Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation
Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation
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DOI：
10.1111/j.1530-0277.2006.00107.x
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发表时间：
2006-06-01
期刊：
影响因子：
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通讯作者：
Edenberg, Howard J.
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文献类型：
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作者：
Dannenberg, Luke O.;Chen, Hui-Ju;Edenberg, Howard J.
人类I类乙醇脱氢酶（ADH）基因（ADH 1A，ADH 1B和ADH 1C）在发育过程中和各种组织中的表达不同。它们在HepG 2人肝癌细胞系中被抑制。我们假设表观遗传修饰在这种抑制中发挥作用，并且I类ADH基因的表达在DNA甲基化和组蛋白脱乙酰化的全面抑制后会增强。Southern印迹法用于评估每个I类ADH基因的甲基化状态。用DNA甲基化抑制剂5-氮杂-2 '-脱氧胞苷（5-aza-dC）、组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）或两者的组合处理HepG 2和HeLa细胞，并分析I类ADH基因表达。染色质免疫沉淀法分析组蛋白H3乙酰化。瞬时转染和凝胶迁移率改变分析甲基化在抑制转录因子结合和启动子功能中的作用，我们发现在HepG 2细胞中ADH 1A、ADH 1B和ADH 1C的上游区域被甲基化。5-氮杂-2 '-脱氧胞苷处理增强了ADH 1B和ADH 1C的表达。曲古抑菌素A处理增加了ADH 1C的表达。ADH 1A的表达不受5-aza-dC或TSA的刺激。与ADH 1B的甲基化上游区域相关的H3组蛋白在TSA处理的HepG 2细胞中被过度乙酰化，但在5-aza-dC处理的HepG 2细胞中没有。在5-aza-dC或TSA处理后，ADH 1C的甲基化上游区域实现组蛋白H3超乙酰化。体外甲基化使ADH 1B近端启动子的活性降低54%，并抑制了其与上游刺激因子的结合，提示在人肝癌细胞中ADH Ⅰ类基因受表观遗传机制的调控。这些基因的时间和组织特异性表达可能部分是由于表观遗传修饰的差异和关键转录因子的可用性。
The human class I alcohol dehydrogenase (ADH) genes (ADH1A, ADH1B, and ADH1C) differ in expression during development and in various tissues. They are repressed in the HepG2 human hepatoma cell line. We hypothesized that epigenetic modifications play a role in this repression and that class I ADH gene expression would be enhanced upon global inhibition of DNA methylation and histone deacetylation.Southern blotting was used to assess the methylation status of each class I ADH gene. HepG2 and HeLa cells were treated with either the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase inhibitor Trichostatin A (TSA), or both in combination, and class I ADH gene expression was analyzed. Chromatin immunoprecipitation assays were performed to analyze histone H3 acetylation. Transient transfections and gel mobility shift assays were used to analyze the role that methylation plays in inhibiting transcription factor binding and promoter function.We show that the upstream regions of ADH1A, ADH1B, and ADH1C are methylated in HepG2 cells. 5-Aza-2'-deoxycytidine treatment enhanced expression of both ADH1B and ADH1C. Trichostatin A treatment elevated expression of ADH1C. ADH1A expression was not stimulated by either 5-aza-dC or TSA. H3 histones associated with a methylated upstream region of ADH1B were hyperacetylated in TSA-treated, but not in 5-aza-dC-treated, HepG2 cells. A methylated upstream region of ADH1C achieved histone H3 hyperacetylation upon either 5-aza-dC or TSA treatment. Methylation of the ADH1B proximal promoter in vitro decreased its activity to 54% and inhibited the binding of the upstream stimulatory factor.These findings suggest that the class I ADH genes are regulated by epigenetic mechanisms in human hepatoma cells. The temporal and tissue-specific expression of these genes may in part result from differences in epigenetic modifications and the availability of key transcription factors.