Acetylated H4K16 by MYST1 protects UROtsa cells from arsenic toxicity and is
decreased following chronic arsenic exposure
William Jaime Joa,1, Xuefeng Renb,1, Feixia Chuc, Maria Aleshinb, Henri Wintza, Alma Burlingamec,
Martyn Thomas Smithb, Chris Dillon Vulpea,⁎, Luoping Zhangb,⁎
aDepartment of Nutritional Sciences and Toxicology, University of California Berkeley, Berkeley, CA 94720, USA
bDepartment of Environmental Health Sciences, School of Public Health, University of California Berkeley, Berkeley, CA 94720, USA
cDepartment of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USA
a b s t r a c ta r t i c l ei n f o
Received 28 May 2009
Revised 20 August 2009
Accepted 24 August 2009
Available online 2 September 2009
Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found
in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the
induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2
gene is required for optimal growth of yeast in the presence of arsenite (AsIII). Yeast Sas2p is orthologous to
human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is
necessary for the resistance of yeast to AsIIIthrough the modulation of chromatin state. We further explored
the role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis in human bladder epithelial
cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has
been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16 and
induced sensitivity to AsIIIand to its more toxic metabolite monomethylarsonous acid (MMAIII) at doses
relevant to high environmental human exposures. In addition, both AsIIIand MMAIIItreatments decreased
global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16
is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure
may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1
gene in human sensitivity to arsenic.
© 2009 Elsevier Inc. All rights reserved.
Bladder cancer is the most common urologic malignancy and the
leading cause of cancer death in patients with urinary tract
malignancies (Jemal et al., 2007). Chronic exposure to arsenic through
drinking water is strongly linked to increased incidence and mortality
of bladder cancer (Smith et al., 1998; Chu and Crawford-Brown, 2007;
Marshall et al., 2007). For example, during 1958–1970, people who
lived in region II of Chile were exposed to high concentrations of
arsenic in water, and excess deaths from lung and bladder cancers
predominated ten years after reduction of exposures (Marshall et al.,
2007; Yuan et al., 2007).
Both DNA hypermethylation and altered histone acetylation have
been observed in tumors from patients with bladder cancer (Chen et
al., 2007; Brait et al., 2008), suggesting that aberrant epigenetic
changes are associated with the development of this disease. There is
increasing evidence indicating that epigenetic dysregulation plays an
important role in the development of bladder cancer induced by
arsenic (Marsit et al., 2006; Chai et al., 2007). Global DNA
hypomethylation and focal DNA hypermethylation are both implicat-
ed in arsenic-induced malignant transformation in vivo and in vitro
(Chen et al., 2001; Benbrahim-Tallaa et al., 2005; Chanda et al., 2006).
Further, chronic exposure to arsenic alters DNA methylation and
induces aberrant gene expression (Zhao et al., 1997; Xie et al., 2007).
Moreover, arsenic induces aberrant acetylation of histone 3 (H3) at
the loci of the proto oncogenes c-jun and c-fos, which in turn
correlates with up-regulation of these genes (Li et al., 2003), and
increases global acetylation of H3 at lysine 9 through inhibition of
histone deacetylases (Ramirez et al., 2007). Therefore, arsenic also
impairs the normal regulation of histone modifications, which may
result in gene expression changes.
In a genome-wide, parallel phenotypic screen of yeast deletion
mutants, we identified several genes associated with epigenetic
changes as essential for optimal growth in the presence of arsenicals
Toxicology and Applied Pharmacology 241 (2009) 294–302
⁎ Corresponding authors. L. Zhang is to be contacted at School of Public Health,
University of California, Berkeley, CA 94720, USA.Fax: +1 510 642 0427. C.D. Vulpe, 317
Morgan Hall, Department of Nutritional Sciences and Toxicology, University of
California, Berkeley, CA 94720, USA. Fax: +1 510 642 0535.
E-mail addresses: firstname.lastname@example.org (W.J. Jo), email@example.com (X. Ren),
firstname.lastname@example.org (F. Chu), email@example.com (M. Aleshin),
firstname.lastname@example.org (H. Wintz), email@example.com (A. Burlingame),
firstname.lastname@example.org (M.T. Smith), email@example.com (C.D. Vulpe),
firstname.lastname@example.org (L. Zhang).
1These authors contributed equally to the work.
0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
(Jo et al., 2009). These results suggest that epigenetic regulation is
required in response to arsenic. One of the identified genes, SAS2, is
required for the resistance of yeast to arsenite (AsIII). This gene
encodes the catalytic subunit of the heterotrimeric something about
silencing (SAS) complex, which is responsible for the acetylation of
histone 4 at lysine 16 (H4K16) (Shia et al., 2005). The SAS complex
(Sas2p–Sas4p–Sas5p) is involved in transcriptional activation and
silencing, chromatin-mediated boundary formation (Kimura et al.,
2002; Suka et al., 2002; Shia et al., 2006a; Shogren-Knaak et al., 2006),
andmay playa rolein DNAdamagerepair andmaintenance of nuclear
integrity (Lafon et al., 2007). The human ortholog of the yeast Sas2p,
MYST1,is a histone acetyltransferase responsible for the acetylation of
H4K16. Loss of MYST1 decreases levels of acetylated H4K16 and the
efficiency of double-strand break repair after induction of DNA
damage with ionizing radiation (Taipale et al., 2005; Gupta et al.,
2008). Loss of MYST1 also leads to G2/M cell cycle arrest, nuclear
morphological defects, spontaneous chromosomal aberrations, and
reduced transcription of certain genes (reviewed in Gupta et al.,
2008). In addition, hypoacetylation of H4K16 is commonly found in
human tumors and cell lines, and loss of acetylation at histone 4 (H4)
appears to occur during malignant transformation (Fraga et al., 2005).
Thus, current evidence suggests a connection between MYST1, H4K16
acetylation status, and human cancer (Lafon et al., 2007; Rea et al.,
Inthecurrent study, UROtsa cells were used toexplore the potential
role of H4K16 acetylation byMYST1 in the response to AsIIIand its more
toxic metabolite, monomethylarsonous acid (MMAIII). Both arsenic
forms are detected in urine and in exfoliated bladder epithelial cells
collected from people exposed to arsenic (Hernandez-Zavala et al.,
2008). Because individuals who excrete a higher proportion of ingested
arsenic as methylated forms are at higher risk of bladder cancer
(Steinmaus et al., 2006), it was considered of particular importance to
evaluate the effects of MMAIII. The UROtsa cell line, originally isolated
from a primary culture of normal human uroepithelium, has previously
been used as a model for bladder epithelium and arsenic-induced
bladder cancer (Sens et al., 2004; Bredfeldt et al., 2006; Eblin et al.,
transformation in this model, partially through alterations in DNA
methylation and histone acetylation (Jensen et al., 2008, 2009). The
also studied in yeast in the current study. The results presented here
show that acetylated H4K16, the status of which is determined by
MYST1 probably in conjunction with other determinants in humans, is
the levels of acetylated H4K16 in bladder epithelial cells could increase
the risk of arsenic carcinogenesis.
