Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2012 Mar 9;260(3):250–259. doi: 10.1016/j.taap.2012.03.002

Sodium Arsenite Represses the Expression of Myogenin in C2C12 Mouse Myoblast Cells Through Histone Modifications and Altered Expression of Ezh2, Glp, and Igf-1

Gia-Ming Hong 1,3, Lisa J Bain 1,2,*
PMCID: PMC3335964  NIHMSID: NIHMS363228  PMID: 22426358

Abstract

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic–exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (−40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (−40 to +42).

Keywords: Arsenite, Myocyte, Myogenin, Histone, Igf-1, Glp

Introduction

Arsenic is a toxicant commonly found in water systems around the world. Chronic arsenic poisoning is a global health problem affecting millions of people (Cherry, 2008; McDonald, 2007; Medrano et al., 2010; Wang et al., 2009) which can result in cancer, central nervous system and sensory deficits, effects on development, and neuromuscular deficits (Andrew et al., 2007; Benbrahim-Tallaa and Waalkes, 2007; Kozul et al., 2009; Mohammad et al., 2009). Unfortunately, the mechanisms responsible for these multiple adverse outcomes remain largely unclear and likely are multi-factorial.

Arsenic is also a developmental toxicant. In humans and rodents, arsenic can traverse the placenta and this exposure results in adverse developmental effects, such as increased neonatal death and stillbirths (Agusa et al., 2010; Concha et al., 1998; Markowski et al., 2011; Raqib et al., 2009; von Ehrenstein et al., 2006). In fish, arsenite-exposed zebrafish embryos have reduced survival and delayed hatching, malformations in the spinal cord and heart, and disordered motor axon projections (Li et al., 2009). Recently, the negative effects of arsenic on early embryonic development have been reported (Flora and Mehta, 2009; Stummann et al., 2008). Results from mouse embryonic stem cells indicate that ~2μM arsenic inhibits cardiac myocyte differentiation and cardiac beating in the embryonic stem cell test (Stummann et al., 2008). Flora and Mehta have observed, using human stem cells, that 1ppb arsenic reduced the pluripotency of stem cells but also caused a significant down regulation of genes indicative of all the three germ layers (Flora and Mehta, 2009).

Arsenic-mediated adverse effects on muscle differentiation have also been reported. In killifish (Fundulus heteroclitus), arsenic exposed parents had offspring with increased trunk curvatures, which was correlated with changes in myosin light chain, type II keratin, tropomyosin, and parvalbumin expression in the hatchlings (Gonzalez et al., 2006). Arsenic exposure to mouse C2C12 myoblasts delayed their differentiation into myotubes, likely due to a reduction in the expression of myogenin (Steffens et al., 2011). In rodent models, arsenic suppresses the regeneration of injured muscles (Yen et al., 2010), alters pulmonary structure and function in utero by increasing the smooth muscle actin in the lung (Lantz et al., 2009), and disrupts the smooth muscle integrity around the blood vessels in the heart (Hays et al., 2008). Collectively, these results suggest that arsenic acts as a developmental toxicant by affecting the development of the musculature.

The development of skeletal muscle is regulated by several myogenic transcription factors, such as Myo D, myogenin, and myocyte enhancer factor 2 (Mef2). In muscle differentiation, MyoD and Mef2 are early markers, which are expressed during myoblast determination, and they then regulate myogenin, which induces terminal differentiation by converting myoblasts into myotubes (Carvajal and Rigby, 2010; Gianakopoulos et al., 2011; Yokoyama and Asahara, 2011). Moreover, other signaling molecules, such as insulin-like growth factor 1 (Igf-1) and myostatin, regulate myogenin expression via the PI3K/AKT pathway during skeletal muscle differentiation (Alzhanov et al., 2010; Artaza et al., 2002; Yang et al., 2007). In addition, chromatin-modifying enzymes also regulate muscle development by epigenetically repressing myogenic transcription factors (Albert and Peters, 2009; McDonald and Owens, 2007; Ohkawa et al., 2007).

Recently, arsenic-induced alterations in DNA methylation and histone modifications have been suggested to play a role in carcinogenesis and the fetal origins of diseases (Zhou et al., 2008; Zhou et al., 2009; Arita and Costa, 2009; Baccarelli and Bollati, 2009; Ren et al., 2010). Altered DNA methylation may occur since the pathway for biotransformation of arsenic also relies on methylation (Baccarelli and Bollati, 2009; Ren et al., 2010; Vahter, 2009). To this end, studies have shown that arsenic exposure results in both hypermethylation and hypomethylation at global and gene specific levels, thereby leading to aberrant gene expression. For example, mice exposed to arsenic have reduced p16 expression in lung tumors due to hypermethylation of the p16 gene and in humans, arsenic induces DNA hypermethylation in the promoters of the p53 and p16 genes (Benbrahim-Tallaa and Waalkes, 2007; Chanda, 2006; Salnikow and Zhitkovich, 2008; Zhou et al., 2008). Moreover, a significant relationship between arsenic exposure and promoter hypermethylation of two tumor suppressor genes, PRSS3 and RASSF1A, was identified in a population-based study of human bladder cancer (Marsit et al., 2006). In addition to DNA methylation, arsenic also has a role in histone modification. H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3), both markers of gene silencing, were induced at the global level in human lung carcinoma A549 cells upon exposure to 1μM arsenic (Zhou et al., 2008). The increased H3-K9 methylation in the arsenic exposed A549 cells was due to the induction of G9a/Ehmt2 (Zhou et al., 2008). G9a heterodimerizes with Glp/Ehmt1 to form a H3-K9 histone methyltransferase (Tachibana et al., 2005). Acetylation of K9 in histone H3 (H3K9 Ac), which represents transcriptional activation, was reduced by 50% at global level in human UROtsa cells upon exposure to arsenic (3μM) for 7 days (Chu et al., 2011). Moreover, arsenic represses steroid hormone-mediated transcription by disrupting acetylation of K18 in histone H3 (H3K18) at the estrogen-responsive pS2 promoter (Barr et al., 2009). Collectively, these and other reports suggest that arsenic can epigenetically alter gene expression via either DNA methylation or histone modifications.

Our previous study indicated that 20nM sodium arsenite delayed the differentiation of C2C12 mouse myoblast cells by repressing myogenin expression, which was likely due to altered DNA methylation patterns on the myogenin promoter and the decreased nuclear translocation of Mef2 (Steffens et al., 2011). Since the potential regulatory mechanisms responsible for the arsenic-induced delay in muscle differentiation remain largely unclear, the objectives of the present study were to examine whether arsenic-induced abnormal methylation patterns on myogenin promoter would lead to changes in chromatin structure and investigate whether other muscle transcription and growth factors were altered by arsenic exposure. The results indicate reduced Igf-1 expression, coupled with altered histone marks, is likely repressing muscle cell differentiation after arsenic exposure.

