Structural Biology of Long Poly-Zinc Finger Proteins

C2H2锌指(ZF)是一个高度保守的蛋白质结构域，参与转录因子的序列特异性DNA识别。700多个人类基因至少有一个，平均每个基因有8个连续的ZF。ZF由保守的连接子TGEKP-(Y/F)分开。这种连接子采用扩展的构象，并允许建立适当的ZF间界面，以适应DNA和分子识别的主槽。作为我们理解一级结构分歧对长聚ZF蛋白结构生物学影响的长期目标的一部分，我们最近在非正则连接体对DNA结合和扫描模式的影响方面取得了重要发现。简而言之，我们已经揭示了磷化模拟(D)取代T和H取代Y/F导致连接子可逆折叠成a-螺旋构象和ZF间界面的大规模调制。这些现象共同对DNA结合亲和力和结合方式产生了深刻的影响。事实上，一方面，连接物的螺旋构象允许ZF在DNA上的局部解离，另一方面，ZF间界面的调制允许识别DNA的弯曲(或卷曲)构象。在这里，我们将继续我们的长期目标，通过解决以下问题：1-CDK1对连接子中T的磷酸化如何导致有丝分裂时所有多聚ZF转录因子从DNA中清除；2-Krab结构域多聚ZF蛋白(KZNFP)识别螺旋和ZF间界面上进化保守且庞大的色氨酸残基的影响。KZNFP(如ZNF91)通过DNA甲基化(胞嘧啶甲基化：MEC)和组蛋白的甲基化来传播异染色质，以控制组织特异性基因的表达和转座元件(如SVA)的表观遗传沉默。更具体地说，我们提出了一系列具体的目标和培训机会来验证以下假设：1-SP1的三个ZF的两个连接子的磷酸化将导致其同源DNA序列完全失去亲和力，而ZF 1和3的局部解离是由于连接子稳定在a-螺旋构象和2-ZNF91中保守的Trp残基是MEC阅读器，可以稳定弯曲(或卷曲)构象中的甲基化DNA，促进核小体的形成。该方法将包括在我们实验室建立的协议中，包括基于核磁共振的这些现象的结构和动力学表征，以及这些结合事件的定量热力学表征。这些假说的验证将显著影响我们对聚ZF蛋白结构生物学的理解，并提供一个坚实的跨学科培训环境，为HQP在自然科学所有部门的基础研究生涯做准备。

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.