p8/nupr1 regulates DNA-repair activity after double-strand gamma irradiation-induced DNA damage.
ABSTRACT The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein, which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA gamma-irradiation. Contrary to the HAT-associated activity, MSL1-dependent DNA-repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1-dependent DNA-repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after gamma-irradiation, it is eventually submitted to sustained down-regulation, presumably to allow development of MSL1-associated DNA-repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1-dependent HAT activity to the vicinity of damaged DNA. MSL1-dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery.
- SourceAvailable from: Adrian Velazquez-Campoy[Show abstract] [Hide abstract]
ABSTRACT: The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered ("fuzzy"), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners.PLoS ONE 10/2013; 8(10):e78101. · 3.53 Impact Factor
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ABSTRACT: hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2(-/-) cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2(-/-) chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.PLoS ONE 07/2013; 8(7):e68549. · 3.53 Impact Factor
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ABSTRACT: NUPR1 (nuclear protein 1) was found to play a key role in the development of several malignancies including pancreas, breast, and prostate cancers. However, the functional role of NUPR1 in nonsmall cell lung cancer (NSCLC) progression and development is little known. Here, lentivirus-mediated small interfering RNA (siRNA) was employed to downregulate endogenous NUPR1 expression to study the function of NUPR1 in growth of nonsmall cell lung cancer. A lentivirus-mediated RNAi technology was used to specifically knock down the expression of NUPR1 in H1299 cells. Quantitative real-time reverse transcriptase polymerase chain reaction, flow cytometry, western blot and cell count assays were studied to characterize NUPR1 expression in vitro. Furthermore, nonsmall cell lung cancer xenograft models in nude mice were established to investigate whether knockdown of NUPR1 reduces the tumor growth in vivo. We found that downregulation of NUPR1 expression significantly inhibited nonsmall cell lung cancer H1299 cells proliferation and colony formation in vitro. Moreover, the specific downregulation of NUPR1 arrested cells in G0 phase of cell cycle and increased apoptosis rate. Silencing of NUPR1 also suppressed tumor growth by tail vein injection of lentivirus encoded shRNA against NUPR1 in vivo. Our findings revealed that the NUPR1 gene represents a promising target for gene silencing therapy in nonsmall cell lung cancer. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 09/2012; · 1.34 Impact Factor
DNA-Repair Activity After
Irradiation-Induced DNA Damage
MERITXELL GIRONELLA,1,2CEDRIC MALICET,1CARLA CANO,1MARIA JOSE´SANDI,1
TEWFIK HAMIDI,1RICARDO MARTIN NEME TAUIL,3MARIELA BASTON,1PIA VALACO,3
SILVIA MORENO,3FREDERIC LOPEZ,4JOSE LUIS NEIRA,5,6JEAN CHARLES DAGORN,1
AND JUAN LUCIO IOVANNA1*
1INSERM U.624, Stress Cellulaire, Case 915, Parc Scientifique et Technologique de Luminy, Marseille Cedex 9, France
2Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Hepa ´ticas y Digestivas (Ciberehd), Barcelona, Spain
3Departamento de Quı´mica Biolo ´gica, Ciudad Universitaria, Buenos Aires, Argentina
4INSERM IFR31, Institut Louis Bugnard, Ho ˆpital Rangueil, Toulouse Cedex 4, France
5Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna ´ndez, Elche, Alicante, Spain
6Biocomputation and Complex Systems Physics Institute, Zaragoza, Spain
exerted throughinteractions withother proteins, whoseactivities arethereby enhancedorrepressed. Inthiswork we describe another
example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein,
which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA g-irradiation. Contrary to the
emerged from our findings is that 53BP1 could be ascaffold that gets the HAT MSL1-dependent DNA-repair activity to thesites of DNA
by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery.
J. Cell. Physiol. 221: 594–602, 2009. ? 2009 Wiley-Liss, Inc.
The p8/nupr1 (p8 herein) gene was first described as over-
of pancreatitis (Mallo et al., 1997). Then, we and others found
that expression of p8 mRNA is rapid, strong, and transient in
response to several injuries, including minimal stresses (Jiang
et al., 1999; Garcia-Montero et al., 2001a; Zinke et al., 2002;
Taieb et al., 2005; Plant et al., 2006) and in response to several
factors such as TGFbeta (Garcia-Montero et al., 2001b)
endothelin (Goruppi et al., 2002). Structurally, p8 is a highly
basic 82-amino-acid polypeptide, with a theoretical molecular
mass of about 8kDa, containing a canonical bipartite domain of
positively charged amino acids typical of nuclear-targeting
signals (NLS) (Vasseur et al., 1999) and its nuclear and/or
cytoplasmic location has been established (Igarashi et al., 2001;
Su et al., 2001; Ito et al., 2003, 2005a,b; Valacco et al., 2006). In
fact, the nuclear or cytoplasmic localization of p8 depends, at
protein contains an N-terminal PEST (Pro/Glu/Ser/Thr-rich)
proteasome system (Goruppi and Kyriakis, 2004).