Material and methods

Cell culture

C2C12 myoblasts were maintained in growth medium (GM) consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin solution. Differentiation medium (DM) was DMEM containing 2% horse serum, 1% L-glutamine and 1% penicillin/streptomycin solution (Kubo, 1991). For differentiation studies, 15×104 cells were seeded in a 150mm dish with or without 20nM arsenic as sodium arsenite (NaAsO2, certified >99.8%; Fisher Scientific, Pittsburg, PA), and then cultured in growth medium for 3 days (GM3). On day 4, the culture medium was changed to differentiation medium (DM) with or without 20nM arsenic to induce myotube differentiation. Cells were cultured for 2 days (DM2) and then harvested.

Igf-1 mRNA Expression

C2C12 cells were cultured with or without 20nM arsenic as sodium arsenite as described above, harvested at differentiation hour 12, 24, 36, and 48 (n=3 per group per time point), and total RNA extracted. The mRNA expression of Igf-1 was quantified using RT2 SYBR Green Supermix (Qiagen) according to manufacturer’s instructions. The oligonucleotides used for qPCR were Igf-1 Forward: 5′-GAC CGA GGG GCT TTT ACT TCA-3′, Reverse: 5′-GGA CGG GGA CTT CTG AGT CTT-3′; and Gapdh Forward: 5′-TGC GAC TTC AAC AGC AAC TC-3′, Reverse: 5′-ATG TAG GCC ATG AGG TCC AC-3′. Samples were run in triplicate, and relative gene expression was calculated using the comparative threshold (Ct) method (Livak and Schmittgen, 2001).

Chromatin Immunoprecipitation (ChIP)

C2C12 cells were cultured with or without 20nM sodium arsenite as described above and harvested on differentiation day 2 (DM2) (n=4 per group per day). Chromatin was extracted and sonicated using a Vibra Cell probe (Sonic and Materials Inc, Danbury, CT) using 20% power output for 120 seconds and run on a gel to ensure appropriate fragmentation. Both chromatin extractions and immunoprecipitations were performed according to standard protocols (Abcam Inc., Cambridge, MA, http://www.abcam.com/ps/pdf/protocols/x_CHip_protocol.pdf). The antibodies used for ChIP assays were: anti-Ezh2 (#17-622, Millipore Inc., Temecula, CA), anti-Mef2 (sc-313, Santa Cruz Biotech Inc., Santa Cruz, CA), anti-Dnmt3a (IMG-268A, Imgenex Inc., San Diego, CA), anti–Dnmt3b (IMG-184A, Imgenex Inc.), and anti-H3K9 Ac (ab4441, Abcam), -H3K9 Me2 (ab1220, Abcam), and -H3K9 Me3 (ab8898, Abcam). Normal rabbit (sc-2027, Santa Cruz) and mouse IgG (sc-2025, Santa Cruz) were used as negative controls. Quantification of precipitated DNA was performed by qPCR using SYBR green and myogenin promoter-specific primers. The oligonucleotides used for ChIP assays were ChIP 1 Forward: 5′-TAA TCA AAT TAC AGC CGA CGG CCT CC-3′, Reverse: 5′-GCT GCA CAT CAA GAC GTT TCC AGT-3′; ChIP 2 Forward: 5′-CGT CTT GAT GTG CAG CAA CAG CTT-3′, Reverse: 5′-CAT TTA AAC CCT CCC TGC TGG CAT-3′; and ChIP 3 Forward: 5′-GGG TTT AAA TGG CAC CCA GCA GTT-3′, Reverse: 5′-TCA TAC AGC TCC ATC AGG TCG GAA-3′ (see Fig. 2 for specific locations of each ChIP assay). Relative enrichment of the myogenin promoter DNA relative to a matched IgG-antibody control was calculated based on difference in threshold (Ct) values (2[Ctantibody–CtIgG])(Nelson et al., 2006).

Fig. 2. Arsenic exposure alters the histone remodeling status of the myogenin promoter in C2C12 cells.

Fig. 2

Open in a new tab

Schematic diagram of the Myogenin promoter, showing the transcription start site (TSS), Mef2 response element, TATA box, E-box (MyoD binding site), CpG sites, and the locations where the three ChIP assays were performed. Black triangles represent two hypermethylated CpGs at −236 and −126, whereas the white triangle represents a hypomethylated CpG at −207 on myogenin promoter after sodium arsenite exposure to differentiating C2C12 cells (Steffans et al. 2011) (A). ChIP assays were performed with antibodies against di-methylated H3K9 (H3K9 Me2), tri-methylated H3K9 (H3K9 Me3), and acetylated H3K9 (H3K9 Ac) using DNA harvested from DM2 C2C12 cells exposed to 0 or 20nM sodium arsenite. Enriched DNA fragments from these ChIP assays were analyzed by qPCR and expressed as relative fold enrichment after subtraction of the matched IgG negative control. The values from replicates experiments were plotted as the mean ± S.D (n=4) and statistical differences (*) were determined by Student’s t-test (p<0.05) (B).

Immunofluorescence analysis of Ezh2, Glp, MyoD, and Igf-1

C2C12 cells were seeded in Lab-tek II 8-well chamber slides (Nunc) at a concentration of 100 cells/well. To determine the expression of Ezh2 and MyoD, cells were cultured with or without 20nM sodium arsenite as described above and examined for specific protein expression on differentiation days 0 (GM3) and 2 (DM2), while Glp expression was determined on DM2 (n=4 wells per protein per group per day). Igf-1 expression was examined on differentiation day 0 (GM3) 1, 1.5, and 2. Cells were fixed with either 4% paraformaldehyde at room temperature for 20 minutes or methanol at −20 ° C for 5 minutes, blocked in 1% bovine serum albumin, 0.1% Triton-X100 in PBS, and incubated with the appropriate primary antibody for 1 hour at a 1:100 dilution. The antibodies used in the immunofluorescence analysis were: anti-Ezh2 (Millipore Inc., Temecula, CA), anti-MyoD (Santa Cruz), anti-Glp (Abcam), and Igf-1 (Santa Cruz). The secondary antibody (1μg/mL) conjugated to Alexa Flour 488 (Invitrogen, Carlsbad, CA) was incubated with the cells, which were counterstained with DAPI (Invitrogen). Cells were examined by conventional immunofluorescence on a Ti Eclipse Inverted Microscope (Nikon, Melville, NY). Ezh2, Glp, and MyoD expression was quantified by determining the intensities of nuclear staining in six randomly selected fields per well, for a total of four independent wells/time point/group. Intensities were expressed as fold-change relative to control groups. For Igf-1 quantification, the number of nuclei expressing Igf-1 was counted from six randomly selected fields per chamber, for a total of four independent wells/time point/group.

Statistical analysis

Student’s t-test was used for all statistical analysis and p-values of <0.05 were considered to be statistically different.

