Ghoshal Kalpana, Datta Jharna, Majumder Sarmila, Bai Shoumei, Dong Xiaocheng, Parthun Mark, Jacob Samson T
Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Mol Cell Biol. 2002 Dec;22(23):8302-19. doi: 10.1128/MCB.22.23.8302-8319.2002.
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 histones 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 histone H3 with the MT-I 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 histone deacetylase 1 (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 Dnmt1 and Dnmt3a were associated with the MT-I 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-I 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 histones, 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.
DNA甲基转移酶（Dnmt）抑制剂和组蛋白脱乙酰酶（HDAC）抑制剂可协同激活小鼠淋巴肉瘤细胞中甲基化的金属硫蛋白I基因（MT-I）启动子。仅通过短暂暴露于5-氮杂胞苷（5-AzaC）使少数CpG二核苷酸去甲基化后，这两类抑制剂对MT-I启动子活性的协同作用就很强，并且即使在用核苷类似物进行长时间处理后这种协同作用仍然持续。HDAC抑制剂（曲古抑菌素A [TSA]和缩肽）单独使用或与5-AzaC联合使用均未促进MT-I启动子的去甲基化。用HDAC抑制剂处理细胞会增加H3和H4组蛋白多乙酰化形式的积累，在用5-AzaC处理后这些多乙酰化形式不受影响。染色质免疫沉淀（ChIP）分析显示，用TSA处理后，乙酰化组蛋白H4和赖氨酸9（K9）-乙酰化H3与MT-I启动子的结合增加，在用5-AzaC处理后这一结合不受影响。相反，在用5-AzaC和TSA处理后，K9-甲基化组蛋白H3与MT-I启动子的结合显著减少。用针对甲基-CpG结合蛋白（MBD）的特异性抗体进行的ChIP分析表明，只有甲基-CpG结合蛋白2（MeCP2）与MT-I启动子相关，在用TSA处理后这种相关性显著增强。在用TSA或5-AzaC处理后，组蛋白脱乙酰酶1（HDAC1）与启动子的结合减少，在用两种抑制剂处理后这种结合被消除。在DNA甲基转移酶中，Dnmt1和Dnmt3a都与淋巴肉瘤细胞中的MT-I启动子相关，在用5-AzaC处理后，Dnmt1与启动子的结合随时间减少。用HDAC抑制剂处理这些细胞还会增加金属转录因子-1（MTF-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-I启动子的金属反应元件的占据增加。通过体内用限制酶进行图谱分析对启动子进行分析表明，与单独使用任何一种试剂处理相比，在用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。这些结果表明，包括修饰的组蛋白、MBD和Dnmt在内的多种因素参与了淋巴肉瘤细胞中甲基化的MT-I启动子的沉默。这两种类型的抑制剂对该启动子的协同激活是由于启动子的去甲基化以及不同因子的结合改变，从而导致染色质重组以及启动子对活化转录因子MTF-1的可及性增加。