with other proteins of known function. However, some of its
biochemical properties are shared by members of the high
mobility group proteins (HMG), particularly by the HMG-I/Y
family (Encinar et al., 2001). NMR and CD analyses of
The protein binds DNA weakly and is a substrate for protein
kinase A. The phosphorylated p8 has a higher content of
secondary structure than the non-phosphorylated protein and
phosphorylated p8 binds DNA strongly (Hoffmeister et al.,
2002). Moreover, secondary structure prediction methods
indicate the presence, within the region showing homology
with HMG proteins, of a basic helix–loop–helix secondary
Abbreviations: HAT, histone acetyl transferase; 53BP1, p53-binding
protein 1;MSL1, male-specific lethal protein 1;MOF, males-absent-
Meritxell Gironella and Cedric Malicet contributed equally to this
Additional Supporting Information may be found in the online
version of this article.
Contract grant sponsor: INSERM.
Contract grant sponsor: Ligue Contre le Cancer.
Contract grant sponsor: Canceropole PACA.
Contract grant sponsor: Ecos-Sud.
Contract grant sponsor: Instituto de Salud Carlos III.
*Correspondence to: Juan Lucio Iovanna, INSERM U.624, Stress
Cellulaire, 163 Avenue de Luminy, CP 915, 13288 Marseille Cedex
9, France. E-mail: firstname.lastname@example.org
Received 8 May 2009; Accepted 26 June 2009
Published online in Wiley InterScience
(www.interscience.wiley.com.), 31 July 2009.
? 2 0 0 9 W I L E Y - L I S S , I N C .
structure motif, characteristic of some classes of transcription
factors. An architectural role in transcription was proposed
(Garcia-Montero et al., 2001b; Hoffmeister et al., 2002; Quirk
the cell (Vasseur et al., 2003; Taieb et al., 2005), of the
endoplasmic reticulum stress response (Carracedo et al.,
2006a,b), and of tumor formation and progression (Vasseur
et al., 2002; Mohammad et al., 2004). In addition, p8 is involved
in cell-cycle regulation, through its interaction with Jab1
(Malicet et al., 2006a), and apoptosis by interacting with
prothymosin alpha (Malicet et al., 2006b,c). To account for
the protein, its lack of specific tridimensional structure, and its
partners to target different signaling pathways.
binds to and inhibits MSL1, a protein with 53BP1-dependent
DNA-repair activity. Through this interaction p8 regulates
DNA repair after double-strand DNA damage induced by
Materials and Methods
Cell lines and cell culture conditions
All cell lines were obtained from American Type Culture
Collection (ATCC, Molsheim, France). HeLa and the Phoenix
amphotropic viral packaging cell lines were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum and 2mM L-glutamine. All cell lines were routinely
cultivated in humidified 5% CO2atmosphere.
Anti-H4K16ac (catalogue # 07-329) and anti-H3K9ac (catalogue #
07-352) antibodies were purchased from Millipore (Molsheim,
France). The anti-MSL1 rabbit polyclonal antibody (catalogue #
ab61008) was purchased from Abcam (Paris, France). The anti-
53BP1 rabbit polyclonal antibody (catalogue # sc-22760), anti-
MRG15 (A-13) goat antibody (catalogue # sc-26529), and anti-
MORF4L1 (N-19) goat antibody (catalogue # sc-26525) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-b-actin mouse monoclonal antibody (catalogue # A5441) and
(Saint-Quentin Fallavier, France), the mouse monoclonal anti-HA
(catalogue # 1666606) was from Roche (Meylan, France)
Diagnostics, and the rabbit polyclonal anti-V5 (catalogue #
MCA1360GA) was from Serotech (Oxford, UK). The p8
Yeast two-hybrid screenings
Using a PCR-based strategy we subcloned the complete coding
sequence of human p8 (Vasseur et al., 1999) into the Mlu1
restriction site of the pSos vector to generate the fusion protein
pSos-p8. MSL1 was PCR amplified and subcloned into the pSos
vector using the same approach as for pSos-p8. These constructs
the manufacturer (Stratagene, La Jolla, CA), a human testes library
constructed into the pMYR vector.
The full-length sequence of MSL1 was cloned into the mammalian
expression vector pcDNA4 using the Gateway approach to
produce the MSL1-V5 recombinant protein following
recommendations of the supplier (Invitrogen, Cergy-Pointoise,
sequence of human p8 into the XhoI and EcoRV restriction sites of
the pcDNA3-Flag vector. DNA constructs were systematically
sequenced to confirm their correct sequence. Plasmids were
transfected into 293T cells using the lipofectamine 2000 reagent
lysis in 50mM Tris–HCl, pH 8.0, 0.5% Nonidet P-40 with protease
inhibitors, p8 was immunoprecipitated by adding rabbit antibodies
against Flag epitope and MSL1 with an anti-V5 polyclonal antibody
by rocking 2h at 48C. Then, 20ml of protein A-Sepharose or
protein G-Sepharose conjugate (Zymed Laboratories Inc, San
Francisco, CA) was added and incubated for an additional 2h at
48C. The Sepharose beads were washed three times followed by
SDS–PAGE and Western blotting by using the anti-V5 or anti-Flag
antibodies. Immunoprecipitation of endogenous proteins was
performed using an anti-p8 rabbit antibody (Vasseur et al., 1999).
