Structural Biology of Long Poly-Zinc Finger Proteins

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The C2H2 Zinc Finger (ZF) is a highly conserved protein domain involved in the sequence specific DNA recognition of transcription factors. More than 700 human genes possess at least one, with an average of 8 consecutive ZFs per gene. ZFs are separated by the conserved linker: TGEKP-(Y/F). This linker adopts an extended conformation and allows the establishment of the proper inter-ZF interface to fit in the major groove of DNA and molecular recognition. As part of our long term goal to comprehend the effect of divergences in the primary structure on the structural biology of long poly-ZF proteins, we have recently made important discoveries about the impact of non-canonical linkers on the mode of DNA binding and scanning. Briefly, we have revealed that the occurrence of phosphomimetics (D) in place of the T and of an H in place of the Y/F leads to the reversible folding of the linker into an a-helical conformation and a large-scale modulation of the inter-ZF interface. Collectively, these phenomena have a profound impact on the DNA binding affinity and mode of binding. Indeed, on the one hand, the helical conformation of the linker allows for local dissociation of ZFs on DNA and on the other hand the modulation of the inter-ZF interface allows for the recognition of bent (or coiled) conformations of DNA. Here, we will continue our long term goal by addressing: 1- how the phosphorylation of the T in the linkers by CDK1 leads to the eviction of all poly-ZF transcription factors from DNA at mitosis and 2- the impact of the evolutionary conserved and bulky Tryptophan residues in the recognition helix and at the inter-ZF interface of Krab domain poly-ZF proteins (KZNFP). KZNFPs (e.g. ZNF91) spread heterochromatin by the provoking methylation of DNA (methylation of cytosines: meC) and histones to control tissue specific gene expression and the epigenetic silencing of transposable elements such as SVAs. More specifically, we propose a series of specific objectives and training opportunities to validate the following hypotheses: 1- That the phosphorylation of the two linkers of the three ZFs of SP1 will lead to the complete loss of affinity of its cognate DNA sequence do the local dissociate of ZFs 1 and 3 due to the stabilization of the linkers in an a-helical conformation and 2- the Trp residues conserved in ZNF91 are meC readers and can stabilize methylated DNA in a bent (or coiled) conformation to promote the formation nucleosomes. The methodology will consist in established protocols in our laboratory and includes NMR-based structural and dynamical characterization of these phenomena as well as a quantitative thermodynamical characterization of these binding events. Validation of these hypotheses will significantly impact on our understanding of the structural biology of poly-ZF proteins as well as to provide a solid inter-disciplinary training environment to prepare HQPs for a career in fundamental research in all sectors of natural sciences.