Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium
Introduction
Cr(VI) is widely utilized in many industries (Levy and Venitt, 1986, Langard, 1990), leading to occupational exposure and contamination of numerous drinking water supplies. Human exposure to Cr(VI) can induce a variety of cytotoxic and genotoxic effects eventually leading to cancer (Dayan and Paine, 2001, Costa and Klein, 2006). For example, epidemiological and risk assessment studies revealed a high incidence of lung cancer following occupational inhalation exposure to Cr(VI) (Gibb et al., 2000). However, the molecular mechanisms of carcinogenicity of Cr(VI) are still an area of intense investigation.
As a potent oxidant, the ability of Cr(VI) to induce oxidative stress and the formation of stable Cr-DNA adducts are thought to mediate a majority of its cytotoxic and genotoxic effects (Zhitkovich, 2005). After entering cells via a sulfate anion transporter, Cr(VI) is subjected to a series of metabolic reductions to form reactive Cr(V) and Cr(IV) intermediates as well as the final stable metabolite Cr(III) (Stearns and Wetterhahn, 1994, Zhitkovich, 2005). These reactive intermediates and final products, in combination with reactive oxygen species (ROS) generated from the reduction process, are able to induce the formation of stable Cr-DNA ternary adducts, protein–DNA cross-links, DNA–DNA cross-links, and DNA single or double-strand breaks (Shi et al., 2004). The Cr(VI)-induced DNA damage can in turn impact upon DNA replication, transcription and translation, resulting in altered gene expression (Sugden and Stearns, 2000). Thus for many years genetic defects mediated by DNA damage have been considered as the key mechanism underlying the genotoxicity and carcinogenicity of Cr(VI).
Recently several studies highlighted the potential epigenetic effects of Cr(VI). The first evidence came from a study in a cell line expressing the bacterial gpt reporter gene (G12) (Klein et al., 2002). Exposure to potassium dichromate was able to induce DNA methylation in the G12 cells and silenced the expression of the gpt transgene (Klein et al., 2002). Recently, treating the Brassica napus L. plants with potassium dichromate induced genome-wide cytosine-hypermethylation in the CCGG-sequence (Labra et al., 2004). Consistent with this finding DNA methylation was increased in the promoter region of the tumor suppressor gene p16 (Kondo et al., 2006) and DNA mismatch repair (hMLH1) (Takahashi et al., 2005) gene in chromate-induced human lung cancers, as compared to the lung cancer in humans without chromate exposure. Since DNA methylation recruits various methylated DNA binding proteins which can inhibit the binding of specific transcription factors to the promoter, it was proposed that Cr(VI) might silence p16 and hMLH1 tumor suppressor genes by inducing DNA methylation in their respective promoter regions. In a more recent study chromate was able to cross-link histone deacetylase 1-DNA methyltransferase 1 complex to chromatin and produce histone silencing marks that prevented aryl hydrocarbon receptor (AHR)-mediated gene transactivation (Schnekenburger et al., 2007). Taken together, these studies suggested that other than genotoxic effects, chromate can also alter epigenetic marks which may contribute to its carcinogenic activity.
As an important epigenetic marker, DNA methylation can be induced in cooperation with other epigenetic modifications (Esteller, 2007). Recent studies revealed that DNA methyltransferases (DNMTs) were associated with the enzymes that modify histone acetylation and methylation (Dobosy and Selker, 2001, Ting et al., 2006), and their ability to induce DNA methylation was associated with the status of specific histone tail modification (Jackson et al., 2002, Tamaru et al., 2003). At least eight histone modifications have been identified. Acetylation, methylation, phosphorylation, and ubiquitination were among the most commonly studied (Strahl and Allis, 2000, Peterson and Laniel, 2004). Unlike DNA methylation in the promoter region that exclusively represses gene transcription, with the exception of histone acetylation, other histone modifications exhibit site-specific but not modification-specific activating or silencing marks. For example, in the promoter region methylation of histone H3 lysine 9 (H3K9) was associated with gene silencing, but methylation of histone H3 lysine 4 (H3K4) was associated with gene activation (Peterson and Laniel, 2004). H3K9 methylation has also been found to be a critical mark for the establishment of DNA methylation and long-term gene silencing (Tamaru et al., 2003, Jackson et al., 2004). Genetic ablation of G9a, a methyltransferase that specifically methylates H3K9, induced genome-wide and locus-specific DNA hypomethylation suggesting a crucial role of H3K9 methylation in the establishment of DNA methylation (Xin et al., 2003). On the other hand methylation of H3K4 protected gene promoters from de novo methylation of DNA (Ooi et al., 2007). Therefore, various histone modifications have distinct impacts on DNA methylation.
While studies on chromate-induced lung cancer suggested that Cr(VI) might silence tumor suppressor genes by inducing DNA methylation in their promoters (Takahashi et al., 2005, Kondo et al., 2006), very little is known about whether Cr(VI) can affect histone marks. In the present study we analyzed both global and gene promoter alterations in histone marks using human bronchial epithelials and lung carcinoma cells exposed to chromate. Our results demonstrated that chromate treatment increased H3K9 dimethylation in the human MLH1 gene promoter, coincident with decreased MLH1 mRNA expression. Thus the capacity of chromate to modulate histone methylation and subsequently silence specific tumor suppressor genes may underlie its carcinogenicity.
Section snippets
Reagents
Potassium chromate (K2CrO4) was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). Antibodies against mono-, di-, tri-methylated H3K9, tri-methylated H3K27, di-methylated H3K4, and G9a were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against di-methylated H3R2, mono- and tri-methylated H3K4 were purchased from Abcam (Cambridge, UK).
Cell culture
Human lung carcinoma A549 cells were cultured in Ham's F-12K medium (Invitrogen, Carlsbad, CA). The medium was supplemented with
Chromate modulates global H3K9 methylation
A number of studies have demonstrated that both H3K9 methylation and DNA methylation were present within the same promoter region of silenced genes suggesting that methylation of H3K9 was associated with DNA methylation and both marks acted to sequentially suppress gene expression. We assessed changes in the global levels of H3K9 methylation induced by Cr(VI).
Human lung carcinoma A549 cells were incubated with 5 or 10 μM of potassium chromate. Cr(VI) can be very rapidly reduced to Cr(III) after
Discussion
It is known that environmental factors have a significant impact upon the epigenetic program of gene expression. Previous studies in our laboratory demonstrated that carcinogenic nickel compounds induced a broad range of epigenetic changes which likely mediate its effects on gene silencing and cell transformation (Zhang et al., 2003, Costa et al., 2005, Chen et al., 2006, Ke et al., 2006). In the current study we report that Cr(VI), another important mutagen and carcinogen also affected various
Acknowledgments
We would like to thank T. Kluz for the technical assistance and Dr. T.P. Ellen for his comments on the manuscript. This work was supported by grant numbers ES000260, ES014454, ES005512, from the National Institutes of Environmental Health Sciences, and grant number CA16087 from the National Cancer Institute.
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These authors contribute equally.