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，这将解锁我们对从酵母、植物到人类的高等生物中基因表达的理解和控制能力，并在生物技术中有广泛的应用。染色质是结合在基因组顶部的数百种蛋白质层。它可以对包括衰老和癌症在内的各种细胞和有机体过程进行监管，并且可以在生物技术应用中加以利用，包括生物修复、环境传感和基于生物的商品化学品生产。染色质也为教学创新和丰富提供了一个强有力的主题。该项目将为初高中学生开发动手实验，将染色质的分子变化与肉眼观察到的效应联系起来，在这种情况下，酵母细胞与琼脂板表面的结合。此外，染色质的数值和生化多样性提倡在大学和博士水平上采用新的教学方法。具体来说，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支持研究者在其职业生涯的关键阶段。染色质在分子水平上具有令人难以置信的多样性。在已知的5种组蛋白上有80多种不同的氨基酸残基，它们经历了20多种不同的翻译后修饰。数百种蛋白质，通常包含被称为“书写者”和“阅读者”的结构域，它们调节并与这种复杂的生化调色板相互作用。通过与生物化学和遗传方法相结合的高含量技术的发展，已经清楚地表明，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，功能分析在规模上仍然受到很大限制。特别是，表征和设计表观遗传书写者的酶活性和特异性在很大程度上仍然依赖于低通量的生化分析，这些生化分析需要重组蛋白的生产和纯化，即使使用机器人液体处理也很难达到规模。该提案旨在建立在最近的合成生物学平台上，研究者和他的团队利用酵母表面展示和表观基因组编辑器开发了解锁染色质多样性的途径。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)绘制所有已知人类HAT的残基特异性；2)通过定向进化方法获取非常大的酶和底物序列空间，将序列与功能连接起来；3)探究分子环境对功能的影响。当研究集中在HATs上时，研究者和他的团队寻求激励这个实验平台的未来扩展和应用到染色质的多种生物化学中。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.