Ubc9 and protein inhibitor of activated STAT 1 activate chicken ovalbumin upstream promoter-transcription factor I-mediated human CYP11B2 gene transcription
Ubc9 and protein inhibitor of activated STAT 1 activate chicken ovalbumin upstream promoter-transcription factor I-mediated human CYP11B2 gene transcription
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DOI：
10.1074/jbc.m411820200
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发表时间：
2005-02-25
影响因子：
4.8
通讯作者：
Saruta, T
中科院分区：
文献类型：
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作者：
Kurihara, I;Shibata, H;Saruta, T
醛固酮合成酶(CYP11B2)参与了醛固酮生物合成的最后几步，仅在肾上腺肾小球带细胞中表达。利用凝胶迁移率改变分析，我们证明了COUP-TFI与人CYP11B2启动子的-129/-114元件(Ad5)结合。瞬时转染H295R肾上腺细胞表明COUP-TFI增强了细胞色素P11B2报告基因的活性。然而，带有突变的Ad5序列的报告结构显示基础活性和COUP-TFI增强活性降低，这表明COUP-TFI与Ad5的结合对于CYP11B2的反式激活是重要的。为了阐明Coup-TFI介导活性的分子机制，我们随后使用酵母双杂交系统从人肾上腺cDNA文库中筛选与Coup-TFI相互作用的蛋白，并分别鉴定了Ubc9和PIAS1，它们分别具有小泛素相关修饰物-1(SUMO-1)结合酶和连接酶活性。免疫共沉淀实验证实COUP-TFI在哺乳动物细胞中与Ubc9和PIAS1形成复合体。免疫组织化学显示Ubc9和PIAS1在大鼠肾上腺肾小球细胞中有明显表达。Ubc9和PIAS1的共表达协同增强了COUP-TFI介导的CYP11B2报告基因的活性，表明这两种蛋白都是COUP-TFI的共激活蛋白。然而，Ubc9(C93S)和PIAS1(C351S)突变株仍然作为COUP-TFI的辅助激活因子发挥作用，这表明SUMP-TFI的SUMP活性与辅助激活活性是分开的。此外，染色质免疫沉淀分析表明，异位表达的COUP-TFI、Ubc9和PIAS1被招募到内源CYP11B2启动子。此外，小干扰RNA降低Ubc9或PIAS1蛋白水平可抑制COUP-TFI对CYP11B2的反式激活。我们的数据支持Ubc9和PIAS1作为转录共激活因子在COUP-TFI介导的CYP11B2转录中的生理作用。
Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmuno-precipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.