Examining how the spatial partitioning of metabolism underlies cell state

检查新陈代谢的空间划分如何成为细胞状态的基础

• 批准号: 10684148
• 负责人: Will H. Bailis
• 金额: $ 44万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684148
• 关键词: Animals; Bar Codes; Binding; Biochemical; Biochemistry; Biological Models; Biology; CRISPR screen; Cell Communication; Cell Differentiation process; Cell Nucleus; Cell membrane; Cells; Cellular Structures; Cellular biology; Cytosol; Development; Enzymes; Epigenetic Process; Eukaryota; Gene Expression Regulation; Genes; Genetic Transcription; Hematopoietic; Hematopoietic System; Histones; In Vitro; Maps; Membrane; Membrane Transport Proteins; Metabolic; Metabolism; Mitochondria; Molecular; Molecular Biology; Movement; Nuclear Envelope; Organelles; Population; Process; Prokaryotic Cells; System; Testing; Tissues; biological research; body system; cellular development; chromatin remodeling; combinatorial; genetic approach; in vivo; insight; novel; programs; response; reverse genetics; transcriptional reprogramming; transmission process

Project Summary Multicellular development requires extensive cell-cell interactions and transcriptional reprogramming accomplished at the level of chromatin remodeling. These processes are classically understood to entail the transmission of information from outside a cell to its nucleus, however this paradigm largely overlooks the fact that biology involves the movement of biochemical information across spaces beyond the plasma membrane and the nuclear envelope. Unlike prokaryotes, in which transcription and metabolism occur in the same membrane- bound compartment, multicellular eukaryotes partition their metabolism and biochemistry at multiple layers: amongst the organelles within a cell, between cells within a tissue, and throughout the organ systems that compose a host. We hypothesize that the spatial partitioning of biochemistry, through cellular metabolism, regulates cell development and function by controlling histone epigenetics and coordinating interacting cells within tissues. We have recently shown that metabolic crosstalk between the mitochondria and cytosol is an essential component of cell differentiation and set out to extend this paradigm to explore how the movement of metabolites across between cellular and tissue compartments dictates their biology. We seek to explore this at two levels: 1) elucidating the molecular mechanism explaining how mitochondrial-cytosolic crosstalk controls histone epigenetics; 2) investigating how cell-cell metabolite exchange influences development and coordinates responses between interacting populations of cells. We will explore these concepts in the context of the hematopoietic system, as its development requires extensive epigenetic remodeling, with each lineage and functional program now understood to be supported by a unique metabolic signature, making it an ideal model system for us to pursue these studies. This will be accomplished by taking advantage a CRISPR screening system we have developed that is compatible with nearly every population of primary hematopoietic cells. We will conduct both in vitro and in vivo unpooled and pooled, barcoded CRISPR screens evaluating all 77 genes encoding mitochondrial transporters as well as all plasma membrane transporters, to investigate how these metabolic transport systems impact epigenetic remodeling and development. These studies will be furthered by tandem sgRNA studies that will allow us to test the epigenetic remodeling enzymes downstream of the metabolic processes we are studying as well as a combinatorial reverse genetic approach in which different genes in the same network will be targeted in different populations of interacting cells, allowing us to map metabolic flow in trans. Altogether these studies will not only help establish a novel paradigm with which to approach molecular biology, but also provide fundamental mechanistic insights into gene regulation and development.

项目摘要 多细胞发育需要广泛的细胞间相互作用和转录重编程 在染色质重塑的水平上完成。这些过程通常被理解为需要 信息从细胞外传递到细胞核，然而这种范式在很大程度上忽略了这样一个事实： 生物学涉及到生物化学信息在质膜之外的空间中的运动， 核膜。不像原核生物，转录和代谢发生在同一个膜上- 结合区室，多细胞真核生物将其代谢和生物化学分配在多个层： 在细胞内的细胞器之间，在组织内的细胞之间，以及在整个器官系统中， 一个主持人我们假设生化的空间分配，通过细胞代谢，调节细胞 通过控制组蛋白表观遗传学和协调组织内相互作用的细胞来实现发育和功能。 我们最近已经表明，线粒体和胞质溶胶之间的代谢串扰是一个重要组成部分， 细胞分化的研究，并着手扩展这一范式，探索代谢物如何在细胞中运动 决定了它们的生物学特性。我们试图在两个层面上探讨这一点：1） 阐明了细胞质-细胞溶质串扰如何控制组蛋白的分子机制 表观遗传学; 2）研究细胞间代谢物交换如何影响发育和协调反应 相互作用的细胞群体之间的关系。我们将在造血的背景下探讨这些概念 系统，因为它的发展需要广泛的表观遗传重塑，每个谱系和功能程序 现在被理解为由独特的代谢特征支持，使其成为我们追求的理想模型系统 这些研究。这将通过利用我们开发的CRISPR筛选系统来实现， 与几乎所有的原代造血细胞群体相容。我们将在体外和体内 评价编码线粒体转运蛋白的所有77个基因的体内未合并和合并条形码CRISPR筛选 以及所有质膜转运蛋白，以研究这些代谢转运系统如何影响 表观遗传重塑和发育。这些研究将通过串联sgRNA研究进一步推进， 我们来测试我们正在研究的代谢过程下游的表观遗传重塑酶， 一种组合反向遗传方法，其中同一网络中的不同基因将以不同的方式被靶向。 相互作用的细胞群，使我们能够绘制反式代谢流。总之，这些研究不仅 有助于建立一个新的模式，以接近分子生物学，但也提供了基本的 对基因调控和发育的机械见解。