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: 10247770
• 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/10247770
• 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; 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 多细胞开发需要广泛的细胞 - 细胞相互作用和转录重编程 在染色质重塑的水平上完成。这些过程经常理解为 信息从细胞外传播到其核，但是这种范式在很大程度上忽略了事实 该生物学涉及跨质膜以外空间的生化信息的运动 核信封。与原核生物不同，其中转录和代谢发生在同一膜中 约束隔室，多细胞真核生物在多层分配其新陈代谢和生物化学： 在细胞内的细胞器中，组织内的细胞之间以及整个器官系统组成 主机。我们假设通过细胞代谢的生物化学的空间划分来调节细胞 通过控制组蛋白表观遗传学并协调组织中的相互作用细胞来开发和功能。 我们最近表明，线粒体和细胞质之间的代谢串扰是必不可少的成分 细胞分化并着手扩展此范式，以探讨代谢物的运动如何跨越 在细胞和组织室之间决定了它们的生物学。我们试图在两个层面上探索这一点：1） 阐明分子机制，解释了线粒体 - 胞质串扰如何控制组蛋白 表观遗传学； 2）研究细胞 - 细胞代谢产物交换如何影响发展并坐标反应 在相互作用的细胞种群之间。我们将在造血的背景下探索这些概念 系统，由于其开发需要大量的表观遗传重塑，每个血统和功能程序 现在据了解，它得到了独特的代谢签名的支持，使其成为我们追求的理想模型系统 这些研究。这将通过利用我们已经开发的CRISPR筛选系统来实现 与几乎每个原发性造血细胞群体兼容。我们将在体外和 Vivo未冷却和合并的条形码CRISPR屏幕评估编码线粒体转运蛋白的所有77个基因 以及所有质膜转运蛋白，以研究这些代谢运输系统如何影响 表观遗传重塑和发育。这些研究将通过串联SGRNA研究进一步进一步 我们要测试我们正在研究的代谢过程下游的表观遗传重塑酶 组合反向遗传学方法，其中同一网络中的不同基因将针对不同 相互作用的细胞种群，使我们能够映射反式的代谢流。这些研究总共将不仅 帮助建立一个新的范式，可以使用分子生物学，但也提供基本 对基因调节和发育的机械洞察力。