Dissecting the Mechanisms of Pioneer Factor Facilitated Chromatin Opening

项目摘要 核小体覆盖了真核生物基因组的大部分，是许多核小体有效结合的障碍 控制基因表达的调节蛋白。一组称为先锋因子的特殊转录因子可以 结合核小体DNA上的靶部位，松解染色质，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。Sox2和Oct4是先驱因素 在胚胎发生和成年神经发生过程中协调细胞命运的变化，并在 将体细胞重新编程为多能状态。这些蛋白质的突变和缺失与 神经紊乱，而它们的丰富与癌症有关。尽管有大量的研究，但人们对此知之甚少。 对Sox2，Oct4等先锋因子在分子水平上发挥先锋活性。我们的目标是 解决有关Sox2和Oct4如何以不同的结合模式与核小体接触的关键悬而未决的问题 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 利用高分辨率核磁共振波谱进行分析以获得特定部位的结构 以及关于先锋因子与核小体相互作用及其引起的扰动的动态信息 DNA和组蛋白来潜在地调节其他因素。在目标1中，我们将利用体外结合试验来 Sox2和Oct4与人工和天然核小体的不同结合模式及其依赖性 DNA序列、辅因子协同作用和组蛋白组成。我们还将采用甲基TROSY核磁共振， 适用于超大分子生物分子的研究，询问特定结合部位的构象和 这些蛋白质的动态选择具有原子细节的核小体。在目标2中，我们将研究Sox2的影响 和Oct4结合在不同核小体位置对核小体DNA构象和可及性的影响 并用核磁共振、化学和酶等方法对结构敏感的无序组蛋白尾巴进行了探测 和动态扰动。我们将进一步评估SOX2和10月4日是否通过直接接触和 核小体扰动，刺激组蛋白甲基转移酶的活性，发现这些酶能 在活体内与这些先锋因素合作。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结合蛋白和建议的先锋因子Kaiso结合并改变核小体，提供 深入了解表观遗传DNA标记在染色质调节中的识别和功能。拟议中的工作 将为未来研究其他先锋因子以及DNA上下文、协同作用、 组蛋白变体和表观遗传修饰进入先锋因子介导的染色质开放。这将会有所改善 我们能够改变先锋因子的功能，并导致治疗靶向的新方法。

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.