Results

Arsenic exposure reduces Igf-1 expression in C2C12 cells

Igf-1 is a potent inducer of muscle differentiation (Musaro et al., 1999) and it has been shown that Igf-1 induces myogenin expression by targeting Mef2 and MyoD to the myogenin promoter in C2C12 cells through the Igf-1/PI3K/AKT pathway (Xu and Wu, 2000). To this end, we examined whether arsenic exposure would alter the expression of Igf-1 in C2C12 cells. Immunofluorescence staining showed no significant alteration in Igf-1 expression in myoblasts (GM3) with or without arsenic exposure (Fig. 1A). A time course study was conducted to examine Igf1 expression at DM1 (24 hours), DM1.5 (36 hours), and DM2 (48 hours). Interestingly, increased Igf-1 protein in the nuclei was observed in differentiating myoblasts, compared with undifferentiated myoblasts (see the arrows in Fig. 1A) at DM1 and DM1.5. At DM2, some nuclear localization of Igf-1 could still be seen, but Igf-1 was expressed in a more diffuse pattern throughout the cell. Additionally, a rather a significant reduction in cellular Igf-1 was observed from cells differentiated in 20nM arsenic (Fig. 1A). An 11.8-fold and 5-fold reduction in the number of nuclei expressing Igf-1 was observed in the arsenic-exposed C2C12 cells at DM1 and DM1.5, respectively (Figure 1B). qPCR corroborated the immunofluorescence, showing a significant reduction of Igf-1 mRNA expression by 2.3-fold, 1.8-fold, and 2.1-fold in the arsenic-treated cells at differentiation hour 24, 36, and 48, respectively (Fig. 1C).

Fig. 1. Arsenic exposure reduces Igf-1 expression and nuclear translocation in C2C12 cells.

Fig. 1

Open in a new tab

C2C12 cells were cultured with or without 20nM sodium arsenite in growth medium for 3 days (GM3) and then differentiation medium (DM) for 1, 1.5, or 2 days to examine Igf1 expression by immunofluorescence. The magnifications for GM3, DM1, and DM1.5 are the same, while DM2 are presented as increased magnifications to better show cellular Igf-1 localization. Arrows indicates nuclei expressing Igf-1 (A). The percentage of nuclei expressing Igf-1 was determined by randomly selecting 6 fields per chamber, for a total of four chambers/time point/group. Results were plotted as the mean±S.D. (n=4) and statistical differences (*) determined by Student’s t-test (p<0.05) (B). Igf-1 mRNA expression from C2C12 cells in the presence and absence of 20 nM arsenic in differentiation medium at DM0.5, DM1, DM1.5, and DM2 was quantified by qPCR. Each sample was run in triplicate (n=3 plates/day/group) and results were normalized to GAPDH and expressed as normalized fold-change to relative to C2C12 DM0.5 control cells. Results were plotted as the mean ± S.D. (n=3). Statistical differences (*) were determined by Student’s t-test (p<0.05) (C).

Arsenic exposure alters histone remodeling status on myogenin promoter near the transcription start site in C2C12 cells

Our previous study indicated that 20nM sodium arsenite exposure to C2C12 mouse myocyte cells resulted in delayed differentiation because of a reduction in myogenin expression. We hypothesized that the repressed myogenin expression was due to abnormal DNA methylation patterns on the myogenin promoter (Steffens et al., 2011). Since DNA methylation patterns may affect gene expression through histone remodeling (Bird, 2002) or altering transcription factor availability (Lucarelli, 2000; Palacios et al., 2010), ChIP assays were performed to examine the histone remodeling status on the myogenin promoter after arsenic exposure to C2C12 cells. Markers of heterochromain (H3K9 Me2 and H3K9 Me3) and euchromatin (H3K9 Ac) were examined by looking at three different areas on the myogenin promoter. ChIP 1 encompasses −114 to −251 of the myogenin gene, and arsenic exposure hyper-and hypo-methylates CpG#1 at −236 and CpG#5 at −207, respectively. ChIP 2 encompasses −31 to −128, which contains a hypermethylated CpG#6 at −126 and Mef2 binding site. ChIP 3 encompassess −40 to +42, and contains an E-box at −13 to −18 and the transcription start site (Fig. 2A). Results from ChIP 1 and ChIP 2 showed no significant differences between control and arsenic groups with the three histone markers (Fig. 2B). However, chromatin precipitated from ChIP 3 (−40 to +42 of the myogenin promoter) indicated that H3K9 Me2 and –Me3 were significantly induced by 3-fold (Fig. 2B), which is indicative of increased heterochromatin formation, while H3K9 Ac was reduced by 0.5-fold (Fig. 2B), which is indicative of reduced euchromatin formation in arsenic exposed differentiating C2C12 cells.

Arsenic exposure increases Glp nuclear expression in C2C12 cells

Histone H3 lysine 9 (H3K9) mono-, di-, or tri-methylation can be mediated by several histone methyltransferases (HKMTs), such as G9a and G9a related protein (Glp) (Martin and Zhang, 2005; Kouzarides, 2007). In mammals, G9a and Glp forms a G9a-Glp heteromeric complex that performs H3K9 methylation by targeting euchromatins (Fritsch et al., 2010; Shinkai and Tachibana, 2011; Tachibana et al., 2005). To this end, Glp expression in C2C12 cells was examined on differentiation day 2, which was the day that arsenic-induced H3K9 di- and tri-methylation on myogenin promoter could be observed (Figure 2B). Immunofluorescence staining indicates that the nuclear expression of Glp was significantly increased by ~1.6-fold in cells treated with 20 nM arsenic (Figs. 3A and 3B).

Fig. 3. Arsenic exposure increases Glp nuclear expression in C2C12 cells.

Fig. 3

Open in a new tab

Glp protein expression was examined by immunofluorescence in C2C12 cells exposed with or without 20nM arsenic in the differentiation medium for 2 days (DM2). Pictures are representative examples from 4 independent wells/group (A). Differences in Glp protein expression between control and arsenic-treated cells were determined by measuring the nuclear intensity in 6 randomly selected fields per chamber, for a total of four independent experiments/group. Intensities are expressed as relative nuclear expression and values were plotted as the mean±S.D. (n=4) and statistical differences (*) were determined by Student’s t-test (p<0.05) (B).

Arsenic exposure increases Ezh2 nuclear expression and recruits Ezh2 to the myogenin promoter at the transcription start site

The polycomb Ezh2 methyltransferase (Ezh2) has been reported to play a role in the repression of terminal muscle differentiation by epigenetic mechanisms (Caretti et al., 2004; Juan et al., 2009). Since histone marks were changed by arsenic exposure on the myogenin promoter at the TSS, we next asked whether arsenic exposure would increase Ezh2 expression during muscle differentiation. Immunofluorescence staining indicates that the nuclear localization of Ezh2 was significantly increased by ~2-fold in cells treated with 20 nM arsenic (Figs. 4A and 4B). Additionally, results from ChIP assays also showed a significant 3.3-fold recruitment of Ezh2 to the myogenin promoter surrounding the transcription start site in the arsenic-exposed cells (Fig. 4C). These data indicate that Ezh2-mediated gene silencing is increased after arsenic exposure.

Fig. 4. Arsenic exposure enhances Ezh2 nuclear expression and recruits Ezh2 to the myogenin promoter near the transcription start site.

