Carcinogenesis vol.30 no.7 pp.1243–1251, 2009
doi:10.1093/carcin/bgp088
Advance Access publication April 17, 2009
Alterations of histone modifications by cobalt compounds
Qin Li, Qingdong Ke and Max Costa
Department of Environmental Medicine, New York University School of
Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA
To whom correspondence should be addressed. Tel: þ1 845 731 3515;
Fax: þ1 845 351 2118;
Email: [email protected]
In the present study, we examined the effects of CoCl2 on multiple
histone modifications at the global level. We found that in both
human lung carcinoma A549 cells and human bronchial epithelial
Beas-2B cells, exposure to CoCl2 (‡200 mM) for 24 h increased
H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A
and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase
in H3K4me3 and H3K27me3 by cobalt ions exposure was probably
through enhancing histone methylation processes, as methioninedeficient medium blocked the induction of H3K4me3 and
H3K27me3 by cobalt ions, whereas cobalt ions increased
H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of
JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B
ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 mM of
CoCl2 for 24 h, A549 cells not only increased but also decreased
expression of hundreds of genes involved in different cellular
functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It
also sheds light on the possible mechanisms involved in cobaltinduced alteration of histone modifications, which may lead to
altered programs of gene expression and carcinogenesis since
cobalt at higher concentrations is a known carcinogen.
Introduction
Post-translational modifications of nucleosomal histones play critical
roles in all aspects of eukaryotic chromosome dynamics, including
replication, recombination, repair, segregation and gene expression.
Such modifications include acetylation and methylation of lysines
(K) and arginines (R), citrullination of arginines, phosphorylation of
serines (S) and threonines (T), sumoylation and ubiquitination of lysines and adenosine diphosphate-ribosylation (1–3). Each modification
can affect chromatin structure that may regulate gene transcription.
Studies have shown that different histone modifications yield distinct
functional consequences (2). For example, in general, trimethylation
of histone H3 at K9 (H3K9me3), K27 (H3K27me3) and K36
(H3K36me3); dimethylation of histone H3 at K9 (H3K9me2); ubiquitination of histone H2A (uH2A) and the lack of histone H3 (AcH3) and
H4 (AcH4) acetylation correlated with transcriptional repression in
higher eukaryotes (4–8), whereas trimethylation of histone H3 at K4
(H3K4me3) and ubiquitination of histone H2B (uH2B) were associated
with transcriptional activation (9). However, these modifications may
interact with each other and their total sum may be the ultimate determinant of chromatin state that governs gene transcription.
The enzyme-catalyzed methylation of histone lysine is controlled
by both methylation and demethylation processes. For example, the
Set1-containing complex, complex proteins associated with Set1
(COMPASS), which is the yeast homolog of the human MLL comAbbreviations: DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate.
plex, is required for mono-, di- and trimethylation of lysine 4 of
histone H3. Both Cps60 and Cps40 components of COMPASS are
required for proper histone H3 trimethylation (10). Suv39h family
enzymes are responsible for trimethylation of H3K9 in vivo (11).
The enhancer of zeste homolog 2 is a methyltransferase known to
trimethylate H3K27 (12). Set2 is responsible for mediating H3 lysine
36 methylation in vivo (6). Two families of enzymes are capable of
removing methyl groups from lysine residues in histone tails. One
requires protonated nitrogen in the substrate for the demethylation
reaction to occur, such as LSD1, which is a flavin-dependent amine
oxidase and removes the methyl group from mono- or dimethylated
H3K4 by catalyzing the oxidation of amine to an imine intermediate
(13). The other does not require protonated nitrogen for demethylation, such as the JARID1 family, JMJD2 family and UTX, which all
are JmjC-containing proteins and use Fe(II) and a-ketoglutarate as
cofactors to mediate an oxidation-based demethylation. JARID1B specifically demethylates H3K4me3 (14). The family of JMJD2 includes
JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1 and JMJD2D, all of
which can demethylate H3K9me3, whereas only JMJD2A and JMJD2C
can also demethylate H3K36me3 (15). UTX has recently been discovered to exhibit H3K27 di- and tridemethylase activity (16).
In spite of its discovery three decades ago (17), histone ubiquitination is still not well characterized, compared with histone acetylation
and methylation. All the four core histones (H2A, H2B, H3 and H4)
can be ubiquitinated, and the most prevalent forms of this modification are monoubiquitinated H2A (uH2A) and H2B (uH2B) (18).
Ubiquitin is catalytically linked to the lysine 119 (K119) of H2A
and lysine 120 (K120) of H2B (19,20).
Ubiquitination is a reversible process, and several studies have recently demonstrated that uH2A can be deubiquitinated by the enzyme
Ubp-M in humans (21), and both uH2A and uH2B can be deubiquitinated by the enzyme complex of ubiquitin protease USP22, ATXN7L3
(ySgf11 homolog) and ENY2 (ySus1 homolog) in humans (22). In
yeast, uH2B can be deubiquitinated by the enzyme Ubp8 (23,24) and
Ubp10/DOT4 (25). Ubp8 is a component of the SAGA histone acetyltransferase. It has been proposed that the Rad6-catalyzed monoubiquitination of histone H2B is followed by the recruitment of SAGA to the
ubiquitinated nucleosome and subsequent deubiquitination of histone
H2B, which is required to initiate transcription. Ubp10/DOT4 also
targets monoubiquitinated histone H2B for deubiquitination. However,
this enzyme exhibits deubiquitination activities involved in telomericassociated gene silencing (26), indicating that such deubiquitinating
enzymes have distinct functions in the regulation of gene expression
via the targeting of histone H2B deubiquitination.