Cultures of yeast strains and human UROtsa cells.
from the BY4743 (Invitrogen, Carlsbad, CA), and RMY200 and
JTY102TU (generous gift from Prof. Michael Grunstein, University of
California, Los Angeles, CA) backgrounds. Growth was conducted in
rich media (yeast extract–peptone–dextrose, YPD) at 30 °C with
shaking at 200 rpm. UROtsa cells (generously provided by Prof. Petia
Simeonova, National Institute for Occupational Safety and Health,
Centers for Disease Control and Prevention) were cultured at a
starting cell density of 4–5×104cells/ml in RPMI 1640 (Mediatech,
Inc, Manassas, VA) with L-glutamine, 10% fetal bovine serum (FBS),
100 IU/ml penicillin, and 100 μg/ml streptomycin (Omega Scientific,
San Diego, CA), under standard human cell culturing conditions.
Yeast strains were
from Sigma-Aldrich (St. Louis, MO). Monomethylarsine oxide
(MMAIIIO, MMAIII) was a generous gift of Prof. Miroslav Styblo
(University of North Carolina, Chapel Hill, NC). MMAIIIO hydrolyzes to
Sodium arsenite (NaAsO2, AsIII) was purchased
MMAIIIin solution(Petricketal.,2001).Stock solutions wereprepared
in sterile Milli-Q water, protected from light and stored at −80 °C
until use. Yeast cells were treated with concentrations ranging from 0
to 300 μM of either AsIIIor MMAIII. UROtsa cells were treated, once
they reached 80%–90% confluence in culture, with AsIIIat 1, 3, and
10 μM or with MMAIIIat 0.3, 1, and 3 μM.
Yeast growth assay.
mid-log phase, diluted in fresh media to an optical density at 595 nm
(OD595) of 0.0165, and inoculated into a 48-well microplate. Stock
solutions of arsenicals were added to the desired final concentrations
with at least three replicate wells per dose. Plates were incubated in a
Tecan GENios spectrophotometer set to 30 °C, intermittent shaking
and OD595measurements at 15-minute intervals for a period of 24 h.
Raw absorbance data were averaged for all replicates, background-
corrected, and plotted as a function of time. The area under the curve
(AUC) was calculated for the cultures in each well using Prism version
5.01 (GraphPad Software, Inc., La Jolla, CA), the treatments averaged,
and expressed as a percentage of the control.
Yeast strains were pre-grown in YPD media to
Retrovirus-mediated MYST1 gene knockdown.
modifications, the top strand cDNA sequence of the MYST1 shRNA
construct (sh-MYST1) was 5′-GATCCGAGACCATAAGATTTACTGTTT-
strand was 5′-AATTCACGCGTAAAAAAGACCATAAGATTTACTGTTCTC-
control was modified from the non-target shRNA control (Sigma-
Aldrich), and the control sequence used for the shRNA (sh-NSC)
was 5′- GATCCGCAACAAGATGAAGAGCACCAATTCAAGAGATTGGTG-
CTCTTCATCTTGTTGCTTTTTTACGCGTG-3′ for top strand, and 5′-
TTGGTGCTCTTCATCTTGTTGCG-3′ for bottom strand. These oligonucle-
otide pairs containing BamHI and EcoRI overhangs were annealed and
ligated to a linearized RNAi-Ready pSIREN-RetroQ-ZsGreen vector
digested with BamHI and EcoRI (BD Biosciences Clontech). The RNAi-
Ready pSIREN-RetroQ-ZsGreen vector is a self-inactivating retroviral
expression vector designed to express a small hairpin RNA using the
human U6 promoter. The resultant constructs were amplified, purified,
and sequenced. The UROtsa cells were transfected with Lipofectamine
2000 reagent following the manufacturer's instruction (Invitrogen).
Afterincubation at37°Cfor8 h,thesupernatant fraction containingthe
retroviral vector was removed and replaced with normal growth
medium. Cells grown for 48–72 h were assessed by fluorescence
microscopy. The ZsGreen fluorescent marker yields a bright green
fluorescence, permitting direct monitoring of the delivery efficiency.
Finally, the cell populations were sorted by the DAKO-Cytomation
MoFlo High Speed Sorter (Dako North America, Carpinteria, CA), and
the green fluorescent cells were purified and collected for continuing
culture. The green fluorescent cells were used for additional
Cytotoxicity assay (MTT assay).
2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to
assess the effect of MYST1 silencing on cell viability after arsenic
treatment. Cells were cultured in 96-well plates in a volume of 100 μl
of media per well at a density of 5×104cells/ml. Twenty-four hours
after incubation with AsIIIor MMAIII(3 replicates/arsenical concen-
tration), 10 μl of sterile MTT dye (Sigma-Aldrich; 5 mg/ml) was added
to each well, and plates were incubated at 37 °C for 4 h. Then, the
culture medium was removed and 200 μl of DMSO was added and
thoroughly mixed in for 10 min. Spectrometric absorbance at 570 nm
was measured in a microplate reader.
Human tissue array and real-time quantitative PCR assay.
based real-time quantitative polymerase chain reaction (RT qPCR)
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
was performed to quantify MYST1 expression in human normal
tissues in the Human Rapid-Scan Plate (OriGene Technologies, Inc.,
Rockville, MD). The primers and probe used for amplification of
MYST1 and β-actin control were ordered from Applied Biosystems
(Foster City, CA). For quantification of transcripts, relative gene
expression was calculated using the ΔΔCTmethod.
