Dissecting the Mechanisms of Pioneer Factor Facilitated Chromatin Opening

项目摘要 核小体覆盖了大部分真核生物基因组，并为许多核小体的有效结合提供了屏障。 控制基因表达的调节蛋白。一组特殊的转录因子称为先锋因子， 结合核小体DNA上的靶位点，使染色质疏松，并促进其他染色质因子的活性 和酶，导致核小体重新定位和基因激活。Sox 2和Oct 4是先锋因子， 在胚胎发生和成体神经发生过程中协调细胞命运的变化，并且是 将体细胞重新编程为多能状态。这些蛋白质的突变和缺陷与 神经系统疾病，而它们的富集与癌症有关。尽管有大量的研究，但人们对如何做到这一点知之甚少。 到Sox 2、Oct 4等先锋因子在分子水平发挥其先锋活性。我们的目标是 解决Sox 2和Oct 4如何以不同的结合模式与核小体结合的关键未解决问题 并改变核小体的结构和动力学，以及这些变化如何影响核小体的下游作用。 染色质修饰和重塑酶。为了实现这一目标，我们建议将化学探测 使用高分辨率核磁共振（NMR）光谱进行测定以获得位点特异性结构 以及先锋因子与核小体相互作用的动态信息，以及它们在细胞中引起的扰动。 DNA和组蛋白可能调节其他因素。在目标1中，我们将利用体外结合试验， 表征Sox 2和Oct 4与人工和天然核小体的不同结合模式及其依赖性 对DNA序列、辅因子协同性和组蛋白组成的影响。我们还将调整甲基TROSY NMR， 适用于超大型生物分子的研究，以询问位点特异性结合构象， 这些蛋白质的动力学来选择具有原子细节的核小体。在目标2中，我们将研究Sox 2的作用。 和Oct 4在核小体不同位置的结合对核小体DNA构象和可及性的影响 和无序的组蛋白尾部通过使用NMR，化学和酶的探测方法敏感的结构 和动态扰动。我们将进一步评估Sox 2和10月4日是否通过直接接触， 核小体扰动，刺激组蛋白甲基转移酶的活性， 与这些先驱因子在体内合作。最后，在目标3中，我们将调整我们的方法来描述如何 一种甲基-DNA结合蛋白和提议的先锋因子Kaiso结合并改变核小体， 深入了解染色质调控中表观遗传DNA标记的识别和功能。拟议工作 将奠定基础，为今后的调查其他先驱因素和作用的DNA背景下，协同作用， 组蛋白变体和表观遗传修饰转化为先锋因子介导的染色质开放。这将改善 我们的能力，修改先锋因子的功能，并导致新的方法，为治疗的目标。

Project Summary Nucleosomes cover most of the eukaryotic genome and present a barrier for the competent binding of many regulatory proteins that control gene expression. A special set of transcription factors called pioneer factors can bind their target sites on nucleosomal DNA, loosen chromatin, and promote the activity of other chromatin factors and enzymes, leading to nucleosome repositioning and gene activation. Sox2 and Oct4 are pioneer factors that orchestrate changes in cell fate during embryogenesis and adult neurogenesis and are major players in the reprogramming of somatic cells into a pluripotent state. Mutation and deficiency in these proteins are linked to neurological disorders, while their enrichment is tied to cancer. Despite ample studies, little is known about how to Sox2, Oct4 and other pioneer factors exert their pioneering activity at the molecular level. Our goal is to address key unresolved questions about how Sox2 and Oct4 engage with nucleosomes in distinct binding modes and alter the structure and dynamics of nucleosomes, and how these changes affect the downstream action of chromatin modifying and remodeling enzymes. Towards this goal, we propose to integrate chemical probing assays with high-resolution nuclear magnetic resonance (NMR) spectroscopy to obtain site-specific structural and dynamic information about pioneer factor interaction with nucleosomes and the perturbations they elicit in DNA and histones to potentially regulate other factors. In Aim 1, we will utilize in vitro binding assays to characterize distinct binding modes of Sox2 and Oct4 to artificial and natural nucleosomes and their dependence on DNA sequence, co-factor cooperativity, and histone composition. We will also adapt methyl TROSY NMR, suitable for studies of super-large biomolecules, to interrogate the site-specific binding conformation and dynamics of these proteins to select nucleosomes with atomic detail. In Aim 2, we will examine the effect of Sox2 and Oct4 binding in various nucleosome positions on the conformation and accessibility of nucleosomal DNA and the disordered histone tails by using NMR, chemical and enzymatic probing methods sensitive to structural and dynamic perturbations. We will further assess whether Sox2 and Oct4, through direct contact and nucleosome perturbations, stimulate the activity of histone methyltransferase enzymes that are found to collaborate in vivo with these pioneer factors. Finally, in Aim 3, we will adapt our approach to characterize how a methyl-DNA binding protein and proposed pioneer factor Kaiso binds to and alters nucleosomes, providing insight into the recognition and function of epigenetic DNA marks in chromatin regulation. The proposed work will lay the basis for future investigations of other pioneer factors and the role of DNA context, synergistic action, histone variants and epigenetic modifications into pioneer factor-mediated chromatin opening. This will improve our ability to modify pioneer factor function and lead to novel approaches for therapeutic targeting.