Fig. 4

Open in a new tab

Ezh2 protein expression was examined by immunofluorescence in C2C12 cells exposed with or without 20nM arsenic in the growth medium for 3 days (GM3) and in the differentiation medium for 2 days (DM2). Pictures are representative examples from 4 independent wells/time point/group (A). Differences in Ezh2 protein expression between control and arsenic-treated cells were determined by measuring the nuclear intensiy in 6 randomly selected fields per chamber, for a total of four independent experiments/time point/group. Intensities are expressed as relative nuclear expression (B). ChIP was performed with antibodies against Ezh2 using C2C12 cells at DM2. Immunoprecipitated DNA was analyzed by qPCR and the data is expressed as relative fold enrichment (C). Results shown in (B) and (C) were plotted as the mean ± S.D.(n=4) and statistical differences (*) were determined by Student’s t-test (p<0.05).

Arsenic exposure recruits Dnmt 3a, but not Dnmt 3b, to the myogenin promoter

Since Ezh2 can silence gene expression by directly recruiting DNA methyltransferases (Dnmts) to the target gene’s promoter (Viré et al., 2006), we next examined the recruitment of Dnmt 3a and Dnmt 3b, which are both de novo DNA methyltransferases (Ling et al., 2004), to the myogenin promoter in cells exposed to 20 nM arsenic. Interestingly, the area surrounding the TSS (ChIP 3) showed a significant 1.9-fold enrichment in Dnmt3a in arsenic exposed cells (Fig. 5A). However, there was no significant difference in the recruitment of Dnmt 3b to the myogenin promoter between control and arsenic treatments (Fig. 5B).

Fig. 5. Ezh2 recruits Dnmt 3a, but not Dnmt 3b, to the myogenin promoter.

Fig. 5

Open in a new tab

ChIP was performed at the TSS (−40 to +42) of the myogenin promoter using antibodies against Dnmt 3a (A) and Dnmt 3b (B) with DNA from C2C12 cells at DM2 exposed to 0 or 20nM arsenic. Enrichment of Dnmts was examined by qPCR and data are expressed as relative fold enrichment. Results were plotted as the mean ± S.D. (n=4) and statistical differences (*) were determined by Student’s t-test (p<0.05).

Arsenic exposure reduces the recruitment of Mef2 to the myogenin promoter

MyoD and Mef2 are two transcription factors that activate myogenin expression by recruiting CBP/p300 co-activator proteins to the myogenin promoter. These co-activators possess histone acetyltransferase (HAT) activity, which results in the relaxation of chromatin structures (Lu et al., 2000; Ohkawa et al., 2006). Our previous study showed a significant reduction in Mef2 nuclear translocation in arsenic-treated C2C12 cells during differentiation (Steffens et al., 2011) and we wanted to determine whether this reduced nuclear translocation was due to reduced recruitment of Mef2 to the myogenin promoter. There is one Mef2 response element in this region (Fig. 2A), so only the primers specific for −31 to −128 (ChIP 2) were used. Mef2 recruitment was indeed reduced by ~70% on the myogenin promoter after arsenic exposure at DM2 (Fig. 6A). The nuclear expression of MyoD was also quantified by immunofluorescence, but there was no change in its expression during the differentiation of arsenic-exposed C2C12 cells (Figs. 6B and 6C).

Fig. 6. Arsenic exposure reduces the recruitment of Mef2 to the myogenin promoter but does not alter MyoD expression.

Fig. 6

Open in a new tab

On DM2 cells cultured with or without 20nM arsenic, ChIP was performed at the ChIP 2 region with antibodies against Mef2. Enriched DNA fragments were analyzed by qPCR and are expressed as relative fold enrichment (A). MyoD expression in C2C12 cells exposed with or without 20nM arsenic at GM3 and DM2 was examined by immunofluorescence (B). MyoD protein expression was quantified by measuring nuclear intensities in 6 randomly selected fields per chamber, for a total of four chambers/time point/group. Intensities were expressed as relative nuclear expression (C). Results shown in (A) and (C) were plotted as the mean ± S.D. (n=4) and statistical differences (*) were determined by Student’s t-test (p<0.05).

Discussion

Results from the present study, using chromatin immunoprecipitation and immunofluorescence staining, illustrate that repressed myogenin expression in arsenic-exposed C2C12 cells is likely due to a combination of reduced Igf-1 expression, increased Glp and Ezh2 nuclear expression, which leads to the altered histone remodeling status on the myogenin promoter (−40 to +42). To our knowledge, this may be the first report that illustrates the effects of nanomolar arsenic concentration on the expression of Glp, Ezh2, and Igf-1 on skeletal muscle development. Additionally, this may also be the first study that has examined the effects of nanomolar arsenic concentration on the histone remodeling status at gene-specific promoter, since to date, studies have examined the effects of arsenic on global histone modifications (Chu et al., 2011; Ramirez et al., 2008; Zhou et al., 2008; Zhou et al., 2009).

Igf-1 is considered to be a myogenic growth factor (Chakravarthy et al., 2000). Results from control C2C12 cells indicate that nuclear Igf-1 expression was increased in a time-dependent manner at DM1 and DM1.5. Two days after the induction of muscle differentiation, the levels of Igf-1 were maximal and the protein was diffusely expressed within the myotubes. However, in arsenic-exposed C2C12 cells, Igf-1 protein and mRNA expression were significantly reduced. There was also pronounced nuclear Igf-1 expression in the control myoblasts at DM1 and DM1.5, which was nearly absent in the arsenic-exposed cells. Indeed, the translocation of Igf-1 into the nuclei has been reported in regenerating human muscle satellite cells, in which Igf-1 protein was detected in the nuclei at 24 hours after the start of regeneration, and then was diffusely expressed in the myofibers after 72 hours (McKay et al., 2008). Other studies have indicated that the Igf-1 receptor (IGF1R) can localize to the nucleus in corneal epithelial cells (Robertson et al., 2012) and in renal and breast cancer cells (Aleksic et al., 2010). Recently, it been shown that nuclear IFG1R functions as a transcriptional co-activator of LEF1/TCF, leading to increases in protein levels of LEF1 target genes (Warsito et al., 2012). Collectively, these studies support the notion that Igf-1 can be trafficked to the nucleus, where it may play a role in initiating skeletal muscle differentiation.

Although the molecular mechanisms about how Igf-1 transcriptionally induces myogenin mRNA are not fully understood, it has been suggested that IGF1/PI3K/AKT target multiple nuclear factors that bind Mef2 and MyoD to PI3K/Akt-responsive elements residing within the 133-bp proximal myogenin promoter (Xu and Wu, 2000). In regenerating human muscle satellite cells, in which Igf-1 nuclear protein expression was increased after 24 hours from the start of regeneration, myogenin mRNA were significantly increased by 2-fold at 24h and ~3-fold after 72 hours (McKay et al., 2008). In contrast, mice lacking Igf-1 have reductions in the number of formed muscle fibers and myogenin expression is significantly lowered (Miyake et al., 2007). Similarly, inhibition of Igf-1 expression in C2C12 cells represses myogenin expression due to recruitment of the polycomb protein Ezh2 and the formation of hypoacetylated and hypermethylated histones on the myogenin promoter between −106 to +91, all of which act to form closed chromatin structures (Serra et al., 2007). In our arsenic-exposed C2C12 cells, we observed the reduction of Igf-1 expression, as well as repressed Mef2 recruitment on the myogenin promoter. Additionally, Ezh2 enrichment, increases in histone marks indicative of heterochromatin and a reduction in histone marks indicative of euchromatin were detected on the myogenin promoter at −40 to +42 following arsenic exposure. Therefore, our results indicate that nanomolar concentrations of arsenic repress myogenin expression through Igf-1-mediated regulatory mechanisms.

