Extensive multiplexing of protein nucleic-acid interactions to comprehensively study gene expression regulation from chromatin to mRNA degradation

尽管体内的每个细胞都含有相同的DNA序列，但哺乳动物的生物体 由不同的基因表达程序定义的数千种细胞类型。如何分子 包括DNA、RNA和蛋白质在内的成分，对控制基因表达的作用由来已久 问题。调节因子通过核扩散并与其相互作用的模型 通过高亲和力相互作用的同源DNA和RNA靶不能解释许多 基因调控所需的数量特征。观察到许多转录因子 而染色质调节器在细胞核内组织成更高级别的结构可以解释 数量方面。然而，我们不知道(I)大多数的DNA结合和空间组织 调节器，(Ii)这些隔间内有哪些具体部件，以及(Iii) 空间组织在基因调控中的作用。挑战在于，我们缺乏方法来 测量这些分子组分的组合组织和破坏能力 划分并衡量其对基因调控的影响。为了解决这个问题，我们正在 开发尖端基因组规模的方法，使我们能够测量多重蛋白质 DNA的结合和空间组织。具体地说，我们将创建全基因组图谱 数百种DNA结合蛋白，包括转录因子、染色质调节因子、组蛋白 (具有各种修饰)和RNA聚合酶。然后，我们将探索对RNA的依赖 通过比较存在和不存在空间组织的调控蛋白定位 核糖核酸酶处理。最后，我们将扰乱特定监管部门的形成 (使用锁定核酸和/或CRISPR方法)了解可能的机制 特定基因调控中的空间组织。我们的结果将提供新的能力和 了解DNA、RNA和蛋白质分子如何在3D空间中协同工作的框架 以有效地调控基因表达。

Although every cell in the body contains the same DNA sequence, mammalian organisms consist of thousands of cell types defined by distinct gene expression programs. How molecular components, including DNA, RNA and proteins, act to control gene expression is a long-standing question. The model whereby regulatory factors diffuse through the nucleus and engage with their cognate DNA and RNA targets through high affinity interactions cannot explain many of the quantitative features required for gene regulation. The observation that many transcription factors and chromatin regulators organize into higher-order structures within the nucleus may explain the quantitative aspects. Yet, we don’t know (i) the DNA binding and spatial organization of most regulators, (ii) what specific components are present within these compartments, and (iii) what role spatial organization plays in gene regulation. The challenge is that we lack methods to measure combinatorial organization of these molecular components and the ability to disrupt compartmentalization and measure its impact on gene regulation. To address this, we are developing cutting-edge genome-scale methods that allow us to measure multiplexed protein binding and spatial organization of DNA. Specifically, we will create genome-wide maps of hundreds of DNA binding proteins including transcription factors, chromatin regulators, histones (with various modifications) and RNA polymerases. We will then explore the RNA-dependence of regulatory proteins localization by comparing spatial organization in the presence and absence of RNase treatment. Finally, we will then disrupt formation of specific regulatory compartments (using Locked Nucleic Acid and/or CRISPR approaches) to understand possible mechanisms of spatial organization in regulation of specific genes. Our results will provide novel capabilities and a framework for understanding how DNA, RNA, and protein molecules work together in 3D space to efficiently regulate gene expression.