Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing - PubMed (original) (raw)
Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing
Haobin Chen et al. Mol Cell Biol. 2006 May.
Abstract
We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2'-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation.
Figures
FIG. 1.
The changes of global histone H3K9 methylation following nickel ion exposure. (a) A549 cells were exposed to 0.5 mM or 0.75 mM NiCl2 for 24 h. (b) A dose-dependent increase of global H3K9 dimethylation by nickel ions. A549 cells were exposed to various concentrations of NiCl2 for 24 h. (c) A time course study on global H3K9 dimethylation following nickel ion exposure. A549 cells were exposed to 1 mM NiCl2 for selected time intervals as indicated. (d) Exposure to 1 mM NiCl2 for 24 h increased H3K9 dimethylation in different cell types. HOS, human osteosarcoma; MES, murine embryonic stem. Histones were extracted and separated in a 15% SDS-polyacrylamide gel and immunoblotted with various antibodies as indicated. Loading of the histones in all gels was assessed using Coomassie blue staining.
FIG. 2.
Nickel ions decreased histone methyltransferase G9a expression and activity. (a) The protein expression of G9a and Suv39h1 in A549 cells following 1 mM NiCl2 exposure for 24 h. After exposure, whole-cell extracts were prepared and analyzed for the expression of G9a and Suv39h1 by immunoblotting. The membranes were restained with α-tubulin antibody to assess the protein loading. (b) A time course study on G9a expression in nuclei of A549 cells following nickel ion exposure. A549 cells were exposed to 1 mM NiCl2, and the nuclear extracts were prepared at the indicated time intervals. Equal amounts of nuclear extracts were separated in an SDS-polyacrylamide gel and analyzed for the expression of G9a using immunoblotting. The membranes were restained with c-Myc antibody to assess the protein loading. (c) Nickel ions decreased G9a methyltransferase activity. HEK 293 cells were transiently transfected with EGFP, EGFP-hG9a, or EGFP-hG9a ΔSET expression vectors and then exposed to 1 mM NiCl2 for 24 h. The cells were lysed in ice-cold RIPA buffer. The expression of fusion proteins in whole-cell lysates was analyzed by immunoblotting with anti-GFP antibody. The methyltransferase activity of overexpressed proteins was measured as described in Materials and Methods.
FIG. 3.
Nickel ions increased H3K9 dimethylation by inhibiting the histone demethylation process. (a) A549 cells were seeded with F-12-K complete medium. On the second day, cells were replenished with complete DMEM or methionine-deficient DMEM. After incubation for 4 h, cells were then exposed to 1 mM NiCl2 for 24 h. Histones were extracted and immunoblotted for dimethyl-H3K9. BT represents the histones extracted from A549 cells in DMEM before the administration of NiCl2 treatment. (b) Nickel ions decreased the removal rate of histone methylation. A549 cells were radiolabeled with
l
-[methyl-3H]methionine in methionine-deficient DMEM for 24 h and then replenished with F-12-K complete medium supplemented with 1 mM hydroxyurea. After incubation for 24 h, cells were exposed to 1 mM NiCl2 for an additional 48 h in the presence of hydroxyurea. Histones were extracted, and the radioactivity was measured with a scintillation counter. The experiment was conducted in triplicate.
FIG. 4.
Nickel ions inhibited the activity of a putative histone H3K9 demethylase. (a) A549 cells were exposed to hypoxia (0.5% O2 and 99.5% N2), 1 mM DMOG, 100 μM DFX, or 1 mM NiCl2 for 24 h. Histones were extracted and subjected to dimethyl-H3K9 immunoblotting. (b) Nickel ions inhibited the activity of a putative Fe(II)- and 2-oxoglutarate-dependent histone H3K9 demethylation in vitro. The histone H3K9 demethylation assay was performed as described in Materials and Methods. Each condition was used in duplicate. Two independent experiments were performed, and one representative set of results is shown here. The asterisks represent the statistically significant decrease (P < 0.05) compared with the readings in samples with the addition of boiled nuclear extract (H).
FIG. 5.
Nickel ions suppressed the expression of the gpt transgene in G12 cells. (a) G12 cells were exposed to various concentrations of NiCl2 for 24 h. (b) G12 cells were exposed to 100 μM NiCl2 for selected time intervals. Total RNA was isolated, and the expression of the gpt transgene was examined using Northern blotting. The ethidium bromide staining of total RNA was performed to assess the RNA loading.
FIG. 6.
Nickel ions silenced the gpt transgene via epigenetic mechanisms. (a) G12 cells were exposed to various concentrations of NiCl2 for selected time intervals. After a 7-day recovery period, the cells were selected in a medium containing 6-TG (10 μg/ml) for the variants with _gpt_− phenotype. The data represent the values of observable colonies normalized by cell survival rates. (b) The mass population of Ni(II)-induced 6-TGr G12 variants as well as G12 cells was treated with or without 4 μM 5-aza-2′-deoxycytidine for 2 days. After a 7-day recovery period, the cells were selected in HAT medium for gpt + phenotype. The surviving colonies were stained with Giemsa stain.
FIG. 7.
Association of H3K9 dimethylation with the gpt locus following nickel ion exposure. (a) The changes in global H3K9 dimethylation in G12 cells during the course of an extended Ni(II) exposure. G12 cells were exposed to either 50 or 100 μM NiCl2 for selected time intervals. The histones were extracted and immunoblotted for dimethyl-H3K9. (b) Ni(II) exposure increased the occurrence of H3K9 dimethylation at the promoter of the gpt transgene. The chromatin from either 1 × 106 or 1 × 107 cells as indicated was subjected to ChIP assays. Input DNA fractions were amplified by PCR to assess the chromatin loading. A set of representative results is shown from two independent experiments. No HAT, G12 cells without HAT selection for 28 days; NiCl2, G12 cells exposed to 100 μM NiCl2 for 28 days; NiCl2 + 6-TG, G12 cells exposed to 100 μM NiCl2 for 28 days and then selected with 6-TG for 10 days.
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