Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions.
Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions.
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DOI：
10.1210/mend.13.8.0323
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发表时间：
1999-08
影响因子：
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通讯作者：
Weili Wang;Lian Dong;B. Saville;S. Safe
中科院分区：
文献类型：
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作者：
Weili Wang;Lian Dong;B. Saville;S. Safe
17β-雌二醇(E_2)刺激人乳腺癌细胞MCF-7增殖和DNA合成，并诱导E2F1基因和蛋白水平的表达。对E2F1基因启动子的分析表明，瞬时转染法中的-146至-54区域是E2应答所必需的，随后的缺失/突变分析表明，E2的反式激活需要一个上游富含GC的位点和两个下游的CCAAT结合位点。多个寡核苷酸和蛋白质抗体的凝胶迁移率改变分析表明，E2F1基因启动子的-146到-54区域结合了MCF-7细胞中的Sp1和NF-Y蛋白。利用野生型和截短型谷胱甘肽S转移酶-Sp1嵌合蛋白，通过蛋白质-DNA结合(开启和关闭速率)、免疫共沉淀和下拉实验，研究了雌激素受体(ER)蛋白促进Sp1与上游GC富集区的相互作用，以及ER、Sp1和ER/Sp1与下游DNA结合的NF-Y的相互作用。结果表明，Sp1蛋白可使NF-Y-DNA结合的Bmax增加5倍以上，与ER共同孵育可使Sp1增强的NF-Y-DNA复合体进一步稳定，解离率(t1/2)降低约50%。SP1抗体与未标记的Sp1蛋白共孵育后免疫共沉淀[35S]NF-YA。因此，E2对MCF-7细胞E2F1基因表达的转录激活是由富含GC的多蛋白ER/Sp1-NF-Y和E2F1基因启动子近端的两个CCAAT元件相互作用调节的。这代表了一个独特的反式作用蛋白复合体，其中内质网依赖于配体的反式激活不依赖于内质网与启动子元件的直接相互作用。
17beta-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the -146 to -54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the -146 to -54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.