Protein-ligand interface engineering for allele-specific regulation of histone demethylases and epigenome editing

在真核生物中，基因可以通过改变基因组在细胞核中的包装方式来开启或关闭。通常情况下，基因组是通过将DNA缠绕在桶状的蛋白质核心上而紧密相连的。紧密的包装会关闭基因，但蛋白质核心的化学修饰可以放松DNA，让基因打开。这些修饰是由一大群酶进行的，但它们如何以及在哪里起作用仍然未知。该项目将采用跨学科的方法，包括有机化学、蛋白质工程和细胞生物学，以揭示这些修饰剂的特异性，并设计能够随意打开特定基因的酶。研究环境将为研究生和本科生提供一个独特的训练场地。此外，化学生物学实验课程将让本科生参与以探究为基础的练习，学生将学习设计和开展现实世界的实验，旨在培养他们的批判性思维和独立学习技能。尽早实施这种以研究为基础的课程，有望通过激励STEM学生从事科学教育和研究，弥合当前教育课程中存在的差距，并带来培养熟练劳动力的长期利益。人类等真核生物基因表达的改变可以通过对染色质组蛋白组分的可逆化学修饰来实现。这个项目的重点是研究一种特殊类型的修饰，称为赖氨酸甲基化，它被一类称为赖氨酸去甲基化酶的酶去除。特异性去甲基化酶如何促进基因表达在很大程度上仍未被探索，部分原因是缺乏能够在精心控制的条件下快速询问完整细胞中给定去甲基化酶的工具。以一种特殊的赖氨酸去甲基化酶为范例，本研究将重点开发一种新的化学-遗传平台，该平台结合药理学和基因工程，通过合理设计的小分子和精确的时间控制来干扰特定的异构体。此外，工程去甲基化装置将与可编程CRISPR-Cas9的空间选择性相结合，开发一种新型的条件表观基因组编辑工具，用于在空间和时间上调节基因转录。该方法将应用于基因的重编程表达，这些基因是忠实的细胞分裂、细胞分化、谱系承诺以及最终的组织发育的基础。这些独特的工具将广泛提供给研究可逆组蛋白甲基化如何调节真核生物的研究人员。该项目由生物科学理事会分子与细胞生物科学部遗传机制组和数学与物理科学理事会化学部生命过程化学计划共同资助。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。

In eukaryotes, genes can be turned on or off by changing the way the genome is packaged in the nucleus. Normally, the genome is compacted by winding the DNA around barrel-like cores of proteins. Tight packaging turns genes off, but chemical modification of the protein cores can relax the DNA and allow genes to be turned on. The modifications are carried out by a large group of enzymes, but how and where they function remains unknown. This project will take an interdisciplinary approach, including organic chemistry, protein engineering, and cell biology--to uncover the specificity of these modifiers and to design enzymes capable of turning on specific genes at will. The research setting will provide a unique training ground for graduate and undergraduate students. Furthermore, a chemical biology laboratory course will engage undergraduate students in inquiry-based exercises where students will learn to design and carry out real-world experiments aimed at developing their critical thinking and independent learning skills. Early implementation of such a research-based course is expected to bridge a gap existing in the current educational curriculum by motivating STEM students in science education and research with the long-term benefit of generating a skilled workforce.Changes in gene expression in eukaryotic organisms like humans can be achieved by reversible chemical modifications on the histone protein components of chromatin. The focus of this project is to study a particular type of modification, called lysine methylation, which is removed by a class of enzymes known as lysine demethylases. How specific demethylases contribute to gene expression has remained largely unexplored, due in part to the lack of tools capable of rapidly interrogating a given demethylase in intact cells under carefully controlled conditions. Using one particular lysine demethylase as paradigm, this research will focus on developing a novel chemical-genetic platform that combines pharmacological and genetic engineering to perturb specific isoforms with rationally designed small molecules and precise temporal control. Furthermore, the engineered demethylation apparatus will be combined with the spatial selectivity of programmable CRISPR-Cas9 to develop a new type of conditional epigenome editing tool for regulating gene transcription in space and time. The approach will be applied to reprogram expression of genes that underlie faithful cell division, cellular differentiation, lineage commitment, and ultimately, organismal development. These unique tools will be made broadly available to researchers interested in addressing how reversible histone methylation regulates eukaryotic biology.This project is funded jointly by the Genetic Mechanisms Cluster, Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences and the Chemistry of Life Processes Program, Division of Chemistry in the Directorate of Mathematical and Physical Sciences.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.