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打开或关闭。正常情况下，基因组是通过将DNA缠绕在蛋白质的桶状核心上来压缩的。严密的包装可以关闭基因，但对蛋白质核心的化学修饰可以放松DNA，允许基因被激活。这些修饰是由一大群酶进行的，但它们如何发挥作用以及在哪里发挥作用尚不清楚。这个项目将采取跨学科的方法，包括有机化学、蛋白质工程和细胞生物学--以揭示这些修饰物的特异性，并设计能够随意启动特定基因的酶。研究环境将为研究生和本科生提供一个独特的培训场所。此外，化学生物实验室课程将让本科生参与以探究为基础的练习，学生将学习设计和进行旨在培养他们批判性思维和独立学习技能的真实世界实验。这种以研究为基础的课程的早日实施，有望通过激励STEM学生从事科学教育和研究，并长期受益于培养一支熟练的劳动力，来弥补当前教育课程中存在的差距。在人类等真核生物中，基因表达的变化可以通过对染色质的组蛋白成分进行可逆的化学修饰来实现。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，它被一类被称为赖氨酸去甲基酶的酶去除。特定的脱甲基酶对基因表达的作用在很大程度上仍未被探索，部分原因是缺乏能够在严格控制的条件下在完整细胞中快速询问给定脱甲基酶的工具。本研究将以一种特定的赖氨酸脱甲基酶为范例，致力于开发一种新型的化学遗传平台，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，工程化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。这种方法将被应用于重新编程基因的表达，这些基因构成了忠实的细胞分裂、细胞分化、世系承诺以及最终生物发育的基础。这些独特的工具将广泛提供给有兴趣研究可逆组蛋白甲基化如何调控真核生物的研究人员。该项目由生物科学局分子和细胞生物科学部遗传机制组和数学和物理科学局化学部生命过程化学计划联合资助。该奖项反映了NSF的法定使命，并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，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.