Mass spectrometry analysis of H4K16 acetylation.
cells were grown in SILAC RPMI 1640 medium containing L-lysine HCl
(light) or L-lysine 2HCl (U-13C6, 98%; U-15N2, 98%, heavy) (Cambridge
Isotope Laboratories Inc., Andover, MA), supplemented with 10%
dialyzed FBS and antibiotics. Cells were cultured for five passages to
ensure complete labeling of proteins before being exposed to AsIIIor
MMAIIIfor 24 h or 7 days. Treatment and control cultures were mixed
in a 1:1 ratio, and the core histone proteins were extracted and
purified as described before (Shechter et al., 2007), separated by 4%–
20% SDS–PAGE, and visualized with Coomassie staining. In-gel
digestions on histone bands were performed utilizing the procedure
described at http://ms-facility.ucsf.edu/ingel.html.
Mass spectrometric analysis was carried out as described
previously (Chu et al., 2006). Briefly, the tryptic digest of core H4
was separated by a 75-μm×15-cm reverse-phase capillary column
at a flow rate of 330 nl/min. The HPLC eluent was connected
directly to the micro-ion electrospray source of a QSTAR XL mass
spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA). LC–
MS data were acquired in an information-dependent acquisition
mode, cycling between 1-s MS acquisition followed by 3-s low-
energy CID data acquisition. The centroided peak lists of the CID
spectra were searched against the National Center for Biotechnology
Information (NCBI) protein database using Batch-Tag (Chalkley et
al., 2005). Protein N-terminus and lysine acetylation; lysine mono-,
di-, and trimethylation; arginine mono- and dimethylation; phos-
phorylation; and lysine ubiquitination were considered as variable
modifications. Quantitative calculation of identified peptides was
carried out by the UCSF Search Compare program in ProteinPros-
pector package, which performs peak list generation, SILAC- and
extracted ion current-based quantitation, expectation value calcu-
lation, statistic analysis of identified peptide, data filtration, and
presentation. SILAC ratio of each peptide was normalized against
the average SILAC ratio of all identified peptides in each histone
cells using 300 μl of radioimmunoprecipitation assay lysis buffer.
Nuclear extracts were collected from 1×107cells using a nuclear
extraction kit (Millipore, Billerica, MA) according to the man-
ufacturer's protocol. Protein concentrations in cell lysates and nuclear
extracts were determined by the DC assay (Bio-Rad Laboratories, Inc.,
Hercules, CA). Equal protein amounts were resolved by SDS–PAGE,
transferred onto nitrocellulose membranes, and immunoblotted for
MYST1 (Novus Biologicals, Littleton, CO) and β-actin (Sigma-Aldrich)
or acetyl-H4, acetyl-H4K16, and H4 pan (Millipore). Proteins were
visualized using the enhanced chemiluminescence method (Amer-
sham Biosciences, United Kingdom) as per the manufacturer's
protocol. Film was exposed and developed using a Konica SRX-101
developer (Konica Minolta Medical Imaging USA, Wayne, NJ). Images
were quantified using the ImageJ software (NIH, Bethesda, MD). Each
measured protein was normalized to either of the loading controls β-
actin or H4 pan.
Total cell lysates were prepared from 5×106
Fig. 1. Comparison of MYST1 gene expression in human tissues. Expression of MYST1 was quantified by RT qPCR in cDNA from a panel of 48 normal human tissues. Transcript levels of
MYST1 were determined using primers designed to amplify a defined region of the gene. MYST1 mRNA was expressed at detectable levels in the majority of the tissues analyzed.
Expression of MYST1 in liver was selected as the reference for comparison with all other tissues.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
analysis of variance followed by a post-hoc test if the group means
were significantly different. Levels of significance were denoted in
graphs as ⁎p valueb0.05, ⁎⁎p valueb0.01, and ⁎⁎⁎p valueb0.001.
Statistical analyses were performed using one-way
mRNA expression of MYST1 in human tissues
MYST1 is a recently characterized gene, and a search of the
expressed sequence tag (EST) database revealed sequences matching
the cDNA of MYST1 in many human tissues with varied expression
levels. To experimentally measure and compare the mRNA expression
of MYST1 across tissues, real-time quantitative PCR analysis was
performed in cDNA from a panel of 48 human tissues contained in a
tissue array (Fig. 1). Using the liver as reference, it was found that
MYST1 is highly expressed in tissues such as the pituitary gland and
placenta; moderately expressed in others including the stomach,
prostate, kidney, lung, skin, and urinary bladder; and weakly
expressed in the mammary gland, colon, and heart. The presence of
detectable levels of MYST1 mRNA in several normal human tissues
indicates a role for this gene in regular cellular metabolism and
supports studying its relation to arsenic sensitivity in humans. Long-
term exposure to AsIIIhas been associated with cancers in the lung,
urinary bladder, and skin (Tchounwou et al., 2003) and, interestingly,
correlates with the lower MYST1 expression levels observed in these
Deletion of yeast SAS2 and silencing of human MYST1 induces arsenic
The growth phenotype of the SAS-deletion mutants was evaluated
in the presence of either AsIIIor MMAIII(Fig. 2). Deletion strains and
their isogenic counterpart BY4743 wild type were treated with
equitoxic doses equivalent to the concentration that induced 20%
growth inhibition (IC20) and 2×IC20, which were 300 and 600 μM for
AsIIIand 150 and 300 μM for MMAIII, respectively. In these treatments,
sas2Δ, sas4Δ, and sas5Δ exhibited decreased growth in AsIIIrelative to
wild type, but not in MMAIII. These results showed a specific
requirement of the SAS genes for optimal growth in AsIII, despite
the higher toxicity of MMAIII.
To investigate the role of MYST1 in arsenic toxicity in mammals,
the expression of MYST1 was knocked down in UROtsa cells by
approximately 75% compared to controls (Fig. 3A). Transfected
UROtsa cells did not have an altered doubling time or appearance in
culture. The UROtsa sh-MYST1 (MYST1 knockdown) and UROtsa sh-
NSC cells (vector control) were then treated with either AsIIIor
MMAIIIat concentrations up to the IC50for 24 h. The dose ranges
used included doses equivalent to high environmental exposures
encountered by humans. Arsenical treatments induced a dose-
dependent decrease in viability in both cell lines. In the absence of
Fig. 2. Deletion of the SAS genes in yeast results in sensitivity to arsenite. The strains sas2Δ, sas4Δ, sas5Δ and their isogenic counterpart BY4743 wild type were treated with equitoxic
doses equivalent to the IC20and 2×IC20, which were 300 and 600 μM for AsIIIand 150 and 300 μM for MMAIII, respectively. Growth curves show the average optical density of the
cultures at 595 nm (OD595) for each treatment as a function of time for a period of 24 h.The bars represent the mean area under the curve (AUC) for three technical replicates with SE.
