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

DNA是生命的蓝图，它提供了在细胞内产生各种功能蛋白质的指令。但是活细胞中的DNA存在于一种称为“染色质”的蛋白质环境/包装系统中，该系统控制着何时产生功能性蛋白质，或者何时沉默来自DNA的产生蛋白质的指令。染色质控制蛋白质产生的方式会受到生物体的年龄、生物体所处的环境以及生物体是否患病的影响。染色质如何调节蛋白质的产生仍有许多未知之处，但更好地了解可能会导致我们在生物体如何适应环境或如何更好地治疗某些疾病方面的知识取得进展。该项目将利用新的分子生物学技术，通过逐步重写(或重建)萌芽酵母中的系统，更好地了解复杂的人类染色质组织的关键成分，这是一个简单得多的实验室模型。这将使我们能够剖析控制人类、其他动物和其他多细胞生物体的DNA产生功能蛋白的复杂调控电路。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方面的培训。研究结果将通过将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”会议上发表演讲来广泛传播。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和不同的细胞类型。这种多用途的能力源于染色体上相互连接的染色质组织途径，这些途径在多个长度尺度上作用于染色体，以调节基因表达，维持细胞特性，并适应环境变化。什么规则会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展揭示了染色质结构背后的模式，但推断因果关系仍然困难。机械化的生化方法需要复杂的蛋白质纯化和使用最少的染色质底物，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，使重叠的表型效应难以分离。在这项研究项目中，人类表观遗传途径将在真核细胞发芽酵母(Saccharmycescerevisiae)中进行四个水平的重组，以提供一种自下而上的方法来解开染色质组织和遗传基因表达的原理。这四个水平是：(1)组蛋白：扩大产生不同人组蛋白变体结构的能力。(2)异染色质：人类途径，如Polycomb Group蛋白，将被导入萌芽酵母中，产生抑制性组蛋白翻译后修饰(PTMS)H3K27me，H3K9me，(3)常染色质：酵母中的COMPASS复合体将被产生活化组蛋白PTM H3K4me的人类途径所取代。(4)拓扑关联结构域(TAD)结构：将使用人类粘附素和CTCF复合体来设计类似人类的TAD结构。该奖项反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.