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

神经系统 3D 染色质调控事件的高通量成像

• 批准号: 10255107
• 负责人: Bogdan Bintu
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10255107
• 关键词: 3-Dimensional; Academia; Address; Animal Behavior; Atlases; Behavior; Biology; California; Catalogs; Cell physiology; Cells; Chromatin; Chromatin Structure; Collaborations; Communities; Complex; Computational Molecular Biology; DNA; DNA Repair; DNA Sequence; DNA Structure; DNA biosynthesis; Development; Developmental Biology; Discipline; Elements; Ensure; Event; Funding; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Family; Gene Proteins; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Image; Individual; Institution; Libraries; Maps; Measurement; Measures; Medicine; Methods; Microscopy; Modeling; Multimodal Imaging; Nervous system structure; Neurons; Neurosciences; Nucleic Acid Regulatory Sequences; Olfactory Epithelium; Olfactory Pathways; Olfactory Receptor Neurons; Organism; Output; Peripheral; Process; RNA; Receptor Gene; Regulation; Regulator Genes; Regulatory Element; Research; Resolution; Resources; Role; Scientist; Series; Smell Perception; Structural Protein; Structure; System; Technology; Testing; Tissues; Transgenic Organisms; Universities; Viral Vector; Virus; Vision; Work; base; cell type; design; epigenomics; experience; expression vector; genetic regulatory protein; genome-wide; genomic locus; imaging modality; imaging platform; multiplexed imaging; olfactory receptor; olfactory sensory neurons; professor; promoter; protein structure; receptor expression; supportive environment; synergism; synthetic biology; tenure track; three dimensional structure; tool; vector

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.

项目总结/摘要 了解基因和染色质调控的机制及其在细胞内的作用， 多细胞生物与许多学科都有关联，如合成生物学，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（GRES），包括 最近的图谱与基因的特异性表达和它们的推定调控区域内， 复杂组织的不同细胞类型4，5.然而，目前尚不清楚3D组织如何 影响基因调控，反之亦然。建立一个机械的理解， 染色质组织和基因调控之间的相互作用， 同时测量所有的关键元素- DNA序列，调节蛋白质， 转录的RNA -在基因组规模，同时保持有关细胞类型身份的信息。 为了应对这一挑战，我将开发一种成像平台， DNA的3D结构以及受调控基因的RNA表达， 与关键结构蛋白的相互作用（Aim 1）。虽然这种方法可以应用于许多 在嗅觉系统中，特别合适的示例是外周嗅觉系统。嗅觉， 哺乳动物的主要感官，是由最大的基因家族控制的， 1000个嗅觉感受器6，7.调控序列的大型网络在整个细胞中相互作用， 基因组建立超过1000种神经元类型，每种表达一种且只有一种 受体8.我将应用这种成像方法来解决一个长期存在的问题： 不同的嗅觉感觉神经元建立其受体表达？这些集成 与染色质组织和调节蛋白结构相关的测量， 转录活性将提供嗅觉基因调控的模型。第二个目标是解剖这个 模型和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。我会感染嗅上皮细胞 组合了不同调控元件和启动子的联合收割机的病毒载体的大库，和 使用多重成像确定这些载体的精确细胞类型表达。有 这两个目标之间的另一个协同作用-第一个目标提供对 内源性染色质结构-转录关系，其将用于设计 特定细胞亚群的转基因控制。我将探索这种能力， 激活/抑制嗅觉受体神经元的特定亚群，并确定 这些操作的后果。