At the doses tested, the SAS mutants displayed reduced growth in AsIIIrelative to the wild type strain but not in MMAIII.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
arsenic treatment, there was no effect of MYST1 knockdown on cell
viability, relative to vector control. Unlike deletion of the SAS genes
in yeast, silencing of MYST1 led to increased sensitivity to both AsIII
and MMAIII(Fig. 3B). MMAIIIwas more potent than AsIIIin producing
toxicity; at the IC50, the viability of UROtsa sh-MYST1 cells was about
70% of the UROtsa sh-NSC cells post-AsIIIexposure (10 μM), as
opposed to only 60% after MMAIIItreatment (3 μM).
AsIIIresistance in yeast is related to acetylation of H4K16 and its
modulation of chromatin state
The growth phenotype of a H4K16→R yeast mutant, containing a
non-acetylatable arginine residue at position 16 in place of lysine
(Suka et al., 2002), was evaluated to further investigate the role of
H4K16 acetylation in AsIIIresistance. This strain exhibited slow
growth compared to the wild type and was sensitive to high doses of
AsIII(Fig. 4A). In addition, the growth of this mutant was evaluated in
the presence of cytotoxic concentrations of AsIII, cadmium, copper and
zinc, and sodium chloride (Fig. 4B). Only treatment with AsIII
significantly decreased growth relative to wild type, therefore
showing that H4K16 acetylation is specifically required for resistance
In yeast, the histone deacetylase Sir2p antagonizes the histone
acetyltransferase activity of Sas2p by removing acetyl groups from
acetylated lysine residues (Landry et al., 2000). These two proteins
normally establish a gradient of histone acetylation in yeast chromo-
regions to an hyperacetylated state in telomeric–distal regions,
associated with heterochromatin and euchromatin, respectively
(Kimura et al., 2002; Suka et al., 2002). Deletion of SIR2 increases
H4K16 acetylation. Deletion of SAS2 reduces H4K16 acetylation and
telomeric–distal regions, which is reversed by further deletion of SIR2
(Suka et al., 2002). In order to determine whether the alteration in
heterochromatic regions influences the sensitivity of yeast to AsIII, the
growth phenotypes of sas2Δ, sir2Δ, and sas2Δsir2Δ were evaluated in
its presence. Deletion of SAS2, as previously shown, resulted in
sensitivity to AsIII. Deletion of SIR2 in the double knock-out strain was
with the proposed model for Sas2p and Sir2p function (Kimura et al.,
2002; Suka et al., 2002). Both sir2Δ and sir2Δsas2Δ strains were
insensitive to AsIIIin liquid media and resistant to AsIIIwhen grown on
agar plates (Figs. 4C and D), indicating that H4K16 acetylation plays a
role in AsIIIresistance. Silencing of genes located in telomeric–
that H4K16 acetylation in yeast influences the response to arsenic
toxicity through its modulation of chromatin state. In an open
chromatin conformation induced by acetylated H4K16, cells are
resistant to AsIII, whereas in a closed state induced by deacetylated
H4K16, cells are more sensitive to arsenic toxicity.
Arsenic exposure and H4K16 acetylation in UROtsa cells
MYST1 is responsible for the acetylation of H4K16 in human cells
(Taipale et al., 2005; Gupta et al., 2008), and silencing of this gene in
Fig. 3. Knockdown of MYST1 in human UROtsa cells induces sensitivity to arsenic. (A) Western blot analysis of whole cell lysates with anti-MYST1 shows a reduction of
approximately 75% in MYST1 protein levels, relative to vector controls, in human UROtsa cells after knockdown with shRNA constructs targeting MYST1. The nonspecific control
(NSC) shRNA had no effect on the protein level of MYST1. β-Actin was used as the loading control. (B) UROtsa sh-NSC and UROtsa sh-MYST1 cells were treated with increasing
concentrations of AsIIIand MMAIIIfor 24 h. Cell viability was evaluated with MTT, a dye that is reduced by viable cells, resulting in an increase in color that can be quantified by
spectrophotometry. Bars represent the average of three independent experiments with SD. Treatment with either AsIIIor MMAIIIresulted in a dose-dependent decrease in viability
between the two cell lines. At the IC50, UROtsa sh-MYST1 cells displayed a reduction in viability of approximately 30% and 50% after AsIII(10 μM) and MMAIII(3 μM) exposure,
respectively, relative to control UROtsa sh-NSC cells. These results indicated that arsenicals were more cytotoxic in UROtsa sh-MYST1 cells. ⁎pb0.05, ⁎⁎pb0.01.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
Acetylation levels of H4 and H4K16 were compared between UROtsa
sh-MYST1 and UROtsa sh-NSC cells and were slightly reduced in H4
and significantly reduced in H4K16 (Fig. 5A). The correlation
between decreased H4K16 acetylation and decreased viability after
AsIIIand MMAIIItreatments provides evidence that this epigenetic
modification is required for resistance against these two arsenicals in
Next, the ability of arsenic to alter H4K16 acetylation levels was
determined. UROtsa cells were cultured in medium containing either
heavy or light lysine to allow quantitative comparison through mass
spectrometry of the epigenetic changes on histones. The analysis
showed that arsenical treatment reduced levels of H4K16 acetylation
in UROtsa cells. There was a dose- and time- dependent decrease in
SILAC ratio (treatment/control) for the tetra-acetylated peptide
corresponding to the N-terminus of histone H4, GKacGGKacGLG-
KacGGAKacR, after AsIIIand MMAIIItreatments (Table 1). Treatment
with MMAIIIfor 24 h resulted in only a slight decrease in SILAC ratio at
0.3 and 1 μM, but a significant decrease at 3 μM (Fig. 5B). Interestingly,
the SILAC ratios were significantly reduced for both 0.3 and 1 μM after
7 days of treatment and were comparable. Similarly, in the case of
AsIII, only the highest dose of 10 μM led to a significant decrease
(about two-fold) in the SILAC ratio for acetylated H4K16 peptide after
24 h of treatment. Acetylation of H4K16 was significantly decreased
after 1 and 3 μM AsIIItreatment when the UROtsa cells were treated
for 7 days.