Cobalt is found in various ores and is universally used in the steel
industry, being a major constituent of hard metal alloys. Its compounds are also used in the production of pigments, drying agents
and catalysts (27). Cobalt is an essential element necessary for the
formation of vitamin B12 (hydroxocobalamin); however, excessive
administration of this trace element produces goiter and reduced
thyroid activity (28). Radioactive isotopes of cobalt are used in
industry, medicine and nuclear research. In nuclear power plants,
59Co-containing alloys can be activated into radioactive 60Co oxides
and may become dispersed in the cooling water and contaminate
workers. 60Co is an important radioactive tracer and cancer-radiation
oncology treatment agent (29). Occupational exposure to Co occurs
mainly via inhalation leading to various lung diseases, such as pneumonitis, fibrosis and asthma (27). When administered by inhalation,
cobalt significantly increased the incidences of combined malignant
and benign tumors at multiple tissue sites in experimental animals. It
caused lung tumors in male and female mice and female rats, as well
as adrenal gland tumors in the female rats (30). However, the mechanism by which cobalt ions induce cancer remains unclear.
Recent research has indicated that disruption of epigenetic homeostatsis affects gene expression that can be inherited through cell
Ó The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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division and is probably an important event in nickel-induced tumorigenesis. Although cobalt is an essential element for life in minute
quantities, at higher levels of exposure it shows mutagenic and carcinogenic effects similar to nickel, including altering gene expression
(31–33) and interfering with the cellular homeostasis of reactive oxygen species (34,35), Ca (33,36) and Fe (37,38). Thus, the possible
effects of CoCl2 on global histone modifications were examined in
human lung carcinoma A549 cells and human bronchial epithelial
Beas-2B cells, which are well-established models to study the epigenetic effects of metals (39–42).
In this study, cobalt ions increased both the levels of chromatin
gene-repressive marks (H3K9me3, H3K27me3, H3K36me3 and
uH2A) as well as gene activation marks (H3K4me3 and uH2B) and
decreased AcH4. The cobalt-induced increase in H3K4me3 and
H3K27me3 may be operating through activation of histone methyltransferases, whereas the increase in H3K9me3 and H3K36me3 was
attributed to the inhibition of a histone demethylase JMJD2A enzyme
activity, which is probably due to the competition of cobalt ions with
iron for binding to the active site of JMJD2A. Moreover, the induced
uH2A and uH2B probably was caused by inhibition of the histonedeubiquitinating enzyme activity. Our study is the first to demonstrate
the effects of cobalt ions on histone modifications and explore the
possible mechanisms by which cobalt ions alter specific histone modifications. The results of this study will broaden our understanding on
cobalt toxicity and carcinogenesis.
Materials and methods
Chemicals
CoCl26H2O and all other reagents, unless specified, were purchased from
Sigma–Aldrich (St Louis, MO).
Cell culture
Human lung carcinoma A549 cells were cultured in Ham’s F-12K medium
(Invitrogen, Frederick, MD) and human bronchial epithelial Beas-2B cells
were grown in Dulbecco’s modified Eagle’s medium (Invitrogen). Both media
were supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana,
CA) and 1% penicillin/streptomycin (Invitrogen). Cells were maintained at
37°C as monolayers in a humidified atmosphere containing 5% CO2. Cells
were passaged at 70–80% confluence by trypsinization. When cell density
reached 70 to 80% confluence, they were treated with the cobalt ions.
Histone extraction
Cells cultured in the 150 or 100 mm dish were washed with ice-cold 1
phosphate-buffered saline (0.13 M NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4
and 0.27 mM KCl, pH 7.4) twice and lysed with ice-cold radioimmunoprecipitation assay buffer [50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% Nadeoxycholate, 150 mM NaCl and 1 mM ethylenediaminetetraacetic acid
(EDTA)] supplemented with a protease inhibitor mixture (Roche Applied
Sciences, Indianapolis, IN) for 10 min on ice. The lysate was then collected
and centrifuged at 10 000g for 10 min. The supernatant was removed and the
pellet washed once in 10 mM Tris/13 mM EDTA buffer (pH 7.4) and spun
down at maximum speed for 5 min. The pellet was then resuspended in 100 ll
0.4 N H2SO4. After at least 1.5 h of incubation on ice, the sample was centrifuged at 14 000g for 15 min. The resulting supernatant was recovered, then
mixed with 1 ml of cold acetone and kept at 20°C overnight. The histones
were then collected by centrifugation at 14 000g for 15 min. After one wash
with acetone, the histones were air-dried and redissolved in 4 M urea.
Whole-cell protein extraction
Cells cultured in six-well plates were washed with ice-cold 1 phosphatebuffered saline twice and lysed in 100 ll of boiling lysis buffer [10 mM
Tris–HCl, pH 7.4, 1% sodium dodecyl sulfate (SDS) and 1 mM sodium orthovanadate] for 15 min. The cell lysates were transferred to an Eppendorf tube
and sonicated to reduce viscosity by applying 10 one second pulses using
a Branson Sonifier 450. The samples were stored at 20°C until use.
Western blot
The protein concentration was determined using Bio-Rad DC protein assay
(Bio-Rad, Hercules, CA). Five micrograms of purified histones or 50 lg
whole-cell proteins (WCL) were separated by SDS–polyacrylamide gel electrophoresis (PAGE) gel and transferred to polyvinylidene difluoride (PVDF)
membranes (Bio-Rad). Antibodies against dimethyl H3K9, trimethyl H3K9,
trimethyl H3K27, trimethyl H3K36, H3, acetyl-H4, H4, ubiquitinated H2A
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antibody (anti-ubiquityl histone H2A, clone E6C5 mouse monoclonal IgM,
catalog # 05-678) and H2B antibody (rabbit polyclonal lgG, catalog # 07371) were purchased from Upstate (Lake Placid, NY). Trimethyl H3K4 antibody was from Abcam (Cambridge, MA), and b-actin antibody was from
Sigma–Aldrich. Anti-JMJD2A antibody was from Bethyl (Montgomery,
TX). Horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The detection was
accomplished by chemical fluorescence following an ECL Western blotting
protocol (Amersham, Piscataway, NJ). After transfer to PVDF membranes, the
gels were stained with Bio-safe Coomassie stain (Bio-Rad) to assess the loading of histones. The immunoblots were scanned and analyzed using ImageJ
software, and values were normalized to those obtained in the control samples.
