An estrogen receptor-Lu splicing variant mediates both positive and negative effects on gene transcription
An estrogen receptor-Lu splicing variant mediates both positive and negative effects on gene transcription
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DOI：
10.1210/me.14.5.634
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发表时间：
2000-05-01
影响因子：
--
通讯作者：
Miksicek, RJ
中科院分区：
文献类型：
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作者：
Bollig, A;Miksicek, RJ
对各种雌激素反应细胞系、乳腺肿瘤标本和正常乳腺组织制备的mRNA的分析表明，雌激素受体-α(ERα)mRNA通常以转录本的混合形式表达。利用PCR扩增，这种异质性已被证明主要是由于不精确的mRNA剪接模式导致了一系列正确处理和跳过外显子的ERα转录本。我们已经重建了代表单个外显子跳过的变异体ER Delta E2到ER Delta E7的ERαcDNA，以使它们能够在定义良好的细胞转染系统中进行功能表征。所有六个ERα剪接变异体都支持稳定蛋白在Cos7细胞中的有效表达，每个剪接变异体都显示出独特的亚细胞分布模式。在体外凝胶迁移率变化分析中，每个变体都显示出与一致的雌激素反应元件(ERE)的DNA结合活性显著降低。虽然ER Delta E2、ER Delta E3、ER Delta E4和ER Delta E6的DNA结合缺陷似乎是完全的，但ER Delta E5和ER Delta E7的DNA结合很弱。Scatchard对激素结合的分析表明，在这些变体中，只有ER Delta E3与17β-雌二醇(E-2)结合，并且与野生型ERα(wt ERα)具有类似的亲和力。与Pere-TK-CAT报告基因[公认的ERE驱动的氯霉素乙酰转移酶(CAT)报告基因，对E-2连接的wt ERα高度反应]共转染的单个变体在ER阴性的HeLa细胞中诱导CAT表达无效。只有ER Delta E5在Pere-TK-CAT报告上显示出阳性转录活性，但这种活性被限制在wt ERα活性的大约5%。当突变体与wt ERα同时表达时，观察到ER Delta E3和ER Delta E5对wt ERα转录活性有显性的负面影响。与野生型受体一样，ER Delta E3和ER Delta E5在体外都与类固醇受体辅活化子-1e(SRC-1e)相互作用；然而，只有ER Delta E3保留了与wt ERα二聚的能力。在佛波酯处理的瞬时转染的HeLa细胞中，来自卵清蛋白启动子区域的转录受到连接的wt ERα和ER Delta E3的正调控，该区域包含一个ERE半位点和一个AP-1基序。在这两种情况下，共转染的cJun都增强了这一活性。这些观察表明，选定的ERα剪接变异体可能发挥重要的转录效应，特别是在受非共识ERE调控和复杂激素控制的基因上。
Analysis of mRNA prepared from a variety of estrogen-responsive cell lines, breast tumor specimens, and normal breast tissue have established that estrogen receptor-alpha (ER alpha) mRNA is typically expressed as a mixture of transcripts. Using PCR amplification, this heterogeneity has been shown to result largely from an imprecise pattern of mRNA splicing that gives rise to a family of correctly processed and exon-skipped ER alpha transcripts. We have reconstructed ER alpha cDNAs representing the single exon-skipped variants ER Delta E2 through ER Delta E7 to enable their functional characterization in a well defined cell transfection system. All six of the ER alpha splicing variants support the efficient expression of stable proteins in Cos7 cells, and each shows a characteristic pattern of subcellular distribution. Each of the variants displays a dramatic reduction in DNA-binding activity with a consensus estrogen response element (ERE) in an in vitro gel mobility shift assay. While this DNA-binding defect appears to be complete for ER Delta E2, ER Delta E3, ER Delta E4, and ER Delta E6, weak DNA binding is observed for ER Delta E5 and ER Delta E7. Scatchard analysis of hormone binding demonstrates that among the variants, only ER Delta E3 binds 17 beta-estradiol (E-2) and does so with an affinity similar to wild-type ER alpha (wt ER alpha). Individual variants cotransfected with the pERE-TK-CAT reporter plasmid [a consensus ERE-driven chloramphenicol acetyltransferase (CAT) reporter gene that is highly responsive to E-2-liganded wt ER alpha] were ineffective at inducing CAT expression in ER-negative HeLa cells. Only ER Delta E5 showed indications of positive transcriptional activity on the pERE-TK-CAT reporter, but this activity was limited to approximately 5% of the activity of wt ER alpha. When variants were expressed simultaneously with wt ER alpha, ER Delta E3 and ER Delta E5 were observed to have a dominant negative effect on wt ER alpha transcriptional activity. Like the wild-type receptor, both ER Delta E3 and ER Delta E5 interact with steroid receptor coactivator-1e (SRC-1e) in vitro; however, only ER Delta E3 retained the ability to dimerize with wt ER alpha. Transcription from a region of the ovalbumin promoter, which contains an ERE half-site and an AP-1 motif, is positively regulated by liganded wt ER alpha and ER Delta E3 in phorbol ester-treated, transiently transfected HeLa cells. In both cases, this activity was enhanced by cotransfected cJun. These observations suggest that selected ER alpha splicing variants are likely to exert important transcriptional effects, especially on genes that are regulated by nonconsensus EREs and subject to complex hormonal control.