These findings were then confirmed by immunodetection of
acetylated H4K16. Compared to the untreated control, the H4K16
acetylation levels were slightly but significantly reduced in histone
extracts from UROtsa cells following 7 days of treatment with either
3 μM AsIIIor 1 μM MMAIII(Fig. 5C). These slight changes are not
surprising, given the presence of relatively low levels of total H4 in the
nuclear extracts, which contained a mixture of histones. Further,
acetylation may not occur at all H4K16 residues in the cell but is likely
targeted to specific chromosomal locations. In addition, as acetylation
of H4K16 is thought to play a role in normal epigenetic processes the
relatively high basal level of H4K16 acetylation makes the detection of
small differences between treatment and control challenging. There
were no apparent differences in H4K16 acetylation after 24 h of
exposure to AsIIIor MMAIII(data not shown).
In spite of the difference in the magnitude of changes observed
between mass spectrometry and immunoblot analyses, the trend of
decreased H4K16 acetylation after arsenical exposure was consistent
between the two. Overall, the data show that both AsIIIand MMAIII
decrease H4K16 acetylation in UROtsa cells.
Millions of people worldwide are exposed to arsenic through
consumption of contaminated drinking water. Although a direct
correlation between exposure to this metalloid and increased cancer
risk is well documented, the mechanisms involved in arsenic
carcinogenesis are not fully understood. Because inorganic arsenic is
metabolized to methylated trivalent species with greater genotoxic
potential (Mass et al., 2001) and are all found in urine, it is not
surprising that the bladder is a target of arsenic carcinogenesis.
Histone acetylation is commonly associated with transcriptionally
active euchromatin. Acetylation of specific lysine residues can
decrease the affinity of histones for DNA, making the DNA more
accessible to transcription factors and components of the transcrip-
tional machinery (Shia et al., 2006b). Arsenic is known to induce
alterations in histone acetylation that are associated with aberrant
gene expression. Studies aiming to characterize the effects of arsenic
Fig. 4. Acetylation of H4K16 is required for yeast resistance to AsIII. Mutant strains and their isogenic counterpart wild type (RMY200) were grown in rich media containing different
AsIIIconcentrations. The optical density of the cultures was measured at 595 nm for 24 h and used to calculate the area under the curve (AUC). The bars represent the mean AUC for
three technical replicates with SE. ⁎⁎pb0.01, ⁎⁎⁎pb0.001. (A) Decreased growth in rich media and sensitivity of H4K16→R mutant, containing a non-acetylatable arginine residue in
place of the lysine, at AsIIIdoses of 25 μM and higher relative to the wild type strain. (B) The presence in media of growth inhibitory concentrations of cadmium chloride, copper
sulfate, zinc chloride, or sodium chloride does not decrease H4K16→R mutant growth relative to the wild type strain. (C) Deletion of the histone deacetylase gene SIR2 induces
resistance to AsIIIand is dominant over the AsIII-sensitive phenotype that results from deletion of the histone acetyltransferase gene SAS2. Growth assays for wild type, sas2Δ, sir2Δ,
and sas2Δsir2Δ strains in 0, 25, and 50 μM AsIIIshow a dose-dependent decrease in growth for wild type and sas2Δ but no effect on sir2Δ and sas2Δsir2Δ. (D) Plate assays show
resistance of sir2Δ and sas2Δsir2Δ to AsIIIcompared to the wild type. Yeast strains were grown for 3 days on YPD agar in the presence or absence of AsIII. The wild type and sas2Δ
strains were unable to grow in medium with AsIIIbut sir2Δ and sas2Δsir2Δ did.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
on N-tail histone modifications and its relation to transcriptional
changes have focused on the acetylation of H3 (Li et al., 2003; Ramirez
et al., 2007). Moreover, the hyperacetylation of H3 at specific gene
promoters in UROtsa cells transformed by chronic exposure to AsIII
and MMAIII(Jensen et al., 2008) further link these changes to
malignant transformation. Therefore, the induction of alterations in
histone acetylation coupled with aberrant gene expression may play a
role in arsenic carcinogenesis.
Fig. 5. Reduction of acetylated H4K16 levels in UROtsa cells after treatment with arsenicals. (A) Silencing of MYST1 leads to a significant reduction in acetylated H4K16 levels. Nuclear
extracts were run on SDS–PAGE gels, transferred onto nitrocellulose membranes, and probed with anti-acetyl H4 and anti-acetyl H4K16. Equal loading was checked by probing the
membrane with anti-H4 pan. Silencing of MYST1 in UROtsa cells (sh-MYST1) significantly reduce acetylated H4K16 relative to the vector (sh-NSC) and non-transfected controls.
Densitometry analysis shows a significant decrease in acetylated H4K16 after knockdown of MYST1 (⁎⁎pb0.01). (B) Mass spectrometry analysis of representative SILAC sample for
MMAIII. UROtsa cell cultures were labeled with either heavy or light lysine and treated with arsenic or left untreated, respectively. Histones were extracted from culture mixtures at a
ratio of 1:1 and analyzed by LC–MS. Treatment with 3 μM MMAIIIfor 24 h decreases the SILAC ratio (treatment/control) for the tetra-acetylated peptide GKacGGKacGLGKacGGAKacR
labeled with light (m/z 719.88, control) or heavy (m/z 735.91, treatment) lysine. MMAIIItreatments at the lower concentrations did not alter the SILAC ratio significantly. (C)
Representative immunoblot images of nuclear extracts from arsenic-treated and control cultures comparing acetylated H4K16 levels. Nuclear extracts were run on SDS–PAGE gels,
transferred onto nitrocellulose membranes, and probed with anti-acetyl H4 and anti-acetyl H4K16. Equal loading was checked by probing the membrane with anti-H4 pan.
Acetylated H4K16 levels in UROtsa cells are reduced after treatments with 3 μM AsIIIand 1 μM MMAIIIfor 7 days. Densitometry analysis shows a significant decrease in acetylated
H4K16 in cells treated with AsIIIand MMAIIIafter knockdown of MYST1 (⁎pb0.05).
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
This study focused on the N-tail histone acetylation at a different
lysine residue, H4K16. We demonstrate that H4K16 acetylation
mediates arsenic resistance in human UROtsa cells. We showed this
through silencing of the MYST1 gene, which encodes the histone
acetylase primarily responsible for H4K16 acetylation. Furthermore,
we show that MMAIIIand AsIIIdecrease H4K16 acetylation in a dose-
and time-dependent manner, suggesting that the functions mediated
by acetylated H4K16 could be impaired after chronic exposure to
arsenic. Hypoacetylated H4K16 appears early and accumulates during
tumor development in a mouse model of multistage skin carcinogen-
esis, suggesting that loss of H4K16 acetylation occurs during
malignant transformation and does not arise as a consequence of it
(Fraga et al., 2005). Because H4K16 is commonly hypoacetylated in
human tumors, loss of H4K16 acetylation may play a role in the
pathogenesis of different human cancers. In bladder epithelial cells, a
reduction in H4K16 acetylation caused by chronic arsenic exposure
could be detrimental and contribute to bladder carcinogenesis. As a
major determinant of H4K16 acetylation status, expression of MYST1
may constitute a potential biomarker of carcinogenesis in tissues
associated with As-induced cancer.
