Structural Biology of Miz-1 and of C2H2 Zinc Fingers

在本研究计划中，我们的目标是为阐明多指C2H2锌指转录因子MiZ-1的结构生物学做出贡献。我们将重点研究MIZ-1与细胞周期抑制因子p15INK4b(P15)和p21CIP1(P21)核心启动子之间的相互作用。MIZ-1直接与这些核心启动子结合，激活p15和p21的转录。人类基因组中有700多个基因编码多指C2H2 ZF蛋白，平均每个基因8个。C2H2 ZF是已知的小模块结构域(~30个残基)，参与识别基因启动子中的三个DNA碱基。另一方面，越来越多的证据表明，C2H2 ZF也参与了蛋白质-蛋白质的相互作用。关于多指ZF转录因子的功能有一种新的范式，这意味着ZF亚群参与DNA结合，其他ZF亚群参与蛋白质-蛋白质相互作用。MIZ-1具有13个C2H2ZF的共有序列，根据这一新的范式，研究表明它的前4个ZF可以与Smad3结合，有助于激活p15和p21的转录。另一方面，目前还不知道哪些ZF与p15和p21核心启动子结合。在此背景下，我们利用DNase I足迹、凝胶迁移率位移分析、核磁共振、等温滴定量热法和原子力显微镜等技术，确定了：1-13个MIZ-1 ZF中哪些ZF与p15和p21核心启动子特异相互作用，2-ZF 1-4和11-13的核磁共振溶液结构(我们已经解决了5-10)以及同源ZF/DNA复合体的结构，3-阐明ZF 1-4与SMAD3的相互作用方式，4-考察MIZ-1结合对p15和p21核心启动子拓扑的影响。这一研究计划将对MiZ-1和C2H2 ZF的结构生物学产生重大影响，并有助于验证多指C2H2 ZF TF的功能新机制。它还将为我们理解p15和p21的转录调控提供关键的新线索，这两个基因可以说是细胞周期中最重要的两个基因。

In this research program, we aim to contribute to the elucidation of the structural biology the polydactyl C2H2 Zinc Finger Transcription Factor Miz-1. We will focus on the interaction between Miz-1 and the core promoters of the cell cycle inhibitors p15INK4b (p15) and p21CIP1 (p21). Miz-1 binds directly to these core promoters and activates the transcription of p15 and p21. More than 700 genes code for polydactyl C2H2 ZF proteins in the human genome with an average of 8 per gene. C2H2 ZFs are small modular domains (~30 residues) known to be involved in the recognition of triplets of DNA bases in gene promoters. On the other hand, there is an increasing body of evidence demonstrating that C2H2 ZFs are also involved in protein-protein interactions. There is an emerging paradigm for the function of polydactyl ZF transcription factors, which implies that subsets of ZFs are involved in DNA binding and others are involved in protein-protein interaction. Miz-1 possesses 13 C2H2 ZFs consensus sequences and in accordance with this new paradigm, it has been show that its first 4 ZFs can bind to Smad3 to contribute in the activation of the transcription of p15 and p21. On the other hand, it is not known which ZFs bind to the p15 and p21 core promoters. In this context and using techniques such as Dnase I footprinting, Electrophoretic Mobility Shift Assays, Nuclear Magnetic Resonance, Isothermal Titration Calorimetry and Atomic Force Microscopy, we propose to determine: 1- Which of the 13 Miz-1 ZFs interact specifically with the p15 and p21 core promoters, 2- The NMR solution structure of ZFs 1 to 4 and 11 to 13 (we have already solved 5-10) and of the cognate ZF/DNA complexes, 3- Elucidate the mode of interaction between ZFs 1 to 4 and Smad3 and 4- investigate of the effect of the binding of Miz-1 on the topology of the p15 and p21 core promoters. This research program will have a significant impact on the structural biology of Miz-1 and C2H2 ZF in general and contribute to validate of a new mechanism for the function of polydactyl C2H2 ZF TFs. It will also shed crucial new light into our understanding of the transcriptional regulation of p15 and p21, arguably, two of the most important genes in the cell cycle.