INSPIRE Track 1: Epigenomic analysis of single cells with controlled perturbations

INSPIRE奖的部分资金来自分子和细胞生物科学部的遗传机制和细胞动力学与功能计划、综合组织系统部的植物基因组研究计划、生物科学局的新兴前沿部门，以及工程局化学、生物工程、环境和运输系统部的生物技术、生化和生物质工程计划。染色质的DNA和蛋白质成分的表观遗传修饰是基因表达的重要决定因素，而动态的表观遗传变化被认为是生物体快速响应环境扰动的能力的基础。关于这种调控的大部分了解是通过所谓的集合方法获得的，这种方法确定从混合组织类型分离的细胞的表观遗传修饰；因此，数据代表了多个细胞的表观遗传和基因表达谱的平均值。最近的技术进步使获得单细胞的DNA序列信息和基因表达谱成为可能，但还不可能从单细胞获得表观遗传信息。在单个细胞中监测动态表观遗传图谱的能力将使表观遗传反应的随机性、稳定性和遗传性研究成为可能，从而使表观遗传学领域发生革命性的变化。该项目旨在开发和实施一条创新的管道，用于控制植物细胞的激素和胁迫扰动，随后对全基因组DNA甲基化图谱进行单细胞分析。将建立一个集成的微纳流控装置，以使单细胞能够从特定类型的细胞中分离出来，并随后进行染色质分选。然后，通过对基于超灵敏FRET的单分子测序产生的实时测序数据进行新的动力学分析，从分离的细胞中提取DNA甲基化信息。如果成功，这项新技术将能够分析单细胞的表观基因组图谱，从而使以一种革命性的新方式解决表观遗传调控的基本问题成为可能，不仅在植物中，而且在哺乳动物和微生物细胞中。除了科学影响外，该项目还将通过在分子生物学和纳米工程之间提供跨学科培训的机会，产生广泛的教育影响。博士后和研究生将直接参与研究，项目中开发的技术将被纳入研究生和本科生的动手实验室培训模块。

This INSPIRE award is partially funded by the Genetic Mechanisms and Cellular Dynamics and Function Programs in the Division of Molecular and Cellular Biosciences, by the Plant Genome Research Program in the Division of Integrative Organismal Systems, and by the Division of Emerging Frontiers in the Directorate for Biological Sciences, and also by the Biotechnology, Biochemical, and Biomass Engineering Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems in the Directorate for Engineering.Epigenetic modifications to the DNA and protein components of chromatin are important determinants of gene expression, and dynamic epigenetic changes are thought to underlie the ability of organisms to rapidly respond to environmental perturbations. Much of what is understood about such regulation has been obtained through so-called ensemble approaches, which determine epigenetic modifications from cells isolated from mixed tissue types; thus, the data represent an average of epigenetic and gene expression profiles of multiple cells. Recent technical advances have made it possible to obtain DNA sequence information and gene expression profiles for single cells, but it is not yet possible to obtain epigenetic information from single cells. The ability to monitor dynamic epigenetic profiles in single cells would revolutionize the field of epigenetics by enabling hitherto impossible studies of stochasticity, stability, and heritability of epigenetic responses. This project seeks to develop and implement an innovative pipeline for controlled hormone and stress perturbation of plant cells, followed by single-cell analysis of genome-wide DNA methylation profiles. An integrated micro- and nano-fluidic device will be built to enable single-cell isolation from specific cell types and subsequent chromatin sorting. Then DNA methylation information will be extracted from the isolated cells via new kinetic analysis of real-time sequencing data produced by ultrasensitive FRET-based single molecule sequencing. If successful, this new technology will enable analysis of epigenomic profiles of single cells, thus making it possible to address fundamental questions about epigenetic regulation in a revolutionary new way, not only in plants, but also in mammalian and microbial cells. In addition to the scientific impact, the project will have broad educational impacts by offering opportunities for cross-disciplinary training at the interfaces of molecular biology and nano-engineering. Postdoctoral and graduate students will be involved directly in carrying out the research, and the technology developed in the project will be incorporated into hands-on laboratory training modules for both graduate and undergraduate students.