High-throughput imaging of 3D chromatin regulation events in the nervous system

项目摘要/摘要 了解基因和染色质调节的机制及其在细胞周期中的作用 多细胞有机体涉及合成生物学、医学、 发育生物学和神经科学1-3.基因组学界的大规模努力 已经确定了许多功能基因和基因调控元件(GRE)，包括 基因及其可能的调控区域的特定表达的最新图谱 不同细胞类型的复杂组织4，5。然而，目前尚不清楚3D组织是如何 染色质会影响基因调控，反之亦然。建立对…的机械性理解 染色质组织和基因调控之间的相互作用，我们最好 同时测量所有关键元素-DNA序列、调节蛋白和 转录的RNA-在基因组水平上，同时保持有关细胞类型身份的信息。 为了应对这一挑战，我将开发一个成像平台，它可以同时测量 DNA的三维结构及其调控基因的RNA表达 与关键结构蛋白的相互作用(目标1)。虽然这种方法可以应用于许多 系统，一个特别合适的例子是外周嗅觉系统。嗅觉，一种 哺乳动物的主要感官，是由最大的基因家族控制的，这个家族包括超过 1000个嗅觉感受器6，7。大的调节序列网络相互作用 建立1000多种神经元类型的基因组，每种类型表达一种且只有一种 接待员8.我将应用这种成像方法来解决长期存在的问题：如何 不同的嗅觉感觉神经元建立它们的受体表达？这些集成在一起 与染色质组织和调节蛋白结构相关的测量 转录活性将为嗅觉基因调控提供一个模型。目标2，就是剖析这个 模型及GRE-启动子相互作用在实现细胞类型特异性表达中的作用 使用高通量合成生物学方法。我要用它感染嗅觉上皮 结合不同调控元件和启动子的大量病毒载体，以及 使用多路成像技术确定这些载体的精确细胞类型表达。的确有 两个目标之间的额外协同作用-第一个目标提供了 内源染色质结构-转录关系将用于设计 特定细胞亚群的转基因控制。我将探索这种能力，以 激活/抑制嗅觉感受器神经元的特定亚群，并确定 这些操纵的行为后果。

Project Summary/Abstract Understanding the mechanisms of gene and chromatin regulation and their roles within a multicellular organism has relevance across many disciples such as synthetic biology, medicine, developmental biology and neuroscience 1–3. Large-scale efforts of the genomics community have identified many of the functional genes and gene regulatory elements (GREs) including recent atlases with the specific expression of genes and their putative regulatory regions within different cell types of complex tissues 4,5. However, it remains unclear how the 3D organization of chromatin impacts gene regulation and vice versa. To build a mechanistic understanding of the interplay between chromatin organization and gene regulation, we would ideally simultaneously measure all the key elements - DNA sequences, regulatory proteins, and the transcribed RNA - at the genomic-scale, while maintaining information about cell type identity. To address this challenge, I will develop an imaging platform that can simultaneously measure the 3D structure of DNA together with the RNA expression of the regulated genes and their interaction with key structural proteins (Aim 1). While this method can be applied to many systems, a particularly suited example is the peripheral olfactory system. Olfaction, one of the main mammalian senses, is controlled by the largest family of genes comprising more than 1000 olfactory receptors 6,7. Large networks of regulatory sequences interact across the genome to establish more than 1000 neuronal types, each expressing one and only one receptor8. I will apply this imaging method to address the longstanding question: how do different olfactory sensory neurons establish their receptor expression? These integrated measurements relating chromatin organization and regulatory protein structures to transcriptional activity will provide a model of olfactory gene regulation. Aim 2, is to dissect this model and the roles of GRE-promoter interactions in achieving cell-type specific expression using a high-throughput synthetic biology approach. I will infect the olfactory epithelium with large pools of viral vectors that combine different regulatory elements and promoters, and determine the precise cell-type expression of these vectors using multiplexed imaging. There is an additional synergy between the two aims - the first aim provides measurements of the endogenous chromatin structure-transcription relationship which will be used to design transgenic control of specific subpopulation of cells. I will explore this capability to activate/inhibit specific sub-populations of olfactory receptor neurons and determine the behavior consequences of these manipulations.