The homologous proteins primarily responsible for the acetylation
of H4K16 in yeast and humans, Sas2p and MYST1, respectively, belong
to the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) family of histone
acetyltransferases. Members of this family share a conserved MYST
domaincontaininga DNA bindinganda zincfinger motif.The putative
role of the MYST family in human cancer has been reviewed recently
(Avvakumov and Cote, 2007; Lafon et al., 2007). Knockdown of MYST1
resulted in a considerable reduction in acetylated H4K16 levels,
indicating that the bulk of H4K16 acetylation is catalyzed by the
MYST1 protein and confirming previous findings in other cell lines
(Taipaleetal.,2005). Theseresultsprovideevidencethatthe observed
sensitivity of UROtsa cells to arsenic after MYST1 knockdown is due to
a decrease in acetylated H4K16.
The sensitivity to both AsIIIand MMAIIIis indicative of common
mechanisms by which these two arsenicals are toxic to humans and
common biological processes in the cellular response to them. In spite
of this similarity, MMAIIIwas more potent than AsIIIboth at decreasing
viability in MYST1 knockdowncells and at reducing H4K16 acetylation
in UROtsa cells. Compared to a previous study, UROtsa cells were
more sensitive to AsIIIthan in the work presented here, while
sensitivity to MMAIIIwas comparable between the 2 (Drobna et al.,
2005). While the exact reasons are unknown, possible contributions
for the observed difference in sensitivity to AsIIImay be related to cell
culture conditions, timing of the treatments during culture, and purity
of the AsIIIused. Deletion of SAS2 in yeast, on the other hand,
increased sensitivity only to AsIII. Yeast has different but overlapping
detoxification mechanisms against MMAIIIand AsIII. For example, the
arsenicresistance genes ARR are exclusivelyinvolvedin detoxification
of inorganic arsenic. Therefore, SAS2 may be associated with an AsIII-
specific protection mechanism, while detoxification of the different
arsenic species in humans may be mediated through a single pathway
in which MYST1 plays a common role.
Acetylation of H4K16 is a reversible, post-translational histone
modification in eukaryotes that is associated with changes in gene
expression. Thus, the requirement of acetylated H4K16 in the
resistance to arsenic could be due to its ability to promote or maintain
the expression of genes involved in response to arsenic stress. In yeast
chromosomes, the H4K16 acetylation state defines heterochromatic
and euchromatic regions and is associated with gene transcription
(Kimura et al., 2002; Suka et al., 2002). Deletion of SAS2 and mutation
of lysine in H4K16 specifically induced sensitivity to arsenicand not to
other chemical stressors. Interestingly, deletion of SIR2, which results
in hyperacetylation of H4K16, induced resistance to AsIIIand further
confirmed the importance of this epigenetic change. The H4K16
acetylation of chromosomal regions in yeast is associated with
euchromatin. Therefore, H4K16 acetylation mediates arsenic resis-
tance by promoting euchromatin formation in yeast chromosomes.
These results show that SAS2 is not the only determinant factor in
arsenic resistance modulated by H4K16 acetylation status. Further-
more, the data suggest that any factor that can affect H4K16
acetylation has the potential to impact yeast sensitivity to arsenic.
Unlike traditional carcinogens that form metabolites covalently
bound to DNA, AsIIIis not a potent mutagen but may act as a
cocarcinogen by enhancing the mutagenicity of certain agents such as
UV radiation (Danaee et al., 2004; Rossman et al., 2004). Decreased
H4K16 acetylation as a consequence of environmental exposure to
arsenic could render bladder epithelial cells more susceptible to
genotoxic agents, such as chemicals found in cigarette smoke.
Interestingly, smokers exposed to arsenic through drinking water
are at a much higher risk of bladder cancer than exposed nonsmokers
(Steinmaus et al., 2003).
There is limited knowledge of the risk factors that predispose
individuals to the adverse health effects associated with long-term
exposure to arsenic. Because knockdown of MYST1 reduced acetyla-
tion of H4K16 and resulted in sensitivity to arsenic, it seems
reasonable to consider a novel role for the MYST1 gene in human
sensitivity to arsenic in UROtsa cells. Furthermore, because MYST1 is
expressed in a variety of human tissues, the effects of arsenic on
acetylated H4K16 levels and of decreased MYST protein expression on
arsenic sensitivity need to be investigated in other cell types. Studies
to determine potential mechanisms, other than MYST1, involved in
the reduction of acetylated H4K16 levels following arsenic exposure
are warranted. Our results suggest that genetic and/or environmental
factors that can alter normal H4K16 acetylation could influence
sensitivity to arsenic in humans. Importantly, the future identification
of genes whose expression levels are epigenetically regulated by
acetylated H4K16 in UROtsa cells will not only identify additional
sensitivity biomarkers of arsenic exposure but also contribute to
understanding the mechanisms of arsenic toxicity and carcinogenicity
in the bladder.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
We thank Prof. Miroslav Styblo for the generous gift of MMAIIIO,
Prof. Michael Grunstein for providing several yeast strains, and Prof.
Petia Simeonova for providing UROtsa cells. We thank Dr. Cliona
McHale for assistance with manuscript preparation. This research was
funded by the Superfund Basic Research Program NIEHS Grant P42
ES004705 to M.T.S., C.D.V., and L.Z. W.J. and X.R. are trainees of the
SBRP at UC Berkeley.
Avvakumov, N., Cote, J., 2007. The MYST family of histone acetyltransferases and their
intimate links to cancer. Oncogene 26, 5395–5407.
SILAC ratiosaof acetylated H4K16 in UROtsa cells exposed to arsenic and controls.