Cell lysate prepared with a buffer containing 1% Triton X-100 was
used for precipitation with Sepharose beads. The precipitated
materialwas immunoblotted withanti-MSL1,anti-MRG15, oranti-
Full-length 53BP1 HA-tagged cDNA cloned into the pCMH6K
plasmid (Iwabuchi et al., 2003) was a gift from Kuniyoshi Iwabuchi
(University of Kanazawa, Japan) and Aidan Doherty (University of
Sussex, UK). Interaction of 53BP1 with MSL1 in vivo was analyzed
by transfecting pCMH6K-53BP1-HA and pcDNA4-MSL1 plasmids
into 293T cells and processed as described above with the
exception that anti-HA antibody was used instead of anti-Flag.
an anti-53BP1 rabbit antibody. Cell lysate prepared with a buffer
containing 1% Triton X-100 was used for precipitation with
Sepharose beads. The precipitated material was immunoblotted
with an anti-MSL1 antibody.
p8 siRNA sequence (sense
50-GGAGGACCCAGGACAGGAUd(TT)-30) and MSL1 siRNA
sequence (sense 50-TGAGATCCGCGGTGTTCAAd(TT)-30)
were chosen among 4 and 3 sequences, respectively, for their
highest efficacies. 53BP1 siRNA (sense 50-
GCCAGGUUCUAGAGGAUGAd(TT)-30) was previously
reported (Wangetal.,2002).Kinetics ofsilencingby thesesiRNAs
showed that maximum knockdown was reached within the first
48–72h, recessing thereafter. These siRNAs were obtained from
Eurogentec (Serain, Belgium), annealed and ready to use after
rehydration. The day before transfection, cells were plated to give
60–80% confluence. After removal of the medium, cells were
washed once with serum-free medium and transfection was done
in serum-free medium by addition of a mix composed of
XtremeGENE (Roche Diagnostics) and p8 siRNA, MSL1 siRNA,
53BP1 siRNA, or control siRNA diluted in serum-free medium.
After 4h of incubation at 378C, the transfection medium was
replaced by fresh medium. Twenty-four hours later cells were
plated for 24h then g-irradiated with 7Gy or HAT activity
measured after an additional 24-h period.
The lentivirus vectors used in this study were based on the pCCL
self-inactivating vector kindly provided by Cedric Raoult
(University of Marseille, France). In this vector, the viral promoter
box in the U3 promoter region of the 30LTR. In brief, lentiviral
vectors were produced by transient transfection into 293T cells.
293T cells were seeded in 10-cm diameter dishes 24h prior to
ina 5%CO2incubator. A totalof 30mg plasmid DNAwasusedfor
the transfection of one dish: 10mg of pMDG-VSV-G envelope
the transfect vector plasmid (pCCL-p8 or pCCL-MSL1). The
lentiviral vectors were constructed as follows: full-length human
p8-EGFP and MSL1-EGFP fusion proteins were subcloned into
BamHI/XhoI restriction sites of the pCCL vector. DNA constructs
JOURNAL OF CELLULAR PHYSIOLOGY
p 8 , M S L 1 , A N D 5 3 B P 1
were sequenced to confirm their correct sequence. The
transfection was done using the polyethyleneimine (PEI) reagent in
acetate filters. Viral supernatant was used to infect HeLa cells
supplemented with 8mg of polybrene (Sigma)/ml. Infection was
done twice. As control, cells were infected with the pCCL empty
vector and with an EGFP-expressing lentivirus (pCCL-EGFP).
EGFP expression was usedto measure the transduction efficiency.
For cross-infections we mixed equal volumes of each lentivirus
Clonogenic survival assay
HeLa cells were transfected with 2mg of total DNA containing
pcDNA3-p8, pcDNA4-MSL1-V5, pcDNA3-p8þpcDNA4-MSL1-
V5 alone or in combination with pCMH6K-53BP1-HA using the
Fugene HD transfection reagent following manufacturer’s
recommendations (Roche Diagnostics). Empty vectors were used
as control or to complete to identical amounts the mass of
45,000 cells were seeded in duplicate on tissue culture dishes
(6cm) maintained in a humidified incubator at 378C and 5% CO2
until cell colonies were formed. After 9 days, colonies were fixed
with 75% (v/v) methanol, 25% (v/v) acetic acid for 30min, rinsed
twice with PBS and once with distilled water, stained with crystal
violet (1mg/ml in distilled water) for 10min and rinsed abundantly
with distilled water. When stained cultures were dried, visible
HAT activity assay
MSL1 lentivirus. After appropriate antibiotic selection, nuclear
extracts were prepared using the Nuclear/Cytosol Fractionation
Kit (BioVision, Mountain View, CA). In separate experiments,
lentivirus-transduced cells were also transfected with
pCMH6K-53BP1-HA plasmid. Bradford method (Bio-Rad protein
assay; Bio-Rad, Hercules, CA) was used to quantify protein
was used to analyze histone acetyl transferase (HAT) activity using
the HAT activity colorimetric assay kit (BioVision) following the
manufacturer’s recommendations. Plates were incubated at 378C
Analysis of p8 and MSL1 mRNA expression after irradiation
HeLa cells were g-irradiated at 3, 7, or 10Gy and total RNA
6, 12, 18, or 24h after irradiation. Analysis of p8 and MSL1 mRNA
expressions was done using qRT-PCR analysis on a LightCycler
detection system (Roche Applied Science, Meylan, France).
