Structural biology of Miz1

Miz1是一种含有13个锌指(Zf)的转录因子。它通过与核心启动子结合，激活关键的细胞抑制基因如p15INK4b(P15)和p21CIP1(P21)的转录。这两个基因是细胞周期停滞程序的基石。然而，在癌蛋白c-Myc存在的情况下，Miz1不能激活p15和p21的转录。结果，细胞周期停滞程序被废除。尽管Miz1在p15和p21的表达中起着关键作用，但尚不清楚13个ZF中的哪些与核心启动子结合，特异性结合如何促进转录激活，以及c-Myc是否(以及如何)影响这一过程。另一方面，研究表明(DNA足迹)p15和p21核心启动子各有两个Miz1结合位点。然而，从简单的建模可以看出，这些位置太远了，不能在不弯曲或卷曲核心启动子的情况下与一个Miz1分子结合。或者，启动子的激活可能需要两个Miz1分子与核心启动子结合。多个ZF转录因子的相互作用也被证明可以弯曲核心启动子。在此背景下，我们提出了一个总体目标如下的研究计划：1-确定与p15和p21核心启动子特异性相互作用的Miz1 ZF，2-阐明特异性的结构决定因素，3-Miz1结合(有和没有c-Myc/Max)对p15和p21核心启动子拓扑的影响的表征。这样的研究计划将产生必要的数据，以揭示Miz1如何与p15和p21核心启动子相互作用，以及c-Myc是否影响Miz1-核心启动子的相互作用。为了达到这些目标，我们将使用蛋白质组学(酶消化-质谱学)、圆二色谱、核磁共振和原子力显微镜。这样一系列实验方法将允许对Miz1-核心启动子相互作用的彻底表征，并为培养高度熟练的结构生物学家提供独特的环境。

Miz1 is a transcription factor that contains 13 Zinc Fingers (ZFs). It activates the transcription of key cytostatic genes such as p15INK4b (p15) et p21CIP1 (p21) by binding to their core promoters. These two genes are cornerstones of the cell cycle arrest program. However, in the presence of the oncoprotein c-Myc, Miz1 is unable to activate the transcription of p15 and p21. As a result, the cell cycle arrest program is abrogated. Despite the critical role of Miz1 in the expression of p15 and p21, it not known which of the 13 ZFs bind to the core promoters, how the specific binding promotes activation of transcription and if (and how) c-Myc influences this process. On the other hand, it has been shown (DNA foot-printing) that the p15 and p21 core promoters possess two Miz1 binding sites each. However, it can be shown from simple modeling that these sites are too far remote to allow the binding of one Miz1 molecule without bending or coiling of the core promoters. Alternately, it is possible that activation of the promoters necessitates that two molecules of Miz1 bind to the core promoters. the interaction of multiple ZF transcription factors has also been shown to bend core promoters. In this context, we propose a research program with the following general Objectives: 1- Determination of the Miz1 ZFs that interact specifically with the p15 and p21 core promoters, 2- Elucidation of the structural determinants of the specificity and 3- Characterization of the effect of Miz1 binding (with and without c-Myc/Max) on the topology of the p15 and p21 core promoters. Such a research program will generate the necessary data to unveil how Miz1 interacts with the p15 and p21 core promoters and if c-Myc affects Miz1-core promoter interactions. In order to reach these objectives, we will use proteomics (enzymatic digests coupled to mass spectrometry), Circular Dichroism, NMR and Atomic force Microscopy. Such a list of experimental approaches will allow for a thorough characterization of the Miz1-core promoter interactions and provide a unique setting for the training of highly proficient structural biologists.