Inhibitors of histone deacetylase and DNA methyltransfcrase synergistically activate the methylated metallothionein I promoter by activating the transcription factor MTF-1 and forming an open chromatin structure
Inhibitors of histone deacetylase and DNA methyltransfcrase synergistically activate the methylated metallothionein I promoter by activating the transcription factor MTF-1 and forming an open chromatin structure
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DOI：
10.1128/mcb.22.23.8302-8319.2002
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发表时间：
2002-12-01
影响因子：
5.3
通讯作者：
Jacob, ST
中科院分区：
文献类型：
--
作者：
Ghoshal, K;Datta, J;Jacob, ST
DNA甲基转移酶（Dnmt）和组蛋白脱乙酰酶（HDAC）抑制剂协同激活小鼠淋巴肉瘤细胞中甲基化金属硫蛋白I基因（MT-I）启动子。这两类抑制剂对MT-I启动子活性的协同作用在通过短暂暴露于5-氮杂胞苷（5-AzaC）仅使少数CpG二核苷酸去甲基化后是稳健的，但即使在用核苷类似物长时间治疗后也持续存在。HDAC抑制剂（阿司他汀A [TSA]和缩酚酸肽）单独或与5-AzaC组合均不促进MT-I启动子的去甲基化。用HDAC抑制剂处理细胞增加了H3和H4历史的多重乙酰化形式的积累，其在用5-AzaC处理后保持不受影响。染色质免疫沉淀（ChIP）测定显示，用TSA处理后，乙酰化组蛋白H4和赖氨酸9（K9）-乙酰基H3与MT-I启动子的结合增加，而用5-AzaC处理后，这种结合没有受到影响。相比之下，在用5-AzaC和TSA处理后，K9-甲基历史113与MT-1启动子的缔合显著降低。用甲基-CpG结合蛋白（MBD）特异性抗体的ChIP测定表明，只有甲基-CpG结合蛋白2（MeCP 2）与MT-I启动子相关，TSA处理后MT-I启动子显著增强。协会的历史脱乙酰基酶I（HDAC 1）与启动子TSA或5-AzaC治疗后减少，并与两种抑制剂治疗后被取消。在DNA甲基转移酶中，Dnmt I和Dnmt 3a都与淋巴肉瘤细胞中的MT-1启动子相关，并且在5-AzaC处理后，Dnmt 1的相关性随着时间的推移而降低。用HDAC抑制剂处理这些细胞也增加了MTF-1（金属转录因子-1）基因的表达及其DNA结合活性。体内基因组足迹研究表明，在用两种抑制剂处理后，MTF-1对MT-1启动子的金属响应元件的占用增加。通过用限制性内切酶在体内作图的启动子的分析表明，与单独用任一种试剂处理相比，用5-AzaC和TSA联合处理后，MT-I启动子获得了更开放的染色质结构。这些结果暗示参与多种因素，包括修改的历史，MBDs，和Dnmts在沉默甲基化的MT-I启动子在淋巴肉瘤细胞。这两种类型的抑制剂对该启动子的协同活化是由于启动子的去甲基化和不同因子的改变的缔合，其导致染色质的重组和启动子对活化的转录因子MTF-1的可及性的结果增加。
Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histories that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl historic 113 with the MT-1 promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of historic deacetylase I (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt I and Dnmt3a were associated with the MT-1 promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of the MT-1 promoter after treatment with both inhibitors. Analysis of the promoter by mapping with restriction enzymes in vivo showed that the MT-I promoter attained a more open chromatin structure after combined treatment with 5-AzaC and TSA as opposed to treatment with either agent alone. These results implicate involvement of multifarious factors including modified histories, MBDs, and Dnmts in silencing the methylated MT-I promoter in lymphosarcoma cells. The synergistic activation of this promoter by these two types of inhibitors is due to demethylation of the promoter and altered association of different factors that leads to reorganization of the chromatin and the resultant increase in accessibility of the promoter to the activated transcription factor MTF-1.