Structural Biology of Miz-1 and of C2H2 Zinc Fingers

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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.