GeneChip assay
Total RNA was isolated from Co-exposed (200 lM, 24 h) and non-exposed
cells using the TRIzol reagent (Gibco BRL, Rockville, MD). Double-stranded
complementary DNA was synthesized with a Superscript cDNA Synthesis kit
(Gibco BRL) by using an oligo(dT)24 primer with a T7 RNA polymerase
promoter site added to its 3# end. The isolated complementary DNA was used
for in vitro transcription using the Ambion T7 Megascript system (Austin, TX)
in the presence of biotin-11-CTP and biotin-16-UTP (Enzo Diagnostics, Farmingdale, NY). A total of 25–50 lg of the complementary RNA product in buffer
(40 mM Tris–acetate, pH 8.1, 100 mM potassium acetate and 30 mM magnesium acetate) was fragmented at 94°C for 35 min. This probe was used for
hybridization and mixed with herring sperm DNA (0.1 mg/ml; Sigma). The test
3 chip served for the evaluation of the probe quality as directed by the manufacturer (Affymetrix, Santa Clara, CA). Aliquots of the complementary RNA
hybridization mixtures (15 lg complementary RNA in 200 ll hybridization
mix) were hybridized to a human GeneChip array, which was then washed
and scanned (Hewlett-Packard, GeneArray Scanner G2500A, Palo Alto, CA)
according to procedures developed by the manufacturer (Affymetrix).
The gene expression data were analyzed as previously (43). The extent of
changes in gene expression was determined by dividing the mean intensity of
each Co-exposed condition by the mean intensity of the control cells.
Reverse transcription–polymerase chain reaction
Either 0.5 or 1 lg of RNA was reversely transcribed to complementary DNA
with the Superscript III First Strand Synthesis Super-Mix Kit for qRT–PCR
(Invitrogen). Semiquantitative polymerase chain reaction (PCR) was then performed to amplify JMJD1A, HMOX1, GADD45A, BNIP3L and b-actin using
the following primer pairs: JMJD1A, 5#-AATCCAGAATGCCCCATCCAGG-3#
(forward) and 5#-CACATACTCCAAACCCACACCG-3# (reverse); HMOX1,
5#-ACATCTGTGGCCCTGGAG-3# (forward) and 5#-TGTTGGGGAAGGTGAAGAAG-3# (reverse); GADD45A, 5#-AAAGGATGGATAAGGTGGGG-3#
(forward) and 5#-TCCCGGCAAAAACAAATAAG-3# (reverse); BNIP3L,
5#-AATGTCGTCCCACCTAGTCG-3# (forward) and 5#-GAGAGCTATGGTCCCTGCTG-3# (reverse) and b-actin, 5#-TCACCCACACTGTGCCCATCTACGA-3# (forward) and 5#-CAGCGGAACCGCTCATTGCCAATGG-3# (reverse).
An aliquot of 2 ll was used in a final volume of 50 ll PCR mixture containing
both the forward and reverse primers, deoxynucleoside triphosphates (10 mM, 1
ll each), 10 reaction buffer (5 ll) and 2 U of Taq DNA polymerase (Roche,
Indianapolis, IN). The PCR conditions were 95°C for 2 min, followed by 25
cycles (20 cycles for b-actin) of 30 s at 94°C, 45 s at 57°C and 1 min at 72°C. An
aliquot of the PCR products was then analyzed by electrophoresis over 1.5%
agarose gels containing ethilium bromide.
Generation and purification of the recombinant JMJD2A
The pFastBac HTb-Flag-JMJD2A bacmid DNA was kindly provided by Dr Yi
Zhang (44). Sf9 insect cells were cultured in suspension in Grace complete
medium (Invitrogen, Carlsbad, CA) supplied with 10% heat-inactivated fetal
bovine serum and 1% penicillin/streptomycin (Grand Island, NY) at 27°C in
dark. Purified recombinant JMJD2A bacmid DNA was transfected into Sf9
insect cells in Sf-900 SFM (Invitrogen) medium by using Cellfectin Reagent
(Invitrogen). Seventy-two hours post-transfection, the cell culture medium
containing virus was collected and clarified by centrifuge at 500g for 5 min.
The titer of the P1 viral stock (supernatant) was estimated and the P1 viral
stock was used to infect Sf9 cells to generate a high-titer P2 baculoviral stock.
The medium was collected and centrifuged at 500g for 5 min. The supernatant
(P2 viral stock) was removed, and 100 ll SDS–PAGE loading buffer was added
into the pellet (containing expressed recombinant protein), boiled at 95–100°C
for 5 min, sonicated by applying 10 one second pulses with a Branson Sonifier
and centrifuged at 10 000g for 10 min. The supernatant was loaded into a 5%
SDS–PAGE gel and subjected to western blot with anti-JMJD2A antibody.
After the recombinant protein was verified, the titer of the P2 viral stock
was determined by performing a viral plaque assay, and the viral stock was
amplified by infecting cells until it reached a suitable titer (1 108 pfu/ml).
Alterations of histone modifications
Then the viral stock was used to infect insect cells to express Flag-JMJD2A
protein on a large scale.
Sf9 cell suspension (1–2 108 cells/ml) was centrifuged at 500g for 5 min,
and the pellet was resuspended in lysis buffer F (20 mM Tris–HCl, pH 8.0, 500
mM NaCl, 4 mM MgCl2, 0.4 mM EDTA and 20% glycerol) supplemented with
protease inhibitor cocktail (Roche) and 2 mM dithiothreitol (DTT). The lysate
was then sonicated and centrifuged at 11 000g for 10 min. The supernatant was
collected and dialyzed against the lysis buffer F without DTT for 2 h at 4°C.
