Structural biology of Miz1

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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.