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Live-cell multiplex super-resolution imaging of chromatin state transitions

染色质状态转变的活细胞多重超分辨率成像

基本信息

批准号：
10264073
负责人：
Lacramioara Bintu
金额：
$ 102.9万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10264073
关键词：
3-Dimensional Adopted Algorithms Amaze Animals Architecture Back Behavior Biological Assay Cell Cycle Cell Differentiation process Cell Fraction Cell Line Cell Lineage Cell physiology Cells Chromatin Chromatin Modeling Chromatin Structure Color Communities Complex DNA Dancing Development Disease Dose ES Cell Line Engineering Enhancers Epigenetic Process Equilibrium Evolution Excision Gene Activation Gene Expression Gene Expression Profiling Gene Expression Regulation Generations Genes Genetic Transcription Genome Genomics Health Hi-C Human Human Genome Image Imaging technology Individual Knock-in Label Ligation Link MYC gene Measurement Measures Mediating Memory Methods Microfluidic Microchips Microscopy Modeling Modification Molecular Mus Nature Neighborhoods Neural Crest Nuclear Optics Phase Polycomb Polymers Population Positioning Attribute Process Proteins RNA Reporter Reporter Genes Research Resolution Role Shapes Signal Transduction Sister Structure System Techniques Technology Testing Theoretical model Time Transcript Transcriptional Regulation Work cellular imaging chromatin remodeling cohesin data modeling embryonic stem cell epigenetic memory epigenome experimental study forgetting genome-wide genomic locus human embryonic stem cell imaging approach innovation long term memory novel novel imaging technology particle promoter reconstruction recruit stem three dimensional structure tool

项目摘要

Project Summary Chromatin structure and transcription regulation are essential for cellular function, and their dynamics are highly correlated both in development and in disease. However, despite decades of amazing work identifying the molecular players involved in these processes, and mapping their interactions genome-wide, we are currently unable to describe the function connecting 3D chromatin structure and transcription dynamics. This limitation stems from the fact that chromatin structure and gene expression emerge from intrinsically stochastic transitions at the single-cell level, and we are missing the critical temporal parameters associated with these transitions. Therefore, new tools to measure both chromatin structure and transcription over time in single cells are critical for understanding how the human genome is read and for predictively controlling the epigenome. Here, we propose to develop a new set of live single-cell imaging technologies to simultaneously measure changes in 3D chromatin structures and their associated dynamics of gene expression across a large range of timescales: from dynamics of individual topologically associated domains and enhancer-promoter interactions, to changes associated with stable epigenetic memory across cell cycles. For the shorter timescales (under a cell cycle), our new imaging approach combines live super-resolution microscopy of fluorescently labeled loci with end-point demultiplexing of loci identity using Optical Reconstruction of Chromatin Architecture (ORCA), in order to track and trace 3-12 points within a functional chromatin unit. This new technique, which we call live-ORCA, will allow us to measure for the first time the temporal dynamics of an entire topologically associated domain in single cells. We will use live-ORCA in conjunction with time-lapse imaging of transcriptional bursting to study the dynamics of promoter-enhancer activity throughout cell differentiation and under perturbations of the chromatin network. For the longer timescale (across multiple cell cycles), our approach will combine time-lapse microscopy of gene expression, monitoring the distance between two tagged genomic loci as a live reporter of chromatin structure, and end-point chromatin tracing of the entire gene neighborhood using ORCA. We will perform these measurements in two systems: at a highly controlled synthetic reporter where we can induce either short-term silencing or long-term epigenetic memory, and at time points in differentiation when genes commit epigenetically to a new transcriptional state. Moreover, in order to further investigate the mechanism of