The ANTI-FLAG M2 Affinity Gel (Sigma–Aldrich) was then used to purify
the recombinant protein. The dialyzed protein solution was incubated with
Flag-resin for 2 h in a rotator at 4°C and the Flag-resin was washed with buffer
W (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 15% glycerol and
0.01% Igepal CA-630) three times on a rotator (5 min each time). The recombinant protein was eluted with 3 Flag peptide in elution buffer (25 mM
Tris–HCl, pH 8.0, 10% glycerol and 100 mM NaCl).
In vitro demethylation assays
Five micrograms (5 lg) of core histones (Upstate) were incubated with purified
recombinant JMJD2A in histone demethylation buffer [50 mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid–KOH (pH 8.0), 100 lM Fe(NH4)2
(SO4)2, 1 mM a-ketoglutarate, 2 mM ascorbic acid and 1 mM phenylmethylsulfonyl fluoride] with or without CoCl2 at 37°C for 30 min. The demethylation
reaction was terminated by the addition of SDS–PAGE loading buffer
and subjected to western blot with anti-H3K9me3 and anti-H3K36me3
antibodies.
Protein extraction for in vitro ubiquitination and deubiquitination assays
Cells cultured in 150 mm dishes were washed with ice-cold 1 phosphatebuffered saline twice and lysed with 1 ml ice-cold Pagano buffer, consisting of
20 mM Tris–HCl (pH 7.4), 2 mM DTT, 0.25 mM EDTA, 10 lg/ml leupeptin
and 10 lg/ml pepstatin. The suspension was kept on ice and sonicated as
described above. After centrifugation at 14 000g for 10 min, the supernatant
was divided into aliquots and stored at 80°C.
In vitro ubiquitination assay
Five micrograms (5 lg) of histones extracted from cells as described above
were incubated with 10 ll ubiquitination mix, containing 40 mM Tris (pH 7.4),
5 mM MgCl2, 10% glycerol, 1 mM DTT, 10 mM creatine phosphate, 0.1 lg/ml
creatine kinase, 0.5 mM adenosine triphosphate, 1 lM ubiquitin aldehyde,
1 mg/ml methyl ubiquitin, 10 lM iodoacetemide, 1 lM okadaic acid, 1 lg
ubiquitin and 20 lg of protein extract. After incubation at 37°C for 1 h, the
reaction was terminated by addition of SDS–PAGE loading buffer. The products were then boiled at 95–100°C for 5 min and resolved in 15% SDS–PAGE
gels, transferred to a PVDF membrane and blotted with the anti-uH2A or antiH2B antibodies.
In vitro deubiquitination assay
Five micrograms (5 lg) of extracted histones dissolved in H2O were incubated
with 10 ll deubiquitination mix containing 40 mM Tris (pH 7.4), 5 mM MgCl2,
10% glycerol, 1 mM DTT and 20 lg of protein extract. After incubation at
37°C for the indicated times or for 1 h if not indicated, the reaction was
terminated by addition of SDS–PAGE loading buffer.
The products were then boiled at 95–100°C for 5 min and resolved on 15%
SDS–PAGE gels, transferred to a PVDF membrane and blotted with the antiuH2A or anti-H2B antibodies.
Results
Cobalt ions alter multiple histone modifications at the global levels
To study whether cobalt ions alter epigenetic marks on histones, we
treated human lung carcinoma A549 cells with 50, 200 and 300 lM of
CoCl2 (for histone methylation and acetylation) or 250 and 500 lM of
CoCl2 (for histone ubiquitination) for 24 h. Histones were then extracted from the cells, and the levels of selected commonly studied
histone modifications, including histone methylation, acetylation and
ubiquitination, were assessed by western blot analysis using very
sensitive and specific antibodies. The results show that the higher
doses (200 lM) of cobalt ions significantly increased global histone
H3K4me3, H3K9me2, H3K9me3, H3K27me3 and H3K36me3
(Fig. 1A), as well as uH2A and uH2B (Fig. 1B). Furthermore, when
the cells were exposed to 0.5 mM CoCl2 for 24 h, di-uH2A appeared.
In contrast, the global level of acetylation at histone H4 (AcH4) was
Fig. 1. Alterations of histone modifications following cobalt ions exposure in A549 cells. (A–C) A549 cells were exposed to the indicated concentrations of CoCl2
for 24 h. Isolated histones (5 lg) were separated in 15% SDS–PAGE gels and subjected to western blotting with the indicated antibodies. The relative intensity of
the bands was quantitated using ImageJ software and normalized to the loading control. The numbers quantitating the intensity of the bands were given below the
corresponding bands. Coomassie blue staining, histone H3 or H4 were used to assess the loading of the histones in western blot analysis. Similar data were
obtained in at least two other independent experiments, and only one representative blot is shown here. me2 5 dimethylation; me3 5 trimethylation; AcH4 5
acetylated histone H4; uH2A/H2B 5 ubiquitinated H2A/H2B; di-uH2A 5 diubiquitinated H2A.
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Q.Li et al.
decreased by cobalt ions exposure (Fig. 1C). Similar effects were also
observed in the non-tumorigenic human bronchial epithelial Beas-2B
cells. As shown in Fig. 2A–C, 24 h of exposure to 25, 50, 100 and 200
lM of CoCl2 increased global histone H3K4me3, H3K9me2 and
H3K27me3, but decreased AcH4 in Beas-2B cells, whereas only higher doses of CoCl2 increased H3K9me3 (200 lM), H3K36me3 (50–
200 lM), uH2A (150 lM) and di-uH2A (200 lM) in Beas-2B cells.
Cobalt ions increase H3K9me3 and H3K36me3 by inhibiting histone
demethylation process
Histone lysines can be mono-, di- or trimethylated (2), and both the
lysine’s position and its degree of methylation may play regulatory
roles in different effector proteins that read chromatin modifications,
which may result in distinct functional outcomes, including heterochromatin formation (45), X-inactivation (46), genomic imprinting
(47) and gene silencing (39). Abnormal histone methylation has been
associated with numerous human diseases (i.e. cancer) (48,49).