The expression of muscle genes during myoblast differentiation is regulated by chromatin structure, in which heterochomatin is present at muscle-regulatory elements in undifferentiated myoblasts and then as the myoblasts enter the differentiation process, the chromatin structure becomes permissive (Guasconi and Puri, 2009; Saccone and Puri, 2010). Our ChIP assays indicate that arsenic-exposed C2C12 cells have a 3-fold increase of repressive chromatins (H3K9 Me2 and H3K9 Me3), and a 50 % reduction of permissive chromatins (H3K9 Ac) at the TSS (−40 to + 42) of the myogenin promoter when they should be undergoing differentiation. GLP is a protein that heterodimerizes with G9a to become an active histone H3-K9 methyltransferase (Fritsch et al., 2010; Shinkai and Tachibana, 2011; Tachibana et al., 2005). Result from our immunofluorescence staining shows that the level of GLP was significantly increased by ~1.6-fold in cells treated with 20 nM arsenic at DM2, which correlates to the increased chromatin H3K9 methylations. Similar finding has been reported in human lung carcinoma A549 cells, in which cells exposed to 1μM sodium arsenite increase their global levels of H3K9 Me2, H3K9 Me3, and G9a (Zhou et al., 2008).

This area of increased heterochomatin is at the 133-bp proximal myogenin promoter region, and encompasses a TATA box, a MyoD site (E-box), and the transcription start site (Buchberger et al., 1994; Yee and Rigby, 1993). The proximal myogenin promoter has been identified as a critical regulatory element for myogenin expression (Buchberger et al., 1994; Deato and Tjian, 2007; Yee and Rigby, 1993). For example, it has been demonstrated that the binding of TRF3/TAF3 (TBP-related factor 3/TATA-binding protein-associated factors 3) to the TATA box and recruitment of RNA Pol II-ser5 to the proximal promoter are both required to activate the myogenin promoter in differentiating C2C12 cells (Deato and Tjian, 2007). Therefore, the altered chromatin conformation by arsenic exposure may affect the accessibility of basal transcription factors to the proximal promoter and thereby result in the reduction of myogenin in differentiating C2C12 cells exposed to arsenic.

To date, several metals have been reported to repress the basal machinery via histone remodeling on promoter regions of some genes. For instance, in mouse Hepa-1 cells, chromium blocks the transcription of the Cyp1a1 gene by inhibiting RNA polymerase II recruitment to the proximal promoter through the reduced acetylation of H3K9 (Schnekenburger et al., 2007). In human lung carcinoma A549 cells and normal bronchial epithelial BEAS-2B cells, chromate silences the expression of the tumor suppressor gene MLH1 via induction of H3K9 methylation in its promoter (Sun et al., 2009). Moreover, nickel silences the gpt (bacterial xanthine guanine phosphoribosyltransferase) transgene in G12 Chinese hamster cells due to increased H3K9 Me2 enrichment (Chen et al., 2006). Therefore, these and other results suggest that arsenic-mediated histone modifications at the myogenin proximal promoter may play a potential regulatory role in reducing myogenin expression.

Ezh2 is another histone methyltransferase enzyme, one whose increased expression has been correlated to reduced myogenin expression in differentiating C2C12 cells (Juan et al., 2009). Consistently, in our arsenic-exposed C2C12 cells, which have been shown to exhibit delayed muscle differentiation due to reductions in myogenin expression (Steffens et al., 2011), the nuclear expression of Ezh2 was significantly enhanced by 2-fold. Therefore, Ezh2 seems to be another potential mechanism involved in abnormal muscle development due to arsenic. Indeed, Ezh2 has been reported as a negative muscle regulator. For example, in dystrophic muscles, muscle regeneration is inhibited by recruitment of Ezh2 and Dnmt3b in muscle satellite cells, which thereby represses Notch-1 expression (Acharyya et al., 2010). Interestingly, results from our ChIPs also demonstrate a significant increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the TSS in arsenic–treated cells. Recruitment of Dnmt3a to the myogenin promoter at the TSS may be a reason why 55% of the CpGs at +3 on the myogenin promoter were methylated in arsenic treatments (Steffens et al., 2011). Such a high methylation rate of CpG at +3 may alter myogenin expression. For example, a recent study has shown that CIBZ, a methyl-CpG-binding protein, suppresses muscle development by directly binding to the myogenin proximal promoter and thus inhibiting myogenin expression (Oikawa et al., 2011). Luo and coworkers have demonstrated, using C2C12 cells, that methyl-CpG-binding protein 2 (MCB2) suppresses muscle terminal differentiation by inducing heterochromatin formation (H3K9 Me2) at the myogenin proximal promoter (Luo et al., 2009). Therefore, these results suggest that the enriched Dnmt3a after arsenic exposure may highly methylate CpG site at +3, thereby recruiting the methyl-CpG-binding proteins, which results in the silencing of the myogenin gene by increasing heterochromatin formation surrounding the TSS of myogenin.

Since Ezh2 has been reported to silence myosin heavy chain (MHC) and muscle creatine kinase (MCK) by enzymatic methylation of H3K27 (Caretti et al., 2004), we also examined the tri-methylation of histone H3K27 on myogenin promoter at TSS. However, there was only a 1.6-fold induction of H3K27 Me3 at TSS in arsenic treatments (data not shown). Therefore, it appears that the induced Ezh2 expression may repress myogenin expression through recruiting Dnmt3a, rather than performing histone H3K27 trimethylation, on myogenin promoter at TSS in arsenic treatments. To date, the regulatory mechanisms about how arsenic exposure induces Ezh2 expression remain largely unclear. Thus, further examinations of arsenic-mediated Ezh2 expression are required in the future.

In conclusion, our results indicate that 20 nM sodium arsenite reduces Igf-1 expression in differentiating C2C12 cells. Such decreased Igf-1 may be responsible for the reduced recruitment of Mef2 on the myogenin promoter, thereby decreasing myogenin expression after arsenic exposure. Additionally, arsenic exposure to C2C12 mouse myocyte cells alters histone remodeling status on the myogenin promoter surrounding the transcription start site, which may reduce the accessibility of basal transcription factors and repress myogenin expression. Moreover, the nuclear expression of Ezh2, which is known as a negative muscle regulator, was enhanced by arsenic exposure in C2C12 cells. Collectively, we conclude that, rather than acting alone, these altered regulatory mechanisms by arsenic exposure seem to be connected and co-contribute to the repressed myogenin gene in C2C12 cells.

Highlights.

* Igf-1 expression is decreased in C2C12 cells after 20nM arsenite exposure.

*Arsenic exposure alters histone remodeling on the myogenin promoter.

*Glp expression, a H3-K9 methyltransferase, was increased in arsenic–exposed cells.

*Ezh2 and Dnmt3a localization to the myogenin promoter is induced by arsenic.

