c-Myc target gene specificity is determined by a post-DNA-binding mechanism
c-Myc target gene specificity is determined by a post-DNA-binding mechanism
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DOI：
10.1073/pnas.95.23.13887
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发表时间：
1998-11-10
影响因子：
11.1
通讯作者：
Farnham, PJ
中科院分区：
文献类型：
--
作者：
Boyd, KE;Wells, J;Farnham, PJ
不确定性的DNA结合转录因子家族的成员调节一个特定的启动子在完整的细胞是一个共同的问题，许多研究人员。确定靶基因特异性需要分析蛋白质与内源性启动子的结合以及转录因子结合的功能后果的表征。通过使用甲醛交联程序和Gal 4融合蛋白，我们分析了Myc和上游刺激因子（USF）1与内源性细胞基因结合的时间和功能后果。我们证明了内源性cad启动子可以与Myc和USF 1的抗体免疫沉淀，我们进一步证明，虽然Myc和USF 1都可以结合到cad，cad启动子可以只响应于Myc反式激活结构域，我们还表明，结合到cad启动子的Myc的量以生长依赖性的方式波动。因此，我们在完整细胞中分析DNA结合和启动子活性的数据表明，cad是Myc靶基因。此外，我们表明Myc结合可以发生在体内的许多位点，但结合位点的位置决定了这种结合的功能后果。我们的数据表明，后DNA结合机制决定Myc靶基因的特异性。重要的是;我们已经证明了分析体内位点特异性转录因子与单拷贝哺乳动物基因结合的可行性。
Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding, By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1, We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain, We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly; we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.