Recent studies show that histone methylation is dynamically regulated by histone methyltransferases and demethylases. To study the
possible mechanisms by which cobalt ions alter specific histone methylations, A549 cells were preincubated in methionine-deficient
medium for 4 h prior to treatment with cobalt ions. Since methionine
is a substrate for S-adenosylmethionine, which is a coenzyme involved in methyl group transfers and has a short half-life in cells
(50), the methionine-deficient medium could decrease intracellular
S-adenosylmethionine pool and consequently inhibit methyl transfer
reactions. As shown in Fig. 3A and B, incubation of cells in
methionine-deficient medium dramatically decreased global H3K4me3,
H3K9me3, H3K27me3 and H3K36me3, indicating that the histone
methylation reaction was inhibited by the withdrawal of methionine
in the medium. The addition of 300 lM cobalt ions did not cause
observable toxicity (e.g. cell detachment) in cells under methioninedepleted conditions, showing similar cellular toxicity as has been
observed with 1 mM nickel ions (39). Under methionine-depleted
conditions, the addition of cobalt ions failed to increase global
H3K4me3 or H3K27me3 (Fig. 3A) but still elevated H3K9me3 and
H3K36me3 compared with untreated cells (Fig. 3B). These results
indicated that cobalt ions probably increased global H3K4me3 and
H3K27me3 by enhancing the methylation process, in contrast, increased global H3K9me3 and H3K36me3 probably occurred by the
inhibition of the demethylation process rather than activating the
methylation process.
Cobalt ions inhibit histone H3K9/H3K36 trimethyldemethylase
JMJD2A enzyme activity in vitro
Histone H3K9/K36 trimethylation had long been considered as a permanent modification until recently when JMJD2A, a major lysine
trimethyl-specific JmjC histone demethylase, was identified (15).
With its cofactors Fe(II) and a-ketoglutarate, JMJD2A exclusively
catalyzes the demethylation of H3K9me3 and H3K36me3, converting
H3K9/36me3 to H3K9/36me2 but it cannot convert H3K9/36me1 or
unmethylated H3K9/K36 (15). To examine the role of JMJD2A in
cobalt-induced H3K9me3/H3K36me3, we first determined the effect
of cobalt exposure on the endogenous JMJD2A protein level. Total
protein lysates were isolated from A549 cells 24 h following cobalt
exposure (50, 200 and 300 lM) and subjected to western blotting
using an antibody against human JMJD2A. The result showed that
JMJD2A protein levels did not appear to change following cobalt
treatment (data not shown). To determine whether cobalt ions inhibited JMJD2A demethylase activity, we generated recombinant
Flag-JMJD2A in insect cells. Five microliters of JMJD2A was used
for western blot with an antibody against JMJD2A to verify the fusion
protein (Fig. 4A left panel). Ten microliters of JMJD2A and different
amounts of bovine serum albumin were separated in 5% SDS–PAGE
gels and stained with Coomassie blue to estimate the concentration of
Flag-JMJD2A protein, which was 1 lg/ll, as shown in the right
panel of Fig. 3A. To confirm the demethylase enzyme activity of
JMJD2A, 5 lg core histones were incubated with different amounts
Fig. 2. Alterations of histone modifications following cobalt ions exposure in Beas-2B cells. (A–C) Beas-2B cells were exposed to the indicated concentrations of
CoCl2 for 24 h. Isolated histones (5 lg) were separated in 15% SDS–PAGE gels and subjected to western blotting with the indicated antibodies. The relative
intensity of the bands was quantitated using ImageJ software and normalized to the loading control. The numbers quantitating the intensity of the bands were given
below the corresponding bands. Histone H3 or H4 was used to assess the loading of the histones in western blot analysis. Similar data were obtained in at least two
other independent experiments, and only one representative blot is shown here. me2 5 dimethylation; me3 5 trimethylation; AcH4 5 acetylated histone H4;
uH2A 5 ubiquitinated H2A; di-uH2A 5 diubiquitinated H2A.
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Alterations of histone modifications
Fig. 3. (A) Cobalt ions induced H3K4 trimethylation and H3K27 trimethylation by activating methyltransferase. (B) Cobalt ions increase H3K9 trimethylaiton
and H3K36 trimethyation by inhibiting histone demethylation process. A549 cells were seeded in F-12K complete medium. On the second day, cells were
replenished with complete Dulbecco’s modified Eagle’s medium or methionine-deficient Dulbecco’s modified Eagle’s medium. After incubation for 4 h, cells were
then exposed to 300 lM CoCl2 for 24 h. Histones were extracted and immunoblotted for H3K4me3, H3K9me3, H3K27me3 and H3K36me3 antibodies. MT,
methionine. Similar data were obtained in at least two other independent experiments, and only one representative blot is shown here.
of JMJD2A, supplemented with 100 Fe(II), 1 mM a-ketoglutarate and
2 mM ascorbic acid. The demethylation reaction was terminated by
the addition of SDS–PAGE loading buffer and subjected to western
blot with the antibody against H3K9me3 and H3K36me3. The results
show that the recombinant Flag-JMJD2A can effectively remove
methyl group from trimethylated histone H3K9 and H3K36
(Fig. 4B). To determine whether cobalt ions inhibited histone demethylase enzyme activity, 10 ll JMJD2A was preincubated with the
indicated concentrations of CoCl2 for 10 min on ice prior to the
addition of other demethylation reaction components. As shown in
Fig. 4C, in the absence of CoCl2, the level of H3K9me3 and
H3K36me3 was decreased 90% after 30 min of demethylation reaction; however, in the presence of 1 lM CoCl2, the decreased histone
methylation was attenuated, and the decreased histone methylation
level recovered to 50% of original level when JMJD2A was preincubated with 6 lM CoCl2, with a molar ratio of Co2þ to JMJD2A of 2.
These results indicate that cobalt ions directly inhibited demethylase
activity of JMJD2A in vitro.
