Site-specific control of human gene regulation for therapeutically applicable mechanistic insights

项目摘要 基因表达失调是人类疾病的一个广泛和疾病不可知的驱动因素。因此 理解和精确控制基因表达的能力有可能彻底改变治疗方法， 景观人类基因的表达是由一个优雅的融合调控， 力，包括染色质的物理压实，组蛋白的翻译后修饰（PTM） 蛋白质，DNA甲基化，以及转录因子和染色质修饰之间的动态接合 蛋白质和人类基因组。虽然这种协调控制保障了健康和 疾病，我们对这些调控力量如何联合起来驱动人类基因表达的机械理解， 以及如何可预见地为新疗法重新设计它们，仍然有限。 基于核酸酶无效的CRISPR/Cas系统的可编程表观基因组编辑工具最近已被开发。 出现了新的方法来控制内源性人类基因表达和共价修饰， 天然染色质。尽管取得了令人兴奋的进展，但主要的技术和概念差距仍然存在。比如说， 从机制上讲，特定基因的表达水平是如何在大范围内精确调节的， 在人类细胞中。此外，还不完全理解为什么相同的转录因子和染色质 当修饰物定位于不同人类细胞类型中的特定调节元件时，修饰物具有不同的作用。 此外，转录因子和染色质修饰剂在不同温度下的时空稳定性和功能持续时间也不同。 不同的基因组基因座没有很好地定义。 这个MIRA项目的目标是通过开发和 将不同的基于CRISPR/Cas的转录因子和染色质修饰剂与本体和单细胞相结合， 表观基因组学和敏感蛋白质组学。我们将使用这种多学科的方法来回答基本的 关于人类基因调控机制的问题包括：（1）人类基因表达如何被位点- 在健康和疾病中观察到的分辨率下进行专门控制？(2)什么是因果函数和 不同染色质修饰的操作稳定性？(3)在多大程度上， 修饰状态、蛋白质复合物组成和空间邻近性影响转录因子的功能 和染色质修饰剂总之，我们的建议有很大的潜力，发现和实现控制的关键 这些机制对人类健康具有广泛而重大的意义。

PROJECT SUMMARY Dysregulated gene expression is a widespread and disease-agnostic driver of human illness. Therefore, the ability to understand and precisely control gene expression has the potential to revolutionize the therapeutic landscape. The expression of human genes is naturally controlled by an elegant convergence of regulatory forces, including the physical compaction of chromatin, post-translational modifications (PTMs) to histone proteins, DNA methylation, and the dynamic engagement between transcription factors and chromatin modifying proteins and the human genome. Although this coordinated control safeguards the balance between health and disease, our mechanistic understanding of how these regulatory forces unite to drive human gene expression, and how they can be predictably redesigned for new therapies, remains limited. Programmable epigenome editing tools based upon nuclease null CRISPR/Cas-based systems have recently emerged and enable new ways to control endogenous human gene expression and covalent modifications to native chromatin. Despite this exciting progress, major technological and conceptual gaps remain. For instance, it is mechanistically unclear how the expression levels of specific genes can be precisely tuned over wide ranges in human cells. In addition, it is incompletely understood why the same transcription factors and chromatin modifiers have different effects when localized to specific regulatory elements in different human cell types. Further, the spatiotemporal stability and functional durations of transcription factors and chromatin modifiers at different genomic loci are not well defined. The goal of this MIRA project is to overcome these conceptual and technological gaps by developing and combining diverse CRISPR/Cas-based transcription factors and chromatin modifiers with bulk and single cell epigenomics and sensitive proteomics. We will use this multidisciplinary approach to answer fundamental questions about human gene regulatory mechanisms including: (1) How can human gene expression be site- specifically controlled at the resolution observed in health and disease? (2) What are the causal functions and operational stabilities of diverse chromatin modifications? (3) To what extent does chromatin compaction, modification state, protein complex composition, and spatial proximity affect the function of transcription factors and chromatin modifiers? Altogether, our proposal has great potential to uncover and enable control over pivotal mechanisms with broad and significant importance to human health.