1 μM3 μM 10 μM 0.3 μM1 μM3 μM
aSILAC ratio (treatment/control) for each experiment has been normalized to the
mean value of unmodified peptides. UROtsa cells were labeled with heavy lysine before
arsenic treatment and with light lysine for the untreated control.
bTreatment period with arsenical.
cN/A: not available. Samples could not be processed due to reduced cell number
caused by cytotoxicity.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302
Benbrahim-Tallaa, L., Waterland, R.A., Styblo, M., Achanzar, W.E., Webber, M.M.,
Waalkes, M.P., 2005. Molecular events associated with arsenic-induced malignant
transformation of human prostatic epithelial cells: aberrant genomic DNA
methylation and K-ras oncogene activation. Toxicol. Appl. Pharmacol. 206,
Brait, M., Begum, S., Carvalho, A.L., Dasgupta, S., Vettore, A.L., Czerniak, B., Caballero, O.
L., Westra, W.H., Sidransky, D., Hoque, M.O., 2008. Aberrant promoter methylation
of multiple genes during pathogenesis of bladder cancer. Cancer Epidemiol.
Biomarkers Prev. 17, 2786–2794.
Bredfeldt, T.G., Jagadish, B., Eblin, K.E., Mash, E.A., Gandolfi, A.J., 2006. Monomethy-
larsonous acid induces transformation of human bladder cells. Toxicol. Appl.
Pharmacol. 216, 69–79.
Chai, C.Y., Huang, Y.C., Hung, W.C., Kang, W.Y., Chen, W.T., 2007. Arsenic salts induced
autophagic cell death and hypermethylation of DAPK promoter in SV-40
immortalized human uroepithelial cells. Toxicol. Lett. 173, 48–56.
Chalkley, R.J., Baker, P.R., Huang, L., Hansen, K.C., Allen, N.P., Rexach, M., Burlingame, A.
L., 2005. Comprehensive analysis of a multidimensional liquid chromatography
mass spectrometry dataset acquired on a quadrupole selecting, quadrupole
collision cell, time-of-flight mass spectrometer: II. New developments in
ProteinProspector allow for reliable and comprehensive automatic analysis of
large datasets. Mol. Cell Proteomics 4, 1194–1204.
Chanda, S., Dasgupta, U.B., Guhamazumder, D., Gupta, M., Chaudhuri, U., Lahiri, S., Das,
S., Ghosh, N., Chatterjee, D., 2006. DNA hypermethylation of promoter of gene p53
and p16 in arsenic-exposed people with and without malignancy. Toxicol. Sci. 89,
Chen, H., Liu, J., Zhao, C.Q., Diwan, B.A., Merrick, B.A., Waalkes, M.P., 2001. Association of
c-myc overexpression and hyperproliferation with arsenite-induced malignant
transformation. Toxicol. Appl. Pharmacol. 175, 260–268.
Chen, W.T., Hung, W.C., Kang, W.Y., Huang, Y.C., Chai, C.Y., 2007. Urothelial carcinomas
arising in arsenic-contaminated areas are associated with hypermethylation of the
gene promoter of the death-associated protein kinase. Histopathology 51, 785–792.
Chu, H.A., Crawford-Brown, D., 2007. Inorganic arsenic in drinking water and bladder
cancer: a meta-analysis for dose–response assessment. Int. J. Environ. Res. Public
Health 4, 340–341.
Chu, F., Nusinow, D.A., Chalkley, R.J., Plath, K., Panning, B., Burlingame, A.L., 2006.
Mapping post-translational modifications of the histone variant MacroH2A1 using
tandem mass spectrometry. Mol. Cell Proteomics 5, 194–203.
Danaee, H., Nelson, H.H., Liber, H., Little, J.B., Kelsey, K.T., 2004. Low dose exposure to
sodium arsenite synergistically interacts with UV radiation to induce mutations
and alter DNA repair in human cells. Mutagenesis 19, 143–148.
Drobna, Z., Waters, S.B., Devesa, V., Harmon, A.W., Thomas, D.J., Styblo, M., 2005.
Metabolism and toxicity of arsenic in human urothelial cells expressing rat arsenic
(+3 oxidation state)-methyltransferase. Toxicol. Appl. Pharmacol. 207, 147–159.
Eblin, K.E., Bredfeldt, T.G., Gandolfi, A.J., 2008. Immortalized human urothelial cells as a
model of arsenic-induced bladder cancer. Toxicology 248, 67–76.
Fraga, M.F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G.,
Bonaldi, T., Haydon, C., Ropero, S., Petrie, K., Iyer, N.G., Perez-Rosado, A., Calvo, E.,
Lopez, J.A., Cano, A., Calasanz, M.J., Colomer, D., Piris, M.A., Ahn, N., Imhof, A., Caldas,
C., Jenuwein, T., Esteller, M., 2005. Loss of acetylation at Lys16 and trimethylation at
Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37,
Gupta, A., Guerin-Peyrou, T.G., Sharma, G.G., Park, C., Agarwal, M., Ganju, R.K., Pandita,
S., Choi, K., Sukumar, S., Pandita, R.K., Ludwig, T., Pandita, T.K., 2008. The
mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is
essential for embryogenesis and oncogenesis. Mol. Cell. Biol. 28, 397–409.
Hernandez-Zavala, A., Valenzuela, O.L., Matousek, T., Drobna, Z., Dedina, J., Garcia-
Vargas, G.G., Thomas, D.J., Del Razo, L.M., Styblo, M., 2008. Speciation of arsenic in
exfoliated urinary bladder epithelial cells from individuals exposed to arsenic in
drinking water. Environ. Health Perspect. 116, 1656–1660.
Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., Thun, M.J., 2007. Cancer statistics, 2007.
CA Cancer J. Clin. 57, 43–66.
Jensen, T.J., Novak, P., Eblin, K.E., Gandolfi, A.J., Futscher, B.W., 2008. Epigenetic
remodeling during arsenical-induced malignant transformation. Carcinogenesis
Jensen, T.J., Wozniak, R.J., Eblin, K.E., Wnek, S.M., Gandolfi, A.J., Futscher, B.W., 2009.
Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-
associated malignant transformation. Toxicol. Appl. Pharmacol. 235, 39–46.
Jo, W.J., Loguinov, A., Wintz, H., Chang, M.,Smith, A.H., Kalman, D., Zhang, L., Smith, M.T.,
Vulpe, C.D., 2009. Comparative functional genomic analysis identifies distinct and
overlapping sets of genes required for resistance to monomethylarsonous acid
(MMAIII) and arsenite (AsIII) in yeast. Toxicol Sci10.1093/toxsci/kfp1162.
Kimura, A., Umehara, T., Horikoshi, M., 2002. Chromosomal gradient of histone
acetylation established by Sas2p and Sir2p functions as a shield against gene
silencing. Nat. Genet. 32, 370–377.
