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

• 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; URoL; Epigenetics; Reverse; Engineering; Human

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，它提供了用于在细胞内产生各种功能蛋白的指令。 但是活细胞中的DNA存在于称为“染色质”的蛋白质环境/包装系统中，该系统可以控制何时产生功能蛋白，或者何时从DNA产生蛋白质的指令时。 染色质控制蛋白质的产生的方式可能会受到生物体的年龄，生物体所生活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，仍然不知道很多，但是更好的理解可能会导致我们对生物如何适应环境或如何更好地治疗某些疾病的知识的进步。该项目将利用新颖的分子生物学技术来更好地理解复杂人类染色质组织的关键组成部分，通过逐步重写（或重建）在萌芽的酵母中，这是一个更简单的实验室模型。 这将使复杂的调节回路能够解剖，该回路控制人类，其他动物和其他多细胞生物的DNA产生功能蛋白。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。这项研究的结果将通过与纽约市“ Biobus”计划合作，以及在新的“ Epegenome Engineering”会议上的演讲来广泛传播酵母艺术计划。 表观基因组使许多真核生物具有从单个相同基因组中产生稳定和不同细胞类型的能力。这种多功能能力源于相互连接的染色质组织途径在跨染色体的多个长度尺度上作用，以调节基因表达，维持细胞身份并适应环境变化。什么规则导致一个细胞的染色质组织与另一个细胞的组织相对于另一个细胞的组织？全基因组方法的进步揭示了染色质结构的基础模式，但是推断因果关系仍然很困难。需要复杂的蛋白质净化并使用最小染色质底物的机械生化方法在概括生活系统方面的能力有限。多基因基因敲除研究可以导致细胞功能障碍和多效性，因此很难将重叠的表型效应解脱。在该研究项目中，将在四个级别的真核生物芽酵母（酿酒酵母）中重新组建人类表观遗传途径，以提供透明染色质组织和遗传基因表达原理的自下而上的方法。这四个级别是：（1）组蛋白：扩大产生多种人类组蛋白变异体系结构的能力。（2）异染色质：杂染色质：诸如Polycomb组蛋白等人类途径将进口到萌芽的酵母中，以生成抑制性组蛋白后的组蛋白后翻译后的翻译后修饰（PTMS）H3K27ME，H3K27ME，H3K9ME，H3K9ME，H2K9ME，H2AK9ME，H2AK99ME，CCP。构素蛋白：酵母中的指南针络合物将被人类通路所取代，该通路会产生激活的组蛋白PTM H3K4ME。（4）在拓扑结构（TAD）结构上：人类般的TAD结构将使用人类的凝聚蛋白和CTCF综合体进行设计，以反映NSF的构建范围。影响审查标准。