E2F1对细胞凋亡及β-catenin/TCF通路作用的研究

【作者】 谢伟；

【导师】 吴缅；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2007， 博士

【摘要】 转录因子E2F1能够调节大量基因的表达，这些E2F1目标基因在细胞周期进程，DNA合成和有丝分裂中都有非常重要的作用。而E2F1的过量表达不仅能强烈驱使细胞进入S期，同时也能诱导细胞凋亡。虽然已有大量文献报道E2F1能够通过上调ARF活性而诱导p53依赖的凋亡，但是对E2F1诱导的p53不依赖的细胞凋亡机制还不清楚。在此，我们发现E2F1能够通过线粒体促凋亡蛋白Smac／DIABLO的启动子区域上两个E2F1结合位点(binding site)BS2(-542～-535bp)和BS3(-200～-193bp)直接结合到Smac／DIABLO的启动子上，并激发其启动子的活性。同时，在我们的实验中发现，BS2和BS3似乎在E2F1的激活和调节中起协同作用。我们还检测了除E2F1以外的其他E2F家族转录激活子，如E2F2和E2F3，发现Smac／DIABLO启动子仅能特异性的被E2F1所激活。然后，我们构建了一个E2F1活性能被条件性激活的细胞H1299ER-E2F1，利用该细胞我们发现，当E2F1活性被激活时，其内源的Smac／DIABLO的mRNA和蛋白水平都随之上调，同时引起线粒体依赖的凋亡。反之，当我们利用RNA干扰技术，抑制了内源性Smac蛋白水平时，相同情况下，E2F1活性上调引起的细胞凋亡也受到削弱。因此，我们的实验结果说明，E2F1能够通过直接上调其下游，如Smac／DIABLO等线粒体凋亡蛋白而诱导不依赖于p53的细胞凋亡通路。转录因子E2F1是细胞活动的重要调节因子，在细胞增殖和凋亡中都具有非常重要的作用。在健康细胞中，E2F1的活性在时空上都受到pRB／E2F通路的严格控制，但在多数肿瘤中，该通路的频繁突变导致E2F1活性的失控，从而引起细胞恶性增殖，因此E2F1被认为是肿瘤生成中的重要原癌基因。同时，E2F1也能诱导细胞凋亡，表现出抑制肿瘤生成的能力。但是，对于E2F通路与细胞中其他重要信号转导通路的相互关系现在还知之甚少。在此，我们发现E2F1能够直接同泛素连接酶Siah1的启动子区域相结合并激活该启动子活性。外源表达的E2F1能够上调Siah1的表达水平而当用RNA干扰技术降低内源E2F1表达水平时，Siah1也随之减少。我们还发现，外源E2F1的表达能够抑制细胞内Wnt／β-catenin／TCF通路活性，该作用在内源Siah1被相应shRNA抑制后明显减弱。综合这些结果，我们认为Siah1是E2F1的目标基因，E2F1能够通过转录上调Siah1的表达量限制Wnt／β-catenin／TCF通路的活性。这可能反应了细胞中该两条通路之间一条潜在的相互约束关系。更多还原

【Abstract】 The E2F1 transcription factor regulates the expression of a large number of genes whose products are important for cell cycle progression, DNA synthesis, and mitosis. Unrestrained expression of E2F1 not only promotes S-phase entry but also induces apoptosis. Although it has been well documented that E2F1 is able to induce p53-dependent apoptosis via raising ARF activity, the mechanism by which E2F induces p53-independent apoptosis remains unclear.Here we report that E2F1 can directly bind to and activate the promoter of Smac/DIABLO, a mitochondrial proapoptotic gene, through the E2F1 binding sites BS2 (-542 ~ -535 bp) and BS3 (-200～-193 bp). BS2 and BS3 appear to be utilized in combination rather than singly by E2F1 in activation of Smac/DIABLO. Activation of BS2 and BS3 are E2F1 -specific, since neither E2F2 nor E2F3 is able to activate BS2 or BS3. Using the H1299 ER-E2F1 cell line where E2F1 activity can be conditionally induced, E2F1 is shown to upregulate the Smac/DIABLO expression at both mRNA and protein levels upon 4-hydroxytamoxifen (4-OHT) treatment, resulting in an enhanced mitochondria-mediated apoptosis. Reversely, reducing the Smac/DIABLO expression by RNA interference significantly diminishes apoptosis induced by E2F1. These results may suggest a novel mechanism by which E2F1 promotes p53-independent apoptosis through directly regulating its downstream mitochondrial apoptosis-inducing factors, such as Smac/DIABLO. Trascrpition factor E2F1 is the key regulator of cell proliferation and apoptosis. In healthy cells, E2F1 activity is strictly controlled by pRB/E2F pathway, which is frequently found mutated in most cancers. Also, E2F1 is reported to take a role in apoptosis. But it is less known what the crosstalks between E2F pathway with other crucial signal transduction pathways are.Here we demonstrate that E2F1 can directly bind to and activate the promoter of a ubiquitin ligase Siah1. The ectopic E2F1 can elevate Siah1 level. The Siah1 expression is reduced when endogenous E2F1 is repressed by shRNA. Ectopic E2F1 can depressβ-catenin/TCF activity, which is attenuated by the repression of endogenous Siah1 expression by shRNA. These results suggest that Siah1 is a bona fide E2F1 target gene and E2F1 can attenuate the activity ofβ-catenin/TCF pathway by upregulating Siah1 expression, which may imply a novel mutual restriction between the important pathways.更多还原