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

点击翻译按钮获取中文摘要

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.