The inhibition of JMJD2A demethylase activity by cobalt ions may
be due to its interference with function of the iron moiety in this
enzyme. To verify this hypothesis, 50, 100, 200 and 300 lM CoCl2
were first mixed with 100 lM Fe2þ, a-ketoglutarate and other demethylation reaction components, and then incubated with 10 ll of
JMJD2A at 37°C for 30 min. As shown in Fig. 4D, 100 lM or higher
concentrations of CoCl2 significantly prevented the removal of the
methyl group from H3K9 and H3K36. However, when 100 lM
Fe2þ and a-ketoglutarate were preincubated with 10 ll of JMJD2A
on ice for 10 min prior to the addition of other demethylation reaction
component and CoCl2 (added last), addition of 100 lM CoCl2 did not
increase the level of H3K9me3 or H3K36me3, compared with the
absence of CoCl2 (Fig. 4E). These results suggest that Co2þ may
compete with Fe2þ for binding to JMJD2A sequentially resulting in
the inhibition of JMJD2A demethylase activity.
Cobalt ions do not increase the level of uH2A in the in vitro histone
ubiquitinating assay
Increased histone ubiquitination may be due to an increased histone
ubiquitinating enzyme activity. Accordingly, an in vitro ubiquitination
assay was developed to assess the effects of cobalt on any histone
ubiquitinating enzyme activity. A schematic of the in vitro ubiquitination assay is illustrated in Fig. 5A. Excessive amounts of histone
and ubiquitin (obtained commercially), supplemented with sufficient
adenosine triphosphate, were incubated with cell lysate extracted
from untreated A549 cells, in the absence or presence of cobalt ions
(0.05 and 0.5 mM) or nickel ions (0.1 and 1 mM). After incubation
at 37°C for 1 h, the reaction was terminated by the addition of the
SDS–PAGE loading buffer. The products were then subjected to western blot using anti-uH2A antibodies. The same blots were also incubated with anti-Ub antibody to detect the level of ubiquitin. As
shown in Fig. 5B, incubation of the substrates with the cell lysate
increased uH2A, concomitant with a decreased level of ubiquitin.
This enhanced uH2A was not affected by the addition of various levels
of CoCl2 or NiCl2 to the reaction mixture. These results indicated that
cobalt or nickel ions-induced histone ubiquitination in the cell did not
activate a histone ubiquitinating enzyme activity.
Cobalt ions inhibit histone-deubiquitinating enzyme activity in vitro
Inhibition of the histone-deubiquitinating enzyme could also give rise
to increased cellular levels of histone ubiquitination. Thus, an in vitro
deubiquitination assay was developed to assess this activity. Histones
extracted from A549 cells that also contain ubiquitinated histones
were incubated with untreated (control) cell lysate at 37°C, in the
absence or presence of cobalt ions or nickel ions. As shown in
Fig. 6A, incubation of histones with the cell lysate for 1 h led to
a dramatic decrease in ubiquitinated H2A levels concominant with
a corresponding increase in free ubiquitin. Addition of CoCl2 or NiCl2
to the reaction prevented this loss. By comparison, when SDS–PAGE
loading buffer was immediately added to the reaction at time zero, the
levels of ubiquitinated H2A and ubiquitin were not affected. In addition, the level of uH2A remained unchanged, regardless of the presence of CoCl2 or NiCl2, when the lysate was excluded from the
reaction (Fig. 6B). These data provide evidence that the altered levels
of ubiquitinated H2A was directly mediated by a histone deubiquiting
enzyme in the cell lysate during the incubation.
In order to confirm that addition of CoCl2 or NiCl2 prevented the
loss of uH2A in the deubiquitination reaction, a time-course study of
deubiquitination reaction was conducted. The reactions were incubated at 37°C for 0.5, 1, 2, and 4 h, in the absence or presence of
CoCl2 or NiCl2. As shown in Fig. 6C, consistent with the previous
results, incubation of histones with control cell lysate led to a dramatic
decrease of ubiquitinated H2A. About 50% of ubiquitinated H2A was
lost at 0.5 h of incubation and it was undetectable at 1, 2 and 4 h. With
the addition of CoCl2 or NiCl2, the levels of ubiquitinated H2A remained unchanged throughout.
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Q.Li et al.
Fig. 4. Cobalt ions inhibit histone H3K9/H3K36 trimethyldemethylase JMJD2A enzyme activity in vitro. (A) Verification and purification of baculovirus
expressed recombinant Flag-JMJD2A. A baculovirus expressing Flag-JMJD2A was used to produce recombinant protein in insect Sf9 cells. JMJD2A was purified
by immunoprecipitation with monoclonal anti-flag M2 Affinity Gel. Five microliters of JMJD2A was used for western blot with antibody against JMJD2A to verify
the fusion protein (left panel). Ten microliters of JMJD2A and different amounts of bovine serum albumin (BSA) were separated in 5% SDS–PAGE gels and
stained with coomassie blue to estimate the concentration of Flag-JMJD2A protein (right panel). (B) Five microgram core histones were incubated with different
amounts of JMJD2A and following the demethylation reaction, histone methylation levels were analyzed by western blot using antibodies that are highly specific
for the histone modification. (C) Ten microliter JMJD2A was preincubated with indicated concentrations of CoCl2 for 10 min on ice before the addition of other
reaction components. The demethylation reaction was terminated by the addition of SDS–PAGE loading buffer and subjected to western blot with the antibody
against H3K9me3 and H3K36me3. The lower panel shows the gel stained with coomassie blue to assess the loading of the histones and JMJD2A in the reaction.
The molar ratios of cobalt ions to estimated JMJD2A are shown in the figure. (D) The indicated concentrations of CoCl2 were preincubated with demethylation
reaction buffer for 10 min on ice followed by addition of 10 ll JMJD2A. After incubation at 37°C for 30 min, the reaction was terminated and samples were
processed as described above. (E) Hundred micromolar Fe2þ and a-ketoglutarate were preincubated with 10 ll of JMJD2A on ice for 10 min before the addition of
other demethylation reaction components and CoCl2 (added last). After incubation at 37°C for 30 min, the reaction was terminated and samples were processed as
described above.
