNA repair (58). Cells expressing catalytically inac-
tive TIP60 have been found to have impaired DNA DSB repair
(30). TIP60 with its cofactor TRRAP directly binds to chro-
matin near DNA DSBs, and depletion of TRAPP impairs
DNA damage-induced H4 acetylation, which results in defec-
tive DSB repair by HR (45). Similar results were observed with
NuA4, a yeast homolog of TIP60, following induction of DSB
by HO endonuclease (17). NuA4 binds directly to sites of DNA
DSBs concomitantly with the appearance of ?-H2AX (17).
Our analysis revealed that MOF influences the formation of
DNA DSB repair-associated foci, but its global association
with chromatin increases postirradiation, as determined by
ChIP assay (Fig. 3E). Furthermore, MOF retention on chro-
matin also increases postirradiation, suggesting that MOF it-
self has a critical role in the later steps of DNA DSB repair,
which is evidenced by the fact that cells expressing mutant
MOF had higher residual DNA DSBs and chromosome aber-
rations (25). Our previous studies have revealed that expres-
sion of mutant hMOF induced neither a significant increase in
G1-phase cells nor a decrease in S-phase entry or accumulation
in G2phase after IR exposure compared to control cells (25,
75). However, cells expressing a HAT-dead mutant of hMOF
had a higher frequency of IR-induced chromosomal aberra-
tions, suggesting that hMOF does influence DNA DSB repair
Further evidence for the role of MOF in DNA DSB repair
is provided by the following observations: (i) MOF forms a
complex with DNA-PKcs, and (ii) MOF localizes on the syn-
aptonemal complexes in spermatocytes. Depletion of MOF
resulted in abrogation of IR-induced ATM-dependent phos-
phorylation of DNA-PKcs. These observations are consistent
with the model proposed earlier that MOF regulates the ATM
function through the status of chromatin (Fig. 7). Further-
more, depletion of MOF delays the accumulation of DNA-
PKcs postirradiation. Delay in the accumulation could be due
to following reasons: (i) loss of ATM-dependent damage-in-
duced DNA-PKcs phosphorylation; (ii) change in chromatin
structure due to loss of H4K16ac; and (iii) decreased associa-
tion of MOF with DNA postirradiation, preventing chromatin
alterations conducive for DSB repair.
hMOF functions upstream of ATM (25), possibly sensing
DNA damage-induced chromatin changes with subsequent sig-
naling to ATM effectors. In contrast, Tip60 has been reported
to activate ATM by acetylation of its lysine residue, and so
ATM activation could be independent of chromatin modifica-
tions (78). Overall, histone acetylation appears to both unwind
chromatin and create a permissive platform that facilitates
recruitment of remodeling complexes. The INO80 complex,
including the INO80 member of the SWI/SNF family, has long
been known to regulate transcription at RNA polymerase II
(Pol II) promoters through chromatin remodeling. More re-
cently, it was observed that INO80 is recruited to ?-H2AX
near DNA DSBs, and yeast mutants of INO80 are hypersen-
sitive to damaging agents and HO endonuclease, providing one
of the first examples of SWI/SNF ATPase participation in
DNA repair (44, 60, 86). Interestingly, the actin-related pro-
tein Arp4 in yeast is present in both the NuA4-HAT complex
and the INO80-SWR1 complex, further suggesting concerted
action of histone-modifying and chromatin-remodeling activi-
ties in the DSB response (17). The exact role of INO80 has yet
to be clearly defined. TRRAP-TIP60 activity may be limited to
local chromatin unwinding since chromatin relaxation alone is
sufficient to rescue the defects caused by TRRAP deficiency
(45). Since the role of MOF through alteration of H4K16ac
levels is upstream in DDR and MOF forms a complex with
DNA-PKcs, essential for NHEJ, our studies also found that
MOF is required for the repair of DNA DSB by the HR
pathway. This is supported by the observations that depletion
of MOF results in the abrogation of Rad51 focus formation
(Fig. 6A) and reduced frequency of sister chromatid exchange
formation (see Fig. S10 in the supplemental material). Further
evidence about the role of MOF comes from the following
observations: (i) depletion of MOF results in reduced fre-
quency of I-SceI-induced DSB repair, and (ii) MOF is local-
ized on the synapetonemal complex during the process of mei-
In contrast to yeast, which has only two copies of histone H4,
studying the direct role of lysine 16 of histone H4 in mamma-
lian cell systems is more difficult due to the presence of mul-
tiple copies of the H4 gene. Moreover, the yeast and mamma-
lian systems are substantially different, since deletion of Sas2,
the main H4K16 acetyltransferase in yeast, does not result in
cellular lethality, whereas deletion of MOF not only results in
embryonic lethality but also has a cytostatic effect in many
human and mouse cell lines examined (24). Although loss of
acetylation at H4K16 does influence transcription in both yeast
(14, 16) and mammalian (32, 90, 91) cells, it is important to
note that chromatin modifications common to yeast and mam-
malian systems do not always lead to similar DDR phenotypes.
Acetylation of H4K16, for example, results in loss of histone
H4 in yeast but does not result in loss of histone H4 in mam-
3592 SHARMA ET AL.MOL. CELL. BIOL.
malian cells. This could be due to the fact that mammalian cells
have a much more complex chromatin organization, so that
H4K16ac could be playing a more critical role in DDR, as is
further supported by the present work.
In summary, these data indicate that H4K16ac (histone
code) constitutes one of the main early signals for generation
of efficient DDR, which influences both of the pathways of
DNA DSB repair. These results are consistent with the finding
that chromatin alterations due to DNA DSB trigger ATM
phosphorylation in mammalian cells, which is abrogated when
cells are depleted of MOF. Abrogation of both DNA DSB
repair pathways at several stages in cells depleted of MOF and
the presence of MOF in the synaptonemal complex are further
strong evidence of the role of MOF in DDR. Since MOF is
indispensable for both NHEJ and HR DNA DSB repair path-
ways, H4K16ac (a histone code) must provide a permissive
chromatin environment critical for DDR and subsequent
mammalian cell survival. Further studies should reveal
whether the H4K16ac levels of any particular region of the
genome are critical for DDR.
We thank Fred Alt for SirT2 mouse embryonic fibroblast cells and
Tom Misteli for reagents. Thanks are due to Clayton Hunt, Sandeep
Burma, Nobuo Horikoshi, and the members of the Pandita laboratory
for suggestions and comments and helpful discussion.
This work was supported by grants NIH CA123232 and CA10445 to
T.K.P.; CA 50519 to D.C.; and CIHR MOP-64289 to J.C., who is also
a Canada Research Chair. N.A. is a CIHR postdoctoral fellow.
We declare that we have no competing financial interests.
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