Acknowledgements

We thank Terry Bruce for her help with the immunofluoresence assays. Funding for this study was provided by NIH (ES016640 and ES016640-1S1).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of Interest The authors declare that there are no conflicts of interest.

References

  1. Acharyya S, Sharma SM, Cheng AS, Ladner KJ, He W, Kline W, Wang H, Ostrowski MC, Huang TH, Guttridge DC. TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in Duchenne muscular dystrophy. PLoS ONE. 2010;5:e12479. doi: 10.1371/journal.pone.0012479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agusa T, Kunito T, Kubota R, Inoue S, Fujihara J, Minh TB, Ha NN, Tu NP, Trang PT, Chamnan C, Takeshita H, Iwata H, Tuyen BC, Viet PH, Tana TS, Tanabe S. Exposure, metabolism, and health effects of arsenic in residents from arsenic-contaminated groundwater areas of Vietnam and Cambodia: a review. Rev. Environ. Health. 2010;25:193–220. doi: 10.1515/reveh.2010.25.3.193. [DOI] [PubMed] [Google Scholar]
  3. Aleksic T, Chitnis MM, Perestenko OV, Gao S, Thomas PH, Turner GD, Protheroe AS, Howarth M, Macaulay VM. Type 1 insulin-like growth factor receptor translocates to the nucleus of human tumor cells. Cancer Res. 2012;70:6412–6419. doi: 10.1158/0008-5472.CAN-10-0052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Albert M, Peters AHFM. Genetic and epigenetic control of early mouse development. Curr. Opin. Genet. Develop. 2009;19:113–121. doi: 10.1016/j.gde.2009.03.004. [DOI] [PubMed] [Google Scholar]
  5. Alzhanov DT, McInerney SF, Rotwein P. Long range interactions regulate Igf2 gene transcription during skeletal muscle differentiation. J. Biol. Chem. 2010;285:38969–38977. doi: 10.1074/jbc.M110.160986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Andrew AS, Bernardo V, Warnke LA, Davey JC, Hampton T, Mason RA, Thorpe JE, Ihnat MA, Hamiltone JW. Exposure to arsenic at levels foumd in U. S. drinking water modifies expression in the mouse lung. Toxicol. Sci. 2007;100:75–87. doi: 10.1093/toxsci/kfm200. [DOI] [PubMed] [Google Scholar]
  7. Arita A, Costa M. Epigenetics in metal carcinogenesis: nickel, arsenic, chromium and cadmium. Metallomics. 2009;1:222–228. doi: 10.1039/b903049b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Artaza JN, Bhasin S, Mallidis C, Taylor W, Ma K, Gonzalez-Cadavid NF. Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells. J. Cell. Physiol. 2002;190:170–179. doi: 10.1002/jcp.10044. [DOI] [PubMed] [Google Scholar]
  9. Baccarelli A, Bollati V. Epigenetics and environmental chemicals. Curr. Opin. Pediatrics. 2009;21:243–251. doi: 10.1097/mop.0b013e32832925cc. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Barr FD, Krohmer LJ, Hamilton JW, Sheldon LA. Disruption of histone modification and CARM1 recruitment by arsenic represses transcription at glucocorticoid receptor-regulated promoters. PLoS ONE. 2009;4:e6766. doi: 10.1371/journal.pone.0006766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Benbrahim-Tallaa L, Waalkes MP. Inorganic arsenic and human prostate cancer. Environ. Health Perspect. 2007;116:158–164. doi: 10.1289/ehp.10423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bird A. DNA methylation patterns and epigenetic memory. Genes Develop. 2002;16:6–21. doi: 10.1101/gad.947102. [DOI] [PubMed] [Google Scholar]
  13. Buchberger A, Ragge K, Arnold HH. The myogenin gene is activated during myocyte differentiation by pre-existing, not newly synthesized transcription factor MEF-2. J. Biol. Chem. 1994;269:17289–17296. [PubMed] [Google Scholar]
  14. Caretti G, Di Padova M, Micales B, Lyons GE, Sartorelli V. The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation. Genes Develop. 2004;18:2627–2638. doi: 10.1101/gad.1241904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Carvajal JJ, Rigby PW. Regulation of gene expression in vertebrate skeletal muscle. Exper. Cell Res. 2010;316:3014–3018. doi: 10.1016/j.yexcr.2010.07.005. [DOI] [PubMed] [Google Scholar]
  16. Chakravarthy MV, Davis BS, Booth FW. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J. Appl. Physiol. 2000;89:1365–1379. doi: 10.1152/jappl.2000.89.4.1365. [DOI] [PubMed] [Google Scholar]
  17. Chanda S. DNA hypermethylation of promoter of gene p53 and p16 in arsenic-exposed people with and without malignancy. Toxicol. Sci. 2006;89:431–437. doi: 10.1093/toxsci/kfj030. [DOI] [PubMed] [Google Scholar]
  18. Chen H, Ke Q, Kluz T, Yan Y, Costa M. Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing. Mol. Cell. Biol. 2006;26:3728–3737. doi: 10.1128/MCB.26.10.3728-3737.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Cherry N. Stillbirth in rural Bangladesh: arsenic exposure and other etiological factors: a report from Gonoshasthaya Kendra. Bull. World Health Org. 2008;86:172–177. doi: 10.2471/BLT.07.043083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Chu F, Ren X, Chasse A, Hickman T, Zhang L, Yuh J, Smith MT, Burlingame AL. Quantitative mass spectrometry reveals the epigenome as a target of arsenic. Chem. Biol. Interact. 2011;192:113–117. doi: 10.1016/j.cbi.2010.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Concha G, Vogler G, Lezcano D, Nermell B, Vahter M. Exposure to inorganic arsenic metabolites during early human development. Toxicol. Sci. 1998;44:185–190. doi: 10.1006/toxs.1998.2486. [DOI] [PubMed] [Google Scholar]
  22. Deato MD, Tjian R. Switching of the core transcription machinery during myogenesis. Genes Develop. 2007;21:2137–2149. doi: 10.1101/gad.1583407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Flora SJS, Mehta A. Monoisoamyl dimercaptosuccinic acid abrogates arsenic-induced developmental toxicity in human embryonic stem cell-derived embryoid bodies: Comparison with in vivo studies. Biochem. Pharmacol. 2009;78:1340–1349. doi: 10.1016/j.bcp.2009.07.003. [DOI] [PubMed] [Google Scholar]
  24. Fritsch L, Robin P, Mathieu JR, Souidi M, Hinaux H, Rougeulle C, Harel-Bellan A, Ameyar-Zazoua M. A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex. Mol. Cell. 2010;37:46–56. doi: 10.1016/j.molcel.2009.12.017. [DOI] [PubMed] [Google Scholar]
  25. Gianakopoulos PJ, Mehta V, Voronova A, Cao Y, Yao Z, Coutu J, Wang X, Waddington MS, Tapscott SJ, Skerjanc IS. MyoD directly up-regulates premyogenic mesoderm factors during induction of skeletal myogenesis in stem cells. J. Biol. Chem. 2011;286:2517–2525. doi: 10.1074/jbc.M110.163709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gonzalez HO, Roling JA, Baldwin WS, Bain LJ. Physiological changes and differential gene expression in mummichogs (Fundulus heteroclitus) exposed to arsenic. Aquat. Toxicol. 2006;77:43–52. doi: 10.1016/j.aquatox.2005.10.014. [DOI] [PubMed] [Google Scholar]
