CAREER: A Synthetic Biology Platform to Map and Engineer the Diverse Epigenetic Space

该奖项全部或部分由2021年美国救援计划法案（公法117-2）资助。该项目将开发新的技术平台和教育创新，以解锁我们对从酵母和植物到人类的高阶生物中基因表达的理解和控制能力，并在生物技术中具有广泛的应用。染色质是数百种蛋白质结合在基因组顶部的层。它赋予不同的细胞和生物体过程，包括衰老和癌症的监管处理，它可以在生物技术应用，包括生物修复，环境传感和生物基生产的商品化学品。染色质也为教学创新和丰富提供了一个强有力的话题。该项目将为初中和高中学生开发动手实验，将染色质中的分子变化与肉眼观察到的效果联系起来，在这种情况下，酵母细胞与琼脂平板表面的结合。此外，染色质的数量和生物化学多样性倡导在大学和博士水平的新的教学方法。具体而言，该项目将开发新的课程模块，培训下一代科学家如何处理和分析大型数据集。 美国救援计划对该项目的资助将在研究者职业生涯的关键阶段提供支持。染色质在分子水平上是难以置信的多样化。已知在五种组蛋白上有超过80种不同的氨基酸残基经历超过20种不同的翻译后修饰。数以百计的蛋白质，通常包含被称为作家和读者的结构域，都调节并与这种复杂的生化调色板相互作用。通过高含量技术的发展，结合生物化学和遗传学方法，很明显，这种多样性的大部分是功能性的。然而，与许多可以高通量表征染色质的方法不同，功能测定在规模上仍然受到很大限制。特别地，表征和工程化表观遗传书写者的酶活性和特异性仍然主要依赖于低通量生物化学测定，其需要重组蛋白质生产和纯化，即使用机器人液体处理也难以规模化。该提案旨在建立在最近的合成生物学平台上，研究人员和他的团队使用酵母表面展示和表观基因组编辑器开发，以解锁染色质的多样性。以组蛋白乙酰转移酶（HAT）为重点，他们将建立原理证明，这些系统可以1）映射所有已知人类HAT的残基特异性; 2）通过定向进化方法访问非常大的酶和底物序列空间，将序列与功能联系起来; 3）探测分子环境对功能的影响。虽然研究的重点是HAT，研究者和他的团队寻求激励未来扩展和应用这个实验平台的染色质的多样化的生物化学。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).This project will develop new technological platforms and educational innovations that will unlock our understanding of and ability to control the expression of genes in higher order organisms from yeast and plants to humans, with broad applications in biotechnology. Chromatin is the layer of hundreds of proteins bound on top of the genome. It confers regulatory handles over diverse cellular and organismal processes including aging and cancer, and it could be leveraged in biotechnological applications including bioremediation, environmental sensing, and bio-based production of commodity chemicals. Chromatin also provides a strong topic for pedagogical innovation and enrichment. This project will develop hands-on experiments for middle and high school students that connect molecular changes in chromatin to effects observable by eye, in this case the binding of yeast cells to the surface of an agar plate. Furthermore, the numerical and biochemical diversity of chromatin advocates for new pedagogical approaches at the collegiate and PhD levels. Specifically, this project will develop new coursework modules that train the next generation of scientists in how to handle and analyze large data sets. American Rescue Plan funding of this project will support the investigator at a critical stage in his career.Chromatin is incredibly diverse at the molecular level. There are more than 80 different amino acid residues on five histone proteins known to undergo over 20 distinct post-translational modifications. Hundreds of proteins, often containing domains referred to as writers and readers, both regulate and interact with this complex biochemical palette. Through the development of high content technologies paired with biochemical and genetic approaches it has become clear that much of this diversity is functional. However, unlike many methods that can characterize chromatin at high throughput, functional assays remain substantially more limited in scale. In particular, characterizing and engineering the enzymatic activities and specificities of epigenetic writers still rely largely on low throughput biochemical assays that require recombinant protein production and purification that are challenging to scale even with robotic liquid handling. This proposal seeks to build upon recent synthetic biology platforms the investigator and his team have developed using yeast surface display and epigenome editors to unlock access to the diversity of chromatin. With a focus on histone acetyltransferases (HAT), they will establish proof-of-principle demonstrations that these systems can 1) map the residue specificities of all known human HATs; 2) connect sequence to function by accessing the very large enzyme and substrate sequence space by directed evolution approaches; and 3) probe the impact of molecular context on function. While the investigations focus on HATs, the investigator and his team seek to motivate the future expansion and application of this experimental platform to the diverse biochemistry of chromatin.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.