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: 10028932
• 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/10028932
• 关键词: Animals; Bar Codes; 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; chromatin remodeling; combinatorial; genetic approach; in vivo; insight; novel; programs; response; reverse genetics; 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。这些过程被经典地理解为需要 从细胞外到细胞核的信息传递，然而，这种范式在很大程度上忽略了这样一个事实 生物学涉及生化信息在质膜之外的空间中的移动，以及 核包膜。与原核生物不同，在原核生物中，转录和新陈代谢发生在同一膜上- 结合的隔间，多细胞真核生物在多个层面上划分其代谢和生化： 在细胞内的细胞器之间、组织内的细胞之间以及组成的整个器官系统中 一位主持人。我们假设生物化学的空间分割通过细胞新陈代谢来调节细胞 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的发育和功能。 我们最近发现，线粒体和胞浆之间的代谢串扰是一个必不可少的组成部分。 并着手扩展这一范式，以探索代谢物如何跨越 细胞和组织之间的隔间决定了它们的生物学特性。我们试图在两个层面上探索这一点：1) 阐明线粒体-胞浆串扰控制组蛋白的分子机制 表观遗传学；2)研究细胞-细胞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。我们将在造血学的背景下探索这些概念 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新陈代谢签名支持，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筛查系统来实现 几乎与每一种原代造血细胞相容。我们将在体外和体内进行 体内未汇集和汇集的条形码CRISPR筛查评估所有77个编码线粒体转运蛋白的基因 以及所有的质膜转运体，来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研究进一步进行，这将使 我们将测试我们正在研究的代谢过程下游的表观遗传重塑酶以及 一种组合反向遗传方法，即同一网络中的不同基因将针对不同的基因 相互作用的细胞群体，使我们能够绘制反式代谢流。总之，这些研究不仅将 帮助建立一种新的研究分子生物学的范例，但也提供了基本的 对基因调控和发育的机械论见解。