Hypoxia induces class III beta-tubulin gene expression by HIF-1α binding to its 3' flanking region
Hypoxia induces class III beta-tubulin gene expression by HIF-1α binding to its 3' flanking region
复制标题
DOI：
10.1016/j.gene.2007.11.015
复制
发表时间：
2008-02-15
期刊：
影响因子：
3.5
通讯作者：
Ferlini, Cristiano
中科院分区：
文献类型：
--
作者：
Raspaglio, Giuseppina;Filippetti, Flavia;Ferlini, Cristiano
III类β-微管蛋白（TUBB 3）过表达代表了对微管相互作用剂如紫杉烷类和苦马豆生物碱的耐药性的主要机制。在这里，我们测试了缺氧作为TUBB 3的可能诱导剂。缺氧对TUBB 3表达的影响在mRNA和蛋白水平上在A2780、其紫杉醇耐药对应物（TC 1）和HeLa细胞中进行监测。缺氧是A2780细胞TUBB 3的强诱导剂，但在TC 1和HeLa细胞中不存在。在A2780中，使用RNA干扰敲低HIF-1 α，并在常氧和缺氧下评估TUBB 3表达。沉默消除了TUBB 3的缺氧依赖性增加，从而证明HIF-1 α介导缺氧中的TUBB 3诱导。为了研究这种现象，将人TUBB 3的5'侧翼区克隆到GFP上游作为报告基因。该区域含有启动子基因，但报告基因的活性不受缺氧的影响。因此，我们观察了3'侧翼区，并且在距终止密码子+168个核苷酸处，使用其中该位点被克隆到GFP下游作为报告基因的构建体，证明了HIF-1 α结合位点在缺氧中是有活性的。在构建体中缺失该位点消除了缺氧时的GFP增强。染色质免疫沉淀揭示了缺氧时该位点与HIF-1 α的结合。该3'增强子的甲基化分析显示，在A2780中70%的细胞中没有甲基化，而在TC 1和HeLa细胞中少于16%，从而表明缺氧时TUBB 3的增加通过3'增强子的甲基化而消除。(C)2007 Elsevier B. V.保留所有权利。
Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1 alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1 alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at + 168 nucleotides from the stop codon, an HIF-1 alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1 alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer. (C) 2007 Elsevier B.V. All rights reserved.