We next assessed the level of uH2B in the in vitro deubiquitination
assay in the presence and absence of CoCl2 or NiCl2. As shown in
Fig. 6D, incubation of histones with the control A549 cell lysate resulted in the loss of uH2B. Addition of CoCl2 or NiCl2 significantly
prevented this loss. The data demonstrated that cobalt ions, like nickel
ions, inhibited deubiquitination of uH2A and uH2B in the in vitro
deubiquitination assay. These results confirm that cobalt and nickel ions
inhibited the histone-deubiquitinating enzyme activity, and thus may
contribute to the earlier-observed increases in histone ubiquitination.
Modulation of gene expression by CoCl2 in A549 cells
As described in the Introduction, alteration of histone modifications is
very important for the regulation of gene expression—gene transcription and gene repression. In this study, the results showed that exposure to Co2þ increased gene repression markers (H3K9me3,
H3K27me3, H3K36me3, H3K9me2, uH2A and lack of AcH4), as
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well as gene activation markers (H3K4me3 and uH2B) in both
A549 and Beas-2B cells. We therefore used the GeneChip microarray
technique to study the effects of Co compounds on global gene expression. A549 cells were exposed to 200 lM of CoCl2 for 24 h
followed by GeneChip analysis. Microarray data showed that in response to Co compounds, cells not only increase but also decrease the
expression of hundreds of genes that are involved in different cellular
functions. Supplementary Table S1 (available at Carcinogenesis Online) lists the genes upregulated by Co2þ, and supplementary Table S2
(available at Carcinogenesis Online) lists the genes downregulated by
Co2þ. For example, Co2þ upregulates the expression of genes functioning in tumorigenesis (i.e. CAIX and TDE2), signal transduction
and trafficking (i.e. SLC2A3, SLC11A2 and SLC2A1), cell defense
(i.e. BNIP3L), protein expression and turn over (i.e. EEF1A2, SUI1,
UBC and USP15), development, differentiation and proliferation
(i.e. GDF15, VEGF, EGFR, ST13 and TNFRSF10B) and gene
Alterations of histone modifications
Fig. 5. Cobalt ions do not affect histone ubiquitinating enzyme activity. (A)
Schematic of the in vitro ubiquitination assay. (B) Histones (5 lg) were
incubated with ubiquitination mix containing 1 lg ubiquitin and 20 lg of cell
lysate from A549 cells, in the absence or presence of NiCl2 (0.1 and 1 mM)
or CoCl2 (0.05 and 0.5 mM). After incubation at 37°C for 1 h, the reaction
was terminated by the addition of SDS–PAGE loading buffer and subjected
to western blot with the antibody against uH2A. The lower panels show the
same membrane reblotted with the antibody against ubiquitin.
transcription (i.e. MAFF and TCF3). Co2þ downregulates the expression of genes involved in tumor suppression (i.e. NBL1 and MTUS1),
as well as other genes such as RAB1B, a member of the Ras oncogene
family, FARSLA Phenylalaine-tRNA synthetase like, NAB1, an EGR1binding protein 1 and USP52.
We also selected some Co2þ-upregulated genes to be validated by
semiquantitative PCR (reverse transcription–PCR). As shown in supplementary Figure 1S (available at Carcinogenesis Online), Co2þ increased the expression of genes in different functional classes, such as
transcriptional activation (i.e. JMJD1A), cell defense (i.e. HMOX1
and BNIP3L) and DNA repair and cell cycle checkpoint control (i.e.
GADD45A).
Discussion
Cobalt compounds are possible human carcinogens, and chemical
toxicity of cobalt has been proven in occupational exposure and oral
intake for medical treatment; however, the underlying mechanisms
are still not clear. Cobalt is a naturally occurring element that has
properties similar to those of iron and nickel. Previous studies have
shown that nickel exposure results in alteration of histone modifications in cells, and thereby disrupts epigenetic homeostasis (39,41). In
this study, we demonstrated that cobalt exposure also changes histone
modifications in A549 and Beas-2B cells. We found that exposure of
A549 and Beas-2B cells to CoCl2 for 24 h increased H3K4me3,
H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B,
but decreased acetylation at histone H4 (AcH4).
Since inhalation is one of the main occupational exposure routes of
cobalt and cobalt exposure causes lung inflammation and tumors in
experimental animals, human lung carcinoma A549 cells and human
bronchial epithelial Beas-2B cells were used in this study. The choice
of the cobalt exposure concentrations used for the treatment of A549
cells was based on previous cell survival data and epidemiological
studies. Davidson et al. (51) demonstrated that after exposed to
400 lM CoCl2 for 24 h, A549 cells still had a viability as high as
87.66 ± 12.55%. Another study reported that the mean of cell viability following exposure of A549 cells to 250 and 500 lM of CoCl2 for
24 h was 86 and 78% (52). To compare the effects between A549
and Beas-2B cells, the same or lower concentrations of Co ions were
used to treat Beas-2B cells. There was no apoptotic effect observed in
either A549 or Beas-2B cells at the concentrations that were utilized.
Urine concentrations have been used to monitor workers’ exposure to
airborne cobalt. Epidemiological studies have shown that in the
cobalt-grinding industry, the average urinary cobalt level among
workers was 0.8–730 lg/l and the maximum was even .10 lM
(53). Animal studies showed that 2 years of exposure to 1 mg/m3
airborne CoSO47H2O significantly increase malignancy in both rats
and mice (30). Therefore, the acute exposure concentration of cobalt
used in this study were reasonable with regard to those levels that
humans are exposed to in the occupational setting.
