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URoL: Epigenetics 2: Reverse Engineering Human Epigenetic Machinery in Yeast

URoL：表观遗传学 2：酵母中的人类表观遗传机制逆向工程

基本信息

批准号：
1921641
负责人：
Jef Boeke
金额：
$ 300万
依托单位：
New York University Medical Center
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1921641&HistoricalAwards=false
关键词：
URoL Epigenetics Reverse Engineering Human

项目摘要

DNA is the blueprint of life that provides instructions for producing various functional proteins within a cell. But DNA in living cells exists in a protein environment/packaging system called "chromatin" that controls when functional proteins are produced, or when the instructions from DNA to produce proteins are silenced. The way chromatin controls the production of proteins can be influenced by age of the organism, environment that the organism is living in, and whether the organism is diseased. Much is still unknown about how chromatin regulates the production of proteins, but a better understanding could lead to advances in our knowledge of how organisms adapt to an environment or how better to treat certain diseases. This project will leverage novel molecular biology techniques to better understand key components of the complex human chromatin organization by progressively rewriting (or rebuilding) the system within budding yeast, a much simpler laboratory model. This will enable the dissection of the complicated regulatory circuits that control the production of functional proteins from DNA in humans, other animals, and other multicellular organisms. Two postdoctoral researchers and one graduate student will be trained in state-of-the-art molecular biology methods and analyses. Results from the research will be disseminated broadly by a yeast art program to be exhibited on the New York City subway, by partnering with the New York City "biobus" program, and by presentations at a new "epigenome engineering" meeting. The epigenome equips many eukaryotes with the ability to generate stable and distinct cell types from a single identical genome. This multipurpose ability stems from interconnected chromatin organizing pathways acting at multiple length scales across chromosomes to regulate gene expression, maintain cell identity, and adapt to environmental changes. What rules lead to one cell's chromatin organization versus another? Advances in genome-wide methodologies have revealed the patterns underlying chromatin architectures, but inferring causal effects remains difficult. Mechanistic biochemical approaches that require complex protein purifications and use minimal chromatin substrates have limited ability in recapitulating living systems. Multigene knockout studies can lead to cellular dysfunction and pleiotropy, making it difficult to decouple overlapping phenotypic effects. In this research project, human epigenetic pathways will be reconstituted within the eukaryote budding yeast (Saccharomyces cerevisiae) at four levels to provide a bottom-up approach for unraveling principles of chromatin organization and inherited gene expression. The four levels are:(1) Histones: expanding the ability to generate diverse human histone variant architectures.(2) Heterochromatin: human pathways such as Polycomb Group Proteins will be imported into budding yeast to generate the repressive histone Post-Translational Modifications (PTMs) H3K27me, H3K9me, H2AK119ub and CpG methylation.(3) Euchromatin: the COMPASS complex in yeast will be replaced by the human pathway that generates the activating histone PTM H3K4me.(4) Topologically Associating Domain (TAD) structures: human-like TAD structures will be engineered using human cohesin and CTCF complexes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

DNA 是生命的蓝图，为细胞内产生各种功能性蛋白质提供指令。但活细胞中的 DNA 存在于一种称为“染色质”的蛋白质环境/包装系统中，该系统控制功能性蛋白质何时产生，或者 DNA 产生蛋白质的指令何时被沉默。染色质控制蛋白质产生的方式可能受到生物体的年龄、生物体所处的环境以及生物体是否患病的影响。关于染色质如何调节蛋白质的产生还有很多未知，但更好的理解可能会导致我们对生物体如何适应环境或如何更好地治疗某些疾病的了解取得进展。该项目将利用新颖的分子生物学技术，通过逐步重写（或重建）芽殖酵母（一个更简单的实验室模型）内的系统，更好地了解复杂的人类染色质组织的关键组成部分。这将能够剖析控制人类、其他动物和其他多细胞生物体中 DNA 产生功能性蛋白质的复杂调节回路。两名博士后研究人员和一名研究生将接受最先进的分子生物学方法和分析的培训。研究结果将通过在纽约市地铁上展出的酵母艺术项目、与纽约市“biobus”项目合作以及在新的“表观基因组工程”会议上的演示来广泛传播。表观基因组使许多真核生物能够从单个相同的基因组中产生稳定且不同的细胞类型。这种多用途能力源于相互关联的染色质组织途径，在染色体的多个长度尺度上发挥作用，以调节基因表达、维持细胞身份并适应环境变化。哪些规则导致一个细胞的染色质组织与另一个细胞的染色质组织不同？全基因组方法学的进步揭示了染色质结构的潜在模式，但推断因果效应仍然很困难。需要复杂的蛋白质纯化并使用最少的染色质底物的机械生化方法在重现生命系统方面的能力有限。多基因敲除研究可能导致细胞功能障碍和多效性，从而难以消除重叠的表型效应。在该研究项目中，人类表观遗传途径将在真核生物芽殖酵母（酿酒酵母）中的四个水平上进行重建，以提供一种自下而上的方法来揭示染色质组织和遗传基因表达的原理。这四个级别是：(1) 组蛋白：扩展生成多种人类组蛋白变体结构的能力。(2) 异染色质：多梳族蛋白等人类途径将被导入芽殖酵母中，以生成抑制性组蛋白翻译后修饰 (PTM) H3K27me、H3K9me、H2AK119ub 和 CpG 甲基化。(3) 常染色质：酵母中的 COMPASS 复合物将被生成激活组蛋白 PTM H3K4me 的人类途径所取代。(4) 拓扑关联域 (TAD) 结构：将使用人类粘连蛋白和 CTCF 复合物设计类人 TAD 结构。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的评估进行评估，认为值得支持。影响审查标准。