  27. Guasconi V, Puri PL. Chromatin: the interface between extrinsic cues and the epigenetic regulation of muscle regeneration. Trends Cell Biol. 2009;19:286–294. doi: 10.1016/j.tcb.2009.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hays AM, Lantz RC, Rodgers LS, Sollome JJ, Vaillancourt RR, Andrew AS, Hamilton JW, Camenisch TD. Arsenic-induced decreases in the vascular matrix. Toxicol. Pathol. 2008;36:805–817. doi: 10.1177/0192623308323919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Juan AH, Kumar RM, Marx JG, Young RA, Sartorelli V. Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. Mol. Cell. 2009;36:61–74. doi: 10.1016/j.molcel.2009.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705. doi: 10.1016/j.cell.2007.02.005. [DOI] [PubMed] [Google Scholar]
  31. Kozul CD, Hampton TH, Davey JC, Gosse JA, Nomikos AP, Eisenhauer PL, Weiss DJ, Thorpe JE, Ihnat MA, Hamilton JW. Chronic exposure to arsenic in the drinking water alters the expression of immune response genes in mouse lung. Environ. Health Perspect. 2009;117:11805–11815. doi: 10.1289/ehp.0800199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kubo Y. Comparison of initial stages of muscle differentiation in rat and mouse myoblastic and mouse mesodermal stem cell lines. J. Physiol. 1991;442:743–759. doi: 10.1113/jphysiol.1991.sp018817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lantz RC, Chau B, Sarihan P, Witten ML, Pivniouk VI, Chen GJ. In utero and postnatal exposure to arsenic alters pulmonary structure and function. Toxicol. Appl. Pharmacol. 2009;235:105–113. doi: 10.1016/j.taap.2008.11.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li D, Lu C, Wang J, Hu W, Cao Z, Sun D, Xia H, Ma X. Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos. Aquatic Toxicol. 2009;91:229–237. doi: 10.1016/j.aquatox.2008.11.007. [DOI] [PubMed] [Google Scholar]
  35. Ling Y, Sankpal UT, Robertson AK, McNally JG, Karpova T, Robertson KD. Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription. Nucleic Acids Res. 2004;32:598–610. doi: 10.1093/nar/gkh195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  37. Lu J, McKinsey TA, Zhang CL, Olson EN. Regulation of skeletal myogenesis by association of the MEF2 transcription factor with class II histone deacetylases. Mol. Cell. 2000;6:233–244. doi: 10.1016/s1097-2765(00)00025-3. [DOI] [PubMed] [Google Scholar]
  38. Lucarelli M. The dynamics of myogenin site-specific demethylation Is strongly correlated with its expression and with muscle differentiation. J. Biol, Chem. 2000;276:7500–7506. doi: 10.1074/jbc.M008234200. [DOI] [PubMed] [Google Scholar]
  39. Luo SW, Zhang C, Zhang B, Kim CH, Qiu YZ, Du QS, Mei L, Xiong WC. Regulation of heterochromatin remodelling and myogenin expression during muscle differentiation by FAK interaction with MBD2. EMBO J. 2009;28:2568–2582. doi: 10.1038/emboj.2009.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Markowski VP, Currie D, Reeve EA, Thompson D, Sr, J.P.W. Tissue-specific and dose-related accumulation of arsenic in mouse offspring following maternal consumption of arsenic-contaminated water. Basic Clin. Pharmacol. Toxicol. 2011;108:326–332. doi: 10.1111/j.1742-7843.2010.00660.x. [DOI] [PubMed] [Google Scholar]
  41. Marsit CJ, Karagas MR, Danaee H, Liu M, Andrew A, Schned A, Nelson HH, Kelsey KT. Carcinogen exposure and gene promoter hypermethylation in bladder cancer. Carcinogenesis. 2006;27:112–116. doi: 10.1093/carcin/bgi172. [DOI] [PubMed] [Google Scholar]
  42. Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nat. Rev. Mol. Cell Biol. 2005;6:838–849. doi: 10.1038/nrm1761. [DOI] [PubMed] [Google Scholar]
  43. McDonald C. Risk of arsenic-related skin lesions in Bangladeshi villages at relatively low exposure: a report from Gonoshasthaya Kendra. Bull. World Health Orga. 2007;85:668–673. doi: 10.2471/BLT.06.036764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. McDonald OG, Owens GK. Programming smooth muscle plasticity with chromatin dynamics. Circ. Res. 2007;100:1428–1441. doi: 10.1161/01.RES.0000266448.30370.a0. [DOI] [PubMed] [Google Scholar]
  45. McKay BR, O’Reilly CE, Phillips SM, Tarnopolsky MA, Parise G. Co-expression of IGF-1 family members with myogenic regulatory factors following acute damaging muscle-lengthening contractions in humans. J. Physiol. 2008;586:5549–5560. doi: 10.1113/jphysiol.2008.160176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Medrano MA, Boix R, Pastor-Barriuso R, Palau M, Damian J, Ramis R, Del Barrio JL, Navas-Acien A. Arsenic in public water supplies and cardiovascular mortality in Spain. Environ.. Res. 2010;110:448–454. doi: 10.1016/j.envres.2009.10.002. [DOI] [PubMed] [Google Scholar]
  47. Miyake M, Hayashi S, Sato T, Taketa Y, Watanabe K, Hayashi S, Tanaka S, Ohwada S, Aso H, Yamaguchi T. Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice. Cell Biol. Int. 2007;10:1274–1279. doi: 10.1016/j.cellbi.2007.05.007. [DOI] [PubMed] [Google Scholar]
  48. Mohammad MM, Jack CN, Ravi N. Chronic exposure of arsenic via drinking water and its adverse health impacts on humans. Environ. Geochem. Health. 2009;31:189–200. doi: 10.1007/s10653-008-9235-0. [DOI] [PubMed] [Google Scholar]
  49. Musaro A, McCullagh KJA, Naya FJ, Olson EN, Rosenthal N. IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature. 1999;400:581–585. doi: 10.1038/23060. [DOI] [PubMed] [Google Scholar]
  50. Nelson JD, Denisenko O, Bomsztyk K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nature Prot. 2006;1:179–185. doi: 10.1038/nprot.2006.27. [DOI] [PubMed] [Google Scholar]
  51. Ohkawa Y, Marfella CG, Imbalzano AN. Skeletal muscle specification by myogenin and Mef2D via the SWISNF ATPase Brg1. EMBO J. 2006;25:490–501. doi: 10.1038/sj.emboj.7600943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ohkawa Y, Yoshimura S, Higashi C, Marfella CG, Dacwag CS, Tachibana T, Imbalzano AN. Skeletal muscle specification by myogenin. J. Biol. Chem. 2007;282:6564–6570. doi: 10.1074/jbc.M608898200. [DOI] [PubMed] [Google Scholar]