First strand cDNA was synthesized from 1mg of total RNA using
random hexamers and expanded by reverse transcriptase
according to the manufacturer’s instructions (ImProm-II Reverse
Transcription System; Promega, Charbonnie `res, France),
subsequently diluted 1:10 with water, and stored at ?208C until
use. MSL1, p8, and GAPDH PCR products were detected by
quantitative real-time PCR using the SYBR Premix Ex Taq (Takara
Bio, Inc., Gennevilliers, France) following the manufacturer’s
instructions. Five microliters ofdiluted cDNAtemplatewas mixed
with 10ml SYBR Premix Ex Taq (including Taq polymerase,
reaction buffer, MgCl2, SYBR green I dye, and deoxynucleotide
triphosphate mix) and 0.4mM forward and reverse primers, in a
final volume of 20ml. The following primers were used: MSL1
forward 50-GCCTCTAAGGGACCCAAATC-30and MSL1
reverse 50-TGGTCGTCCAAATGCTACAA-30; p8 forward
50-TAGAGACGGGACTGCG-30and p8 50-
GCGTGTCTATTTATTGTTGC-30reverse; GAPDH forward
reverse 50-CATGTGGGCCATGAGGTCCACCAC-30. After an
initial Taq activation for 10sec at 958C,LightCycler PCR was done
588C for 7sec, and 728C for 14sec. Each sample was analyzed in
duplicate and the experiment was repeated three times. Results
were analyzed using RealQuant data analysis software (Roche
Applied Science, Meylan, France).
p8 and MSL-1 recombinant protein productions
Human p8-His6 was obtained as previously described (Encinar et
al., 2001). Human MSL1 full-length cDNA was subcloned into the
downstream of the glutathione-S-transferase (GST) sequence and
used to transform E. coli BL21 strain in order to produce
recombinant MSL1-GST protein. Recombinant proteins were
produced as described in Encinar et al. (2001).
Surface plasmon resonance
immobilized by amine coupling onto a CM5 chip (BIAcore AB) as
previously described (Bousquet et al., 2006). Following
immobilization, the chip was washed for 30min with HBS buffer.
The net increases in signal for p8-His6 were 800RU, where
1,000RU is equivalent to ?1ng of protein/mm2. For interaction
measurements, MSL1-GST was injected at a flow rate of 30ml/min
during 3min, dissociation was then evaluated by passing HBS
run, the chip was regenerated with one short pulse of 0.005% SDS
and two pulses of HBS. One activated/deactivated channel was
used as a negative reference.
Acid extraction of proteins from HeLa cells
Twenty-four hours after transfection with p8-Flag and MSL1-V5,
alone or in combination, HeLa cells were pelleted and lysed with
lysis buffer (10mM HEPES, pH 7.9, 1.5mM MgCl2, 10mM KCl,
0.5mM DTT, 1.5mM PMSF, HCl 0.2M) and incubated on ice for
30min. After centrifugation at 11,000g for 10min at 48C, the
supernatant fraction of acid-soluble proteins was recovered and
dialyzed twice against 0.1M acetic acid for 2h and three times
against H2O for 1h, 3h, and overnight. Finally, concentration of
acid-soluble protein extracts was quantified by the Bradford
SDS–PAGE and Western blotting
Bio-Rad standard proteins with markers covering a 7–240kDa
range. Non-specific binding to the membrane was blocked by 5%
BSA in TBS for 1h at 48C. Blots were incubated overnight at 48C
with polyclonal anti-H4K16ac and anti-H3K9ac antibodies diluted
in 5% BSA. Then, membranes were washed with TBS–0.1% Triton
and incubated with a secondary goat-anti-rabbit-HRPO antibody
(1:3,000) obtained from Santa Cruz Biotechnology diluted in 5%
dry non-fat milk in TBS for 1h at room temperature. Finally,
membranes were washed with TBS–0.1% Triton, developed with
the ECL-detection system (Santa Cruz Biotechnology), quickly
dried, and exposed to ECL film.
