Yan Yan, Kluz Thomas, Zhang Ping, Chen Hao-bin, Costa Max
Department of Environmental Medicine and The NYU Cancer Institute, New York University School of Medicine, Tuxedo 10987, USA.
Toxicol Appl Pharmacol. 2003 Aug 1;190(3):272-7. doi: 10.1016/s0041-008x(03)00169-8.
We have previously reported that the gpt transgene in G12 Chinese hamster cells could be silenced by water-insoluble nickel compounds nickel sulfide (NiS) or nickel subsulfide (Ni(3)S(2)) and showed that the transgene was silenced by de novo DNA methylation and chromatin condensation. To further understand the nature of this silencing, we used the chromatin immunoprecipitation assay to elucidate the chromatin structure in nickel-induced silenced G12 clones. We also analyzed the effects of the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) and a histone deacetylase inhibitor trichostatin A (TSA) on histone H3 and H4 acetylation and gpt gene expression in selected nickel-silenced clones. We observed that both histone H3 and H4 were hypoacetylated and a methyl DNA-binding protein MeCP2 was targeted to the gpt gene locus, resulting in a localized inactive chromatin configuration in nickel-silenced cell clones. The histone H3K9 was also found methylated in three of four nickel- silenced cell clones, whereas the histone H3K9 was deacetylated in all four cell clones, indicating that the H3K9 methylation was involved in nickel-induced gene silencing. The acetylation of the gpt gene could be increased by a combination of 5-AzaC and TSA treatment, but not by either 5-AzaC or TSA alone. The gpt transcript was studied by either Northern blot or by semiquantitative RT-PCR following treatment of the silenced clones with TSA or 5-AzaC. An increase in gpt mRNA could be detected by RT-PCR in the clones that regained acetylation of H3 and H4. These data show that gene silencing induced by nickel in the gpt transgenic cell line involved a loss of histone acetylation and an activation of histone methylation. Both H4 and H3 histone acetylation were lost in the silenced clones and these clones exhibited an increase in the methylation of the lysine 9 in histone H3.
我们之前报道过，G12中国仓鼠细胞中的gpt转基因可被水不溶性镍化合物硫化镍（NiS）或亚硫化镍（Ni₃S₂）沉默，并且表明该转基因是通过从头DNA甲基化和染色质凝聚而沉默的。为了进一步了解这种沉默的本质，我们使用染色质免疫沉淀试验来阐明镍诱导沉默的G12克隆中的染色质结构。我们还分析了DNA甲基转移酶抑制剂5-氮杂胞苷（5-AzaC）和组蛋白脱乙酰酶抑制剂曲古抑菌素A（TSA）对所选镍沉默克隆中组蛋白H3和H4乙酰化以及gpt基因表达的影响。我们观察到组蛋白H3和H4均低乙酰化，并且一种甲基DNA结合蛋白MeCP2靶向gpt基因位点，导致镍沉默细胞克隆中出现局部无活性染色质构型。在四个镍沉默细胞克隆中的三个中还发现组蛋白H3K9甲基化，而在所有四个细胞克隆中组蛋白H3K9均脱乙酰化，这表明H3K9甲基化参与了镍诱导的基因沉默。5-AzaC和TSA联合处理可增加gpt基因的乙酰化，但单独使用5-AzaC或TSA则不能。在用TSA或5-AzaC处理沉默克隆后，通过Northern印迹或半定量RT-PCR研究gpt转录本。通过RT-PCR可在恢复H3和H4乙酰化的克隆中检测到gpt mRNA增加。这些数据表明，镍在gpt转基因细胞系中诱导的基因沉默涉及组蛋白乙酰化的丧失和组蛋白甲基化的激活。在沉默克隆中，H4和H3组蛋白乙酰化均丧失，并且这些克隆中组蛋白H3赖氨酸9的甲基化增加。