To study the mechanisms by which cobalt ions increase histone
methylation, A549 cells were preincubated in the methioninedeficient medium prior to exposure to cobalt. Our data demonstrated
that cobalt ions increased H3K4me3 and H3K27me3 by enhancing
the methylation process, as was evident from the finding that cobalt
ions did not increase H3K4me3 or H3K27me3 when the intracellular
methylation process was transiently inhibited by the withdrawal of
methionine from the medium. In contrast, cobalt ions elevated
H3K9me3 and H3K36me3 by inhibiting the demethylation process
since cobalt ions increased H3K9me3 and H3K36me3 even when the
intracellular methylation process was suppressed by the absence of
methionine.
As a major trimethylated H3K9/H3K36 demethylase, the role of
JMJD2A in cobalt-induced H3K9me3 and H3K36me3 was investigated. Our data demonstrate that cobalt ions did not affect JMJD2A protein level but directly inhibited its demethylase activity. JmjC-containing
JMJD2A binds to Fe2þ and a-ketoglutarate at its catalytic-core region
(residues 1–350) and is an iron- and a-ketoglutarate-dependent oxygenase (54). Co and Fe are in the same group in the chemical periodic
table, and they share a number of similar chemical properties. Studies
have suggested that cobalt may replace essential divalent metal ions,
such as magnesium, calcium, iron, copper and zinc, thus affecting
important cellular functions (55). Therefore, it is reasonable to assume that the presence of cobalt ions may also interfere with the
binding and/or function of Fe(II) in JMJD2A. When Co2þ was preincubated with JMJD2A or Fe2þ prior to the addition of other demethylation reaction components, Co2þ inhibited JMJD2A demethylase
activity and prevented the removal of the methyl group from
H3K9me3/H3K36me3 and increased levels of H3K9me3/
H3K36me3, suggesting that cobalt ions compete with iron for binding
to JMJD2A. However, when 100 lM Fe2þ and a-ketoglutarate were
preincubated with 10 ll of JMJD2A on ice for 10 min before the
addition of other demethylation reaction components and CoCl2
(added last), the same amount of Co2þ (100 lM) failed to inhibit
JMJD2A demethylase activity, suggesting that iron and a-ketoglutarate had bound to JMJD2A and when this complex confronted the
substrate (histone with trimethylated H3K9/H3K36), the demethylation reaction had already been initiated, such that the same amount of
cobalt ions failed to replace iron and inhibit the demethylation reaction. These results indicated that cobalt ions increased the levels of
H3K9me3/H3K36me3 by competing with iron for binding to JMJD2A
thus directly inhibiting JMJD2A demethylase activity. Since there are
several demethylases for H3K9me3 and H3K36me3 and JMJD2A is one
of these, cobalt ions may also inhibit other histone demethylases, such as
JMJD2C, to increase the levels of H3K9me3 and H3K36me3.
In this study, exposure of cells to cobalt resulted in a remarkable
increase of H2A and H2B histone ubiquitination demonstrating similar effects as have been found for nickel ions (41). The mechanism by
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Q.Li et al.
Fig. 6. Cobalt ions inhibit histone-deubiquitinating enzyme activity in vitro. (A) Total histones (5 lg) extracted from A549 cells were incubated with
deubiquitination mix containing 20 lg of cell lysate from A549 cells, in the absence or presence of NiCl2 (0.1, 1 and 10 mM) or CoCl2 (0.05 and 0.5 mM). After
incubation at 37°C for 1 h, the reaction was terminated by the addition of SDS–PAGE loading buffer and subjected to western blot with the antibody against uH2A.
The same blots were reblotted with the antibody against ubiquitin (Ub). (B) Histones were incubated with deubiquitination mix without the cell lysate, in the
absence or presence of NiCl2 (0.1 and 1 mM) or CoCl2 (0.05 and 0.5 mM). After incubation at 37°C for 1 h, the reaction was terminated and samples were
processed as above. (C) Histones were incubated with 20 lg of the cell lysate in the absence or presence of NiCl2 (0.1 mM) or CoCl2 (0.05 mM). After incubation
at 37°C for 0.5, 1, 2 or 4 h, the reaction was terminated and samples processed as above. (D) Total histones (5 lg) were incubated with 20 lg of protein extracts
from A549 cells in the absence or presence of NiCl2 (0.1 mM) or CoCl2 (0.05 mM). After incubation at 37°C for 1 h, the reaction was terminated by addition of
SDS–PAGE loading buffer and subjected to western blot with the antibody against ubiquitinated H2B.
which cobalt increases global histone ubiquitination was investigated
further. The results from the in vitro ubiquitination and deubiquitination assays showed that cobalt did not affect histone ubiquitination but
prevented deubiquitination. Cobalt ions may inhibit one of aforementioned histone-deubiquitinating enzymes by unknown mechanisms.
Further studies should focus on the use of recombinant deubiquitinating enzyme to assess the effect of cobalt ions on enzyme deubiquitinating activity in vitro.
The enzyme-catalyzed, post-translational modification of histones
plays an important role in transcriptional processes. Disruption of the
balance of histone modifications in Co-exposed cells could affect the
normal expression of genes and contribute to Co-mediated carcinogenesis. In summary, this study provides the first evidence that cobalt
disrupts epigenetic events by altering histone modifications, providing
a novel view of cobalt’s potential mechanism of carcinogenesis. Further studies should be directed at how cobalt-induced alterations of
histone modifications are involved in Co-mediated carcinogenesis and
which critical genes are involved.
Supplementary material
Supplementary Figure 1S and Tables S1 and S2 can be found at http://
carcin.oxfordjournals.org/
Funding
National Institutes of Environmental Health Sciences (ES014454,
ES005512, ES000260); National Cancer Institute (CA16087).
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Acknowledgements
The authors would like to thank Dr Yi Zhang from the University of North
Carolina at Chapel Hill for providing us with pFastBac HTb-Flag-JMJD2A
bacmid DNA used in this study.
Conflict of Interest Statement: None declared.
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Received September 25, 2008; revised March 31, 2009; accepted April 7, 2008
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