ImageJ 1.32 software from http://rsbweb.nih.gov/ij/download.html
was used to quantify the intensities of the bands obtained in
JOURNAL OF CELLULAR PHYSIOLOGY
G I R O N E L L A E T A L .
with Student–Newman–Keuls test. Results shown represent
Identification of MSL1 as a partner of p8 by a two-hybrid
conventional yeast two-hybrid screening system was not
suitable because p8 being a co-transcriptional factor would not
need a partnerto induce transcription ofselection factors. The
CytoTrap Sos system generates fusion proteins whose
interaction allows cell growth through activation of the Ras
pathway. The p8 cDNA subcloned into pSos provided the bait
toscreen atestescDNAlibrary constructed inpMyr. After co-
transfection into Saccharomyces cerevisiae, strain cdc25H,
5?106clones were screened and 51 positives identified. All
comparison with the GenBank repertoire (Table 1,
The interaction between p8 and MSL1 was confirmed by
transforming S. cerevisiae with both pMyr-MSL1 and pSos-p8
drop-out (SD) glucose and galactose agar plates lacking leucine
and uracil [SD/glu(?LU) and SD/gal(?LU)] at the stringent
temperature of 378C. Clones growing on SD/gal(?LU) plates
but notonSD/glu(?LU) plates at378C areinteraction-positive
clones. Growth was observed when pSos-p8 and pMyr-MSL1
were both present, but not when pSos-p8 or pMyr-MSL1 was
used separately. Negative and positive controls were grown as
suggested by the manufacturer with expected results. These
pSos-p8-1-46 and pSos-p8-41-82 constructs encode the N-
were co-transfected with the pMyr-MSL1 plasmid to identify
the interacting region. However, whereas complete p8
interacts with MSL1, neither the N-terminal or C-terminal
parts of the protein was able to interact with MSL1 indicating
that the complete p8 protein is necessary for this interaction
(data not shown).
Interaction of p8 and MSL1 in cells
Interaction between p8 and MSL1 was controlled by co-
immunoprecipitation assays. 293T cells were transfected with
MSL1-V5 and p8-Flag, alone or in combination. MSL1-V5 and
p8-Flag were immunoprecipitated from cell extracts with
anti-V5 or anti-Flag antibodies, respectively, and analyzed by
Western blot. Anti-V5 antibody was used to detect tagged
MSL1 and anti-Flag was used to detect tagged p8. MSL1-V5 was
detected in the complex containing p8 while p8-Flag was
detected in the complex containing MSL1-V5. Tags were
not detected in the negative control (Fig. 1). These results
confirm that MSL1 interacts with p8 in 293T cells.
Nonidet P-40 were used for precipitations with Sepharose beads. The precipitated material was immunoblotted with anti-V5 or anti-Flag
antibodies. TCL, total cell lysate. B: BIAcore analysis of the p8/MSL1 complex: Indicated concentrations of MSL1-GST were injected over
immobilized p8-His6. Differential sensograms are obtained by subtracting the response from a free channel to the response from the p8-His6
channel. Affinity constant of the interaction was evaluated using the Biaeval 4.0.1 software (BIAcore AB). RU stands for resonance units. C:
Endogenousp8andMSL1interaction: Immunoprecipitationofendogenousproteins wasperformedusing ananti-p8rabbitantibody. Cell lysate
with anti-MSL1, anti-MRG15, or anti-MORF4L1 antibodies. [Color figure can be viewed in the online issue, which is available at
Interaction of p8 with MSL1: (A) 293T cells were co-transfected with 2mg of pcDNA4-MSL1-V5 and p8-Flag as indicated.
JOURNAL OF CELLULAR PHYSIOLOGY
p 8 , M S L 1 , A N D 5 3 B P 1
To test if a direct interaction exists between MSL1 and p8, we
the Materials and Methods Section. Recombinant protein p8-
His6 was coated on the BIAcore sensor chip. Figure 1 shows
that the purified MSL1-GST recombinant protein, used as an
analyte, interacts with the immobilized p8-His6. Binding of
recombinant MSL1 to p8-His6 was dose-dependent allowing
assessment of their affinity (kd¼0.92mM). By contrast, no
significant interaction was observed between p8-His6
recombinant protein and GST (not shown), indicating the
specificity of the interaction between p8-His6 and MSL1-GST.
Interaction with endogenous proteins
We controlled the interaction of endogenous p8 and MSL1 in
antibodies (Fig. 1). As expected, when p8 was
immunoprecipitated the MSL1 protein was found in the
complex, but not when an irrelevant antibody was used.
Interestingly, we also found that MRG15 and MORF4L1
proteins, two components of the HAT hMSL complex (Smith
et al., 2005) co-precipitate with p8 (Fig. 1), indicating that p8 is
p8 and MSL1 mRNA expressions after g-irradiation
p8 and MSL1 mRNA expressions were measured by qRT-PCR
in HeLa cells, after 3, 7, or 10Gy irradiation. As shown in
Figure 2, p8 mRNA expression was activated within 2h after
treatment with 7 or 10Gy, in a dose-dependent manner. Then,
a progressive and sustained decrease and inhibition of its
expression was observed. p8 mRNA activation was not
sensitive to 3Gy treatment. MSL1 mRNA expression was also
transiently activated by g-irradiation in a dose-dependent
manner. Activation was observed after exposure to 3Gy,
suggesting that MSL1 expression is more sensitive than p8
expression to g-irradiation.
p8 and MSL1 expressions alter HAT activity
(pCCL-p8), MSL1-EGFP (pCCL-MSL1) or p8-EGFP and MSL1-
EGFP, to generate cells constitutively over-expressing these
proteins. p8-EGFP and MSL1-EGFP protein expressions were
confirmed by Western blot (data not shown). Total HAT
activity in nuclear extracts from these cells was also analyzed.
only with a slight increase in HAT activity, compared to
cells infected with control lentivirus. In contrast, cells over-
expressing MSL1-EGFP presented with about 35% increase in
p8-EGFP and MSL1-EGFP showed a strong reduction in HAT
activity when compared to cells over-expressing MSL1-EGFP
alone, suggesting a negative role for p8 expression on MSL1
HeLa cells slightly decreased HAT activity, whereas MSL1
siRNA decreased that activity by about 30% as shown in
Figure 3. Altogether, these results indicate that MSL1
participates in the regulation of HAT activity that the direct
effect of p8 is minor but that p8 interaction with MSL1 inhibits
the effect of MSL1 on HAT activity.
Specificity of p8 and MSL1 activities on histone
To study the specificity of this HAT activity, we assessed by
after forced expression of p8-Flag and MSL1-V5, alone or in
combination. Whereas p8 over-expression did not induce
significant changes, MSL1 over-expression increased
acetylation of H4K16 but not of H3K9. However, over-
H4K16 acetylation as shown in Figure 3. These results suggest
Clonogenic test after g-irradiation
To assess the effect of p8 and MSL1 on survival after g-
irradiation, we examined the ability of p8 and MSL1 alone or
irradiation. Cells transfected with the empty vector could
generate colonies after irradiation. Their number was not
modified by p8 expression but it increased by 50% in cells
expressing MSL1. However, when MSL1 was transfected
together with p8 its activity on colony formation was
completely suppressed, as shown in Figure 4. To confirm these
and found that p8 knockdown did not alter colony formation,
whereas MSL1 suppression decreased it by about 45%. Taken
resistance to g-irradiation induced by MSL1 expression. To
verify that this represents a bone fide effect, we analyzed the
effect of knocking down p8 and MSL1 mRNAs, alone or
together and found no significant effect on cell growth
(Supplementary Fig. 1).
Two-hybrid screening reveals that 53BP1 is a
partner of MSL1
To find a mechanistic explanation for the protective role of
MSL1 against g-irradiation, we used the CytoTrap approach to
Supplementary Table 2). Among them, 53BP1 was the most
DNA repair after exogenous induction of DNA damage. The
interaction between MSL1 and 53BP1 was confirmed by
transforming S. cerevisiae with both pMyr-53BP1 and pSos-
MSL1 constructs as described above.
were g-irradiated at 3, 7, or 10Gy and total RNA extraction was
performed at 0, 2, 6, 12, 18, or 24h after irradiation. p8 and MSL1
mRNA expressions were monitored by qRT-PCR analysis on a
LightCycler detection system as described in the Materials and
Methods Section. mRNA values are represented as percent of values
obtained with untreated cells and expressed as the meanWSD of
combined resultsfrom three independent experiments performedin
duplicate (MP<0.05). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
p8 andMSL1mRNA expressionsafterirradiation: HeLa cells
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G I R O N E L L A E T A L .
MSL1 interacts with 53BP1 in cells
The interaction between MSL1 and 53BP1 observed in yeast
was confirmed by co-immunoprecipitation assays. 293T cells
were transfected with 53BP1-HA and MSL1-V5, alone or in
combination. MSL1-V5 and 53BP1-HA were
antibodies, respectively. Anti-V5 antibody was used to detect
MSL1-V5 was detected in the complex containing 53BP1 while
BP53-HA was detected in the complex containing MSL1-V5.
Tags were not detected in negative controls (Fig. 5). We
controlled the interaction of endogenous MSL1 and 53BP1 in
antibodies (Fig. 5). As expected, when 53BP1 was
immunoprecipitated the MSL1 protein was found in the
MSL1 DNA-repair activity is 53BP1-dependent
We speculated that the MSL1-dependent DNA-repair activity
should be dependent on 53BP1 expression. To test this
hypothesis we treated HeLa cells with a siRNA against 53BP1
mRNA while over-expressing p8 and MSL1, alone or in
combination. We found that knocking down 53BP1 decreased
DNA-repair activity, as evidenced by a decrease in the number
of colonies observed after g-irradiation. Interestingly, that
phenomenon was not enhanced after forced expression of p8
that MSL1 activity on DNA repair is dependent on 53BP1
HeLa cells were transfected with p8 and MSL1 siRNAs. Forty-eight hours later nuclear extracts were prepared and HAT activity measured as
molecular weight markers. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
MSL1: HeLa cells were transfected with pcDNA3-p8 and pcDNA4-
MSL1-V5, alone or in combination (A,C). p8 and MSL1 siRNAs were
transfected to HeLa cells (B,D). After 24h (A,C) or 48h (B,D), cells
were g-irradiated (7Gy) and seeded on tissue culture dishes until cell
colonies were formed. After 9 days, colonies were fixed, stained, and
duplicate. C,D: Representative images of the results. Values are
expressed as the meanWSD of combined results from three
independent experiments performed in duplicate (MP<0.05).