  53. Oikawa Y, Omori R, Nishii T, Ishida Y, Kawaichi M, Matsuda E. The methyl-CpG-binding protein CIBZ suppresses myogenic differentiation by directly inhibiting myogenin expression. Cell Res. 2011:1–13. doi: 10.1038/cr.2011.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Palacios D, Summerbell D, Rigby PWJ, Boyes J. Interplay between DNA methylation and transcription factor availability: implications for developmental activation of the mouse myogenin gene. Mol. Cell.Biol. 2010;30:3805–3815. doi: 10.1128/MCB.00050-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Ramirez T, Brocher J, Stopper H, Hock R. Sodium arsenite modulates histone acetylation, histone deacetylase activity and HMGN protein dynamics in human cells. Chromosoma. 2008;117:147–157. doi: 10.1007/s00412-007-0133-5. [DOI] [PubMed] [Google Scholar]
  56. Raqib R, Ahmed S, Sultana R, Wagatsuma Y, Mondal D, Hoque AM, Nermell B, Yunus M, Roy S, Persson LA, Arifeen SE, Moore S, Vahter M. Effects of in utero arsenic exposure on child immunity and morbidity in rural Bangladesh. Toxicol. Lett. 2009;185:197–202. doi: 10.1016/j.toxlet.2009.01.001. [DOI] [PubMed] [Google Scholar]
  57. Ren X, McHale CM, Skibola CF, Smith AH, Smith MT, Zhang L. An emerging role for epigenetic dysregulation in arsenic toxicity and carcinogenesis. Environ. Health Perspect. 2010;119:11–19. doi: 10.1289/ehp.1002114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Robertson DM, Zhu M, Wu Y-C. Cellular distribution of the IGF-1R in corneal epithelial cells. Exp. Eye Res. 2012;94:179–186. doi: 10.1016/j.exer.2011.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Saccone V, Puri PL. Epigenetic regulation of skeletal myogenesis. Organogenesis. 2010;6:48–53. doi: 10.4161/org.6.1.11293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Salnikow K, Zhitkovich A. Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium. Chem. Res. Toxicol. 2008;21:28–44. doi: 10.1021/tx700198a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Schnekenburger M, Talaska G, Puga A. Chromium cross-links histone deacetylase 1-DNA methyltransferase 1 complexes to chromatin, inhibiting histone-remodeling marks critical for transcriptional activation. Mol. Cell. Biol. 2007;27:7089–7101. doi: 10.1128/MCB.00838-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Serra C, Palacios D, Mozzetta C, Forcales SV, Morantte I, Ripani M, Jones DR, Du K, Jhala US, Simone C, Puri PL. Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation. Mol. Cell. 2007;28:200–213. doi: 10.1016/j.molcel.2007.08.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Shinkai Y, Tachibana M. H3K9 methyltransferase G9a and the related molecule GLP. Genes Develop. 2011;25:781–788. doi: 10.1101/gad.2027411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Steffens AA, Hong GM, Bain LJ. Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin. Toxicol. Appl. Pharmacol. 2011;250:154–161. doi: 10.1016/j.taap.2010.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Stummann T, Hareng L, Bremer S. Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells. Toxicol. 2008;252:118–122. doi: 10.1016/j.tox.2008.08.001. [DOI] [PubMed] [Google Scholar]
  66. Sun H, Zhou X, Chen H, Li Q, Costa M. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium. Toxicol. Appl. Pharmacol. 2009;237:258–266. doi: 10.1016/j.taap.2009.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Tachibana M, Ueda M, Takeda N, Ohta T, Iwanari H, Sakihama T, Kodama T, Hamakubo T, Shinkai Y. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Develop. 2005;19:815–826. doi: 10.1101/gad.1284005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Vahter M. Effects of arsenic on maternal and fetal health. Ann. Rev. Nutrition. 2009;29:381–399. doi: 10.1146/annurev-nutr-080508-141102. [DOI] [PubMed] [Google Scholar]
  69. Viré E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden J-M, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F. The Polycomb group protein EZH2 directly controls DNA methylation. Nature. 2006;439:871–874. doi: 10.1038/nature04431. [DOI] [PubMed] [Google Scholar]
  70. von Ehrenstein OS, Guha Mazumder DN, Hira-Smith M, Ghosh N, Yuan Y, Windham G, Ghosh A, Haque R, Lahiri S, Kalman D, Das S, Smith AH. Pregnancy outcomes, infant mortality, and arsenic in drinking water in West Bengal, India. Amer. J. Epidemiol. 2006;163:662–669. doi: 10.1093/aje/kwj089. [DOI] [PubMed] [Google Scholar]
  71. Wang CH, Chen CL, Hsiao CK, Chiang FT, Hsu LI, Chiou HY, Hsueh YM, Wu MM, Chen CJ. Increased risk of QT prolongation associated with atherosclerotic diseases in arseniasis-endemic area in southwestern coast of Taiwan. Toxicol. Appl. Pharmacol. 2009;239:320–324. doi: 10.1016/j.taap.2009.06.017. [DOI] [PubMed] [Google Scholar]
  72. Warsito D, Sjostrom S, Andersson S, Larsson O, Sehat B. Nuclear IGF1R is a transcriptional co-activator of LEF1/TCF. EMBO Rep. 2012 doi: 10.1038/embor.2011.251. Epub. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Xu Q, Wu Z. The insulin-like growth factor-phosphatidylinositol 3-kinase-Akt signaling pathway regulates myogenin expression in normal myogenic cells but not in rhabdomyosarcoma-derived RD cells. J. Biol. Chem. 2000;275:36750–36757. doi: 10.1074/jbc.M005030200. [DOI] [PubMed] [Google Scholar]
  74. Yang W, Zhang Y, Li Y, Wu Z, Zhu D. Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3β pathway and is antagonized by insulin-like growth factor 1. J. Biol. Chem. 2007;282:3799–3808. doi: 10.1074/jbc.M610185200. [DOI] [PubMed] [Google Scholar]
  75. Yee SP, Rigby PW. The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Develop. 1993;7:1277–1289. doi: 10.1101/gad.7.7a.1277. [DOI] [PubMed] [Google Scholar]
  76. Yen YP, Tsai KS, Chen YW, Huang CF, Yang RS, Liu SH. Arsenic inhibits myogenic differentiation and muscle regeneration. Environ. Health Perspect. 2010;118:949–956. doi: 10.1289/ehp.0901525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Yokoyama S, Asahara H. The myogenic transcriptional network. Cell. Mol. Life Sci. 2011 doi: 10.1007/s00018-011-0629-2. DOI 10.1007/s00018-00011-00629-00012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Zhou X, Sun H, Ellen TP, Chen H, Costa M. Arsenite alters global histone H3 methylation. Carcinogenesis. 2008;29:1831–1836. doi: 10.1093/carcin/bgn063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Zhou X, Li Q, Arita A, Sun H, Costa M. Effects of nickel, chromate, and arsenite on histone 3 lysine methylation. Toxicol. Appl. Pharmacol. 2009;236:78–84. doi: 10.1016/j.taap.2009.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES