Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene - Revision of the ARE consensus sequence
Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene - Revision of the ARE consensus sequence
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DOI：
10.1074/jbc.m205225200
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发表时间：
2002-08-23
影响因子：
4.8
通讯作者：
Mulcahy, RT
中科院分区：
文献类型：
--
作者：
Erickson, AM;Nevarea, Z;Mulcahy, RT
编码人谷氨酸半胱氨酸连接酶 (GCLM) 调节亚基的基因的组成型和诱导型表达由启动子的两个区域之一调节； -302:-291 处的抗氧化反应元件 (ARE) 和 ARE 上游的 44 bp 片段 (-346:-303)。第二个区域包括一个共有的 AP-1 位点，之前被认为负责上游片段的增强子活性。包括 ARE 和上游 44 bp 片段在内的 165 bp 片段 (-348:-183) 的删除完全消除了 GCLM 启动子/荧光素酶转基因的叔丁基氢醌 (tBHQ) 诱导能力。突变分析证实，ARE 和 -346:-303 片段都可以支持 tBHQ 暴露后的诱导，但证明后一种情况下的诱导不涉及位于 -341:-335 的 AP-1 位点。在紧邻 AP-1 位点的 44 bp 片段的 5' 端，鉴定出与共有 ARE 序列具有显着同源性的区域，但 -330 处的单核苷酸错配（5'-TTACnnnGCA-3' 与 5'-TGACnnnGCA-3'）除外。该位置上的 G 被认为是功能性 ARE 序列的不变要求。 T -330 突变为 A（已知可消除 ARE 功能的取代）或 C 消除了基础表达和诱导表达。相对于野生型序列，-330 处的 G 取代增强了基础表达，但 tBHQ 暴露后的诱导是可比较的，表明任一序列（5'-TTACnnnGCA-3' 与 5'-TGACnnnGCA-3'）都可以起到 ARE 的作用，尽管前一个序列在指导基础表达方面效果较差。对原型 ARE hNQO1 核心序列的类似突变分析证实了这种可能性。电泳迁移率变动竞争测定表明，5'-TTACnnnGCA-3'序列可以与hNQO1 ARE竞争蛋白质结合，但不如含有5'-TGACnnnGCA-3'基序的类似探针有效。包含 T(-330)A 或 T(-330)C 突变的探针无效。这些结果表明 GCLM 启动子包含两个功能性 ARE，其中一个具有变异序列。结果表明共有ARE序列应修改为5'-RTKAYnnnGCR-3'。
Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T -330 to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.