Colony formation after g-irradiation is regulated by p8 and
JOURNAL OF CELLULAR PHYSIOLOGY
p 8 , M S L 1 , A N D 5 3 B P 1
Then, we forced the expression of 53BP1 and, as expected,
found an increased resistance to cell death induced by g-
irradiation. The number of colonies formed was twice that of
control cells. A further increase was observed when MSL1 was
alone. The effect of MSL1 was abolished if p8 was also
expressed. Taken together, these results suggest that MSL1
activity on cell resistance to DNA damage is dependent on
Regulation of HAT activity by MSL1 is independent of
To check whether HAT MSL1-dependent activity was
dependent on 53BP1 expression, we over-expressed MSL1,
alone or with p8, in HeLa cells in which 53BP1 was either over-
expressed by plasmid transfection or knocked down by siRNA
treatment. No significant change in HAT activity was observed
after over-expressing or knocking down 53BP1 (Fig. 7),
The stress protein p8 is a small, highly basic, unfolded, and
its functions are exerted through interactions with other
proteins, whose activities are thereby enhanced or repressed
describe another example of such mechanism, by which p8
binds and negatively regulates MSL1, a HAT-associated protein
belonging to the MSL complex (Smith et al., 2005), which in
turn binds to the DNA-damage-associated 53BP1 protein to
facilitate DNA repair. We screened a cDNA library to detect
new p8 partners and found that MSL1 binds to p8. This
interaction was confirmedby co-immunoprecipitation andSPR
analysis. Drosophila MSL complex is found exclusively on X
chromosome in males, where it targets activated genes (Sass
et al., 2003) and enhances the level of gene expression by
humans, this complex does not associate with a specific
chromosome and appears to have a more dispersed and
ubiquitous genomicdistribution (Smith et al., 2005). It hasbeen
described that the acetylase present in the MSL complex is
named MOF andis responsible forH4K16 acetylation ina wide
range of higher eukaryotes (Smith et al., 2005). Several studies
have shown that loss of MOF in mammalian cells has several
consequences such as G2/M cell-cycle arrest, nuclear
morphological defects, spontaneous chromosomal
aberrations, reduced transcription of certain genes, and an
impaired DNA-repair response upon ionizing irradiation
(reviewed in Rea et al., 2007). Moreover, MOF is involved in
p53by MOFinfluences thecell’s decisiontoundergoapoptosis
instead of cell-cycle arrest (Sykes et al., 2006). Here, we show
that p8 acts as a negative regulator of the MSL complex;
interacting with MSL1 is able to abrogate the MSL1-dependent
A second yeast two-hybrid strategy against MSL1 as bait
revealed us that MSL1 binds to 53BP1. We confirmed this
interaction by co-immunoprecipitation experiments. Human
53BP1 is a polypeptide of 1,972 amino acids that contains two
tandem BRCA1 (BRCT) motifs and a tudor domain. 53BP1
binds to the DNA-binding domain of p53 and rapidly forms
2004). Double-strand breaks activate signaling responses at
cell-cycle checkpoints, which monitor DNA damage and
transduce signals to co-ordinate repair and cell-cycle
progression. At the cellular level, damaged DNA that is not
properly repaired can lead to genomic instability, apoptosis, or
senescence, which can greatly affect the organism’s
development and aging process. Therefore, it is essential for
cells to efficiently respond to DNA damage through co-
ordinated and integrated DNA-damage checkpoints and repair
The role of chromatin acetylation by HAT complexes in
transcriptional regulation is well established (Carrozza et al.,
2003; Peterson and Cote, 2004). Recent studies have also
implicated HATs in DNA-damage detection and DNA repair
(Bird et al., 2002; Utley et al., 2005; Murr et al., 2006), but the
precise underlying mechanism remains to be established. We
hypothesized that MSL1-containing HAT complexes may
participate in DNA repair through histone acetylation and
reconfiguration of chromatin at break sites. To test this
hypothesis we measured, by cell colony assay after g-
irradiation, the role of MSL1 on DNA repair after its forced
expression, alone or in combination with p8. As expected,
MSL1 over-expression induced a significant increase in cell
resistance to DNAdamage that was completely inhibited by p8
over-expression. On the contrary, knocking down p8 had no
effect whereas knocking down MSL1 decreased cell survival
after DNA damage. These results strongly suggest that MSL1
interaction with p8. Furthermore, we found that MSL1-
dependent DNA-repair activity is dependent on 53BP1
expression as it is inhibited by siRNA-mediated 53BP1
knockdown and, on the contrary, over-expression of 53BP1
strongly increased that activity. In contrast, we found no
significant changesinMSL1-dependentHATactivityafter over-
expression or knockdown of 53BP1, suggesting that 53BP1 is
not involved in MSL1-dependent HAT activity.
co-transfected with 2mg of pcDNA4-MSL1-V5 and 53BP1-HA as
HA-specific antibodies. Cell lysates prepared with a buffer containing
0.5% Nonidet P-40 were used for precipitations with Sepharose bead
conjugates. The precipitated material was immunoblotted with anti-
V5 or anti-HA antibodies. C: Endogenous MSL1 and 53BP1
interaction: Immunoprecipitation of endogenous proteins was
performed using an anti-53BP1 rabbit antibody. Cell lysate prepared
with a buffer containing 1% Triton X-100 was used for precipitation
with an anti-MSL1 antibody. TCL, total cell lysate.
Interaction of MSL1 with 53BP1: (A,B) 293T cells were
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G I R O N E L L A E T A L .
It has been shown that cellular exposure to irradiation
of ATM function but that MOF inactivation abrogates ATM
activation after irradiation, indicating that MOF acts upstream
of ATM (Gupta et al., 2005). It has been described that H4K16
acetylation destabilizes nucleosomes leading to chromatin
decondensation (Shogren-Knaak et al., 2006). However, ATM
activation needs H4K16 to be specifically acetylated by MOF
(Gupta et al., 2005), suggesting that not only changes in the
chromatin structure provoked by irradiation-induced H4K16
acetylation are sufficient to activate ATM-DNA-repair
pathway; some scaffold proteins would be necessary to link
double-strand breaks to ATM activation. Here we show a
further link between the MSL complex and the ATM-DNA-
repair pathway as MSL1 interacts physically and functionally
with 53BP1, suggesting a strong relationship between both
phenomena in DNA-damage response. From our findings we
suggest that, when irradiation-induced double-strand breaks
happen to DNA, 53BP1 brings MSL1 to the vicinity of damaged
DNA through its proven interaction. MSL1-containing MSL
complexes increase H4K16 acetylation due to increase in HAT
activity and this histone modification leads to changes in
chromatin structure inducing chromatin remodeling and
to damaged DNA sites. Depending on the cell context, p8
would act as a negative regulator of this process by interacting
with MSL1 and preventing the formation of the MSL complex.
consequent DNA-repair decrease as we could see with the
survival assay after irradiation. In this sense, high doses of
irradiation provoke a sustained p8 mRNA down-regulation
preceded by a rapid short increase in p8 expression. This fast
later 53BP1 mRNA expression was measured by qRT-PCR and expressed as percent of control siRNA-transfected cells. C: HeLa cells were
transfected with pcDNA3-p8 or pcDNA4-MSL1-V5 alone or in combination in cells over-expressing 53BP1. Cells were g-irradiated (7Gy) and
seeded on tissue culturedishes until cell colonies were formed. After 9days, colonies werefixed, stained, and visible colonies were counted. The
results from three independent experiments performed in duplicate (MP<0.05).
Colony formation after g-irradiation requires 53BP1 expression: (A) HeLa cells in which 53BP1 was knocked down with specific siRNA
cells were transduced with lentivirus expressing p8-EGFP, MSL1-
EGFP or p8-EGFP and MSL1-EGFP. In these cells, 53BP1 was over-
expressed by transfection of a 53BP1-HA DNA construct (A) or
knocked down with a specific siRNA (B). Nuclear extracts were
prepared to analyze HAT activity as described in the legend of
Figure 3. HAT activity was expressed as percent of value in control
cells and expressed as the meanWSD of combined results from two
independent experiments performed in triplicate (MP<0.05).
HAT activity is not influenced by 53BP1 expression: HeLa
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p 8 , M S L 1 , A N D 5 3 B P 1
DNA repair of too much damaged cells, allowing them to
undergo apoptosis. The subsequent inhibition of p8expression
would allow surviving cells to be DNA repaired by the MSL1
pathway. Although these results strongly suggest that MSL1-
to our knowledge, specific inhibitors of this activity are not
available. However, Sun et al. (2006) established that global
inhibition of HAT activity with the non-specific inhibitor
anacardi acid increased cell sensitivity to ionizing radiation,
indicating that HAT activity is very important for DNA repair.
In summary, our results show that MSL1 plays an important
role in mediating irradiation-induced DNA repair through
formation of HAT complexes and interaction with 53BP1, and
p8 would act as a negative regulator of this process by
interacting with MSL1 and preventing its role on HAT activity.
and S.M. M.G. was supported by INSERM, C.C. was from Ligue
Contre le Cancer. Ciberehd is funded by the Instituto de Salud
Lacombe for their technical help.
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