Lafon, A., Chang, C.S., Scott, E.M., Jacobson, S.J., Pillus, L., 2007. MYST opportunities for
growth control: yeast genes illuminate human cancer gene functions. Oncogene 26,
Landry, J., Sutton, A., Tafrov, S.T., Heller, R.C., Stebbins, J., Pillus, L., Sternglanz, R., 2000.
The silencing protein SIR2 and its homologs are NAD-dependent protein
deacetylases. Proc. Natl. Acad. Sci. U. S. A. 97, 5807–5811.
Li, J., Gorospe, M., Barnes, J., Liu, Y., 2003. Tumor promoter arsenite stimulates histone
H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human
diploid fibroblasts. J. Biol. Chem. 278, 13183–13191.
Marshall, G., Ferreccio, C., Yuan, Y., Bates, M.N., Steinmaus, C., Selvin, S., Liaw, J., Smith,
A.H., 2007. Fifty-year study of lung and bladder cancer mortality in Chile related to
arsenic in drinking water. J. Natl. Cancer Inst. 99, 920–928.
Marsit, C.J., Karagas, M.R., Danaee, H., Liu, M., Andrew, A., Schned, A., Nelson, H.H.,
Kelsey, K.T., 2006. Carcinogen exposure and gene promoter hypermethylation in
bladder cancer. Carcinogenesis 27, 112–116.
Mass, M.J., Tennant, A., Roop, B.C., Cullen, W.R., Styblo, M., Thomas, D.J., Kligerman, A.D.,
2001. Methylated trivalent arsenic species are genotoxic. Chem. Res. Toxicol. 14,
Petrick, J.S., Jagadish, B., Mash, E.A., Aposhian, H.V., 2001. Monomethylarsonous acid
(MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate
dehydrogenase. Chem. Res. Toxicol. 14, 651–656.
Ramirez, T., Brocher, J., Stopper, H., Hock, R., 2007. Sodium arsenite modulates histone
acetylation, histone deacetylase activity and HMGN protein dynamics in human
Rea, S., Xouri, G., Akhtar, A., 2007. Males absent on the first (MOF): from flies to
humans. Oncogene 26, 5385–5394.
Rossman, T.G., Uddin, A.N., Burns, F.J., 2004. Evidence that arsenite acts as a
cocarcinogen in skin cancer. Toxicol. Appl. Pharmacol. 198, 394–404.
Sens, D.A., Park, S., Gurel, V., Sens, M.A., Garrett, S.H., Somji, S., 2004. Inorganic
cadmium- and arsenite-induced malignant transformation of human bladder
urothelial cells. Toxicol. Sci. 79, 56–63.
Shechter, D., Dormann, H.L., Allis, C.D., Hake, S.B., 2007. Extraction, purification and
analysis of histones. Nature Protocols 2, 1445–1457.
Shia, W.J., Osada, S., Florens, L., Swanson, S.K., Washburn, M.P., Workman, J.L., 2005.
Characterization of the yeast trimeric–SAS acetyltransferase complex. J. Biol. Chem.
Shia, W.J., Li, B., Workman, J.L., 2006a. SAS-mediated acetylation of histone H4 Lys 16 is
required for H2A.Z incorporation at subtelomeric regions in Saccharomyces
cerevisiae. Genes Dev. 20, 2507–2512.
Shia, W.J., Pattenden, S.G., Workman, J.L., 2006b. Histone H4 lysine 16 acetylation
breaks the genome's silence. Genome Biol. 7, 217.
Shogren-Knaak, M., Ishii, H., Sun, J.M., Pazin, M.J., Davie, J.R., Peterson, C.L., 2006.
Histone H4-K16 acetylation controls chromatin structure and protein interactions.
Science (New York, NY) 311, 844–847.
Smith, A.H., Goycolea, M., Haque, R., Biggs, M.L., 1998. Marked increase in bladder and
lung cancer mortality in a region of Northern Chile due to arsenic in drinking water.
Am. J. Epidemiol. 147, 660–669.
Steinmaus, C., Yuan, Y., Bates, M.N., Smith, A.H., 2003. Case–control study of bladder
cancer and drinking water arsenic in the western United States. Am. J. Epidemiol.
Steinmaus, C., Bates, M.N., Yuan, Y., Kalman, D., Atallah, R., Rey, O.A., Biggs, M.L.,
Hopenhayn, C., Moore, L.E., Hoang, B.K., Smith, A.H., 2006. Arsenic methylation and
bladder cancer risk in case–control studies in Argentina and the United States. J.
Occup. Environ. Med. / Am. Coll. Occup. Environ. Med. 48, 478–488.
Suka, N., Luo, K., Grunstein, M., 2002. Sir2p and Sas2p opposingly regulate acetylation of
yeast histone H4 lysine16 and spreading of heterochromatin. Nat. Genet. 32,
Taipale, M., Rea, S., Richter, K., Vilar, A., Lichter, P., Imhof, A., Akhtar, A., 2005. hMOF
histone acetyltransferase is required for histone H4 lysine 16 acetylation in
mammalian cells. Mol. Cell. Biol. 25, 6798–6810.
Tchounwou, P.B., Patlolla, A.K., Centeno, J.A., 2003. Carcinogenic and systemic health
effects associated with arsenic exposure—a critical review. Toxicol. Pathol. 31,
Xie, Y., Liu, J., Benbrahim-Tallaa, L., Ward, J.M., Logsdon, D., Diwan, B.A., Waalkes, M.P.,
2007. Aberrant DNA methylation and gene expression in livers of newborn mice
transplacentally exposed to a hepatocarcinogenic dose of inorganic arsenic.
Toxicology 236, 7–15.
Yuan, Y., Marshall, G., Ferreccio, C., Steinmaus, C., Selvin, S., Liaw, J., Bates, M.N., Smith,
A.H., 2007. Acute myocardial infarction mortality in comparison with lung and
bladder cancer mortality in arsenic-exposed region II of Chile from 1950 to 2000.
Am. J. Epidemiol. 166, 1381–1391.
Zhao, C.Q., Young, M.R., Diwan, B.A., Coogan, T.P., Waalkes, M.P., 1997. Association of
arsenic-induced malignant transformation with DNA hypomethylation and
aberrant gene expression. Proc. Natl. Acad. Sci. U. S. A. 94, 10907–10912.
W.J. Jo et al. / Toxicology and Applied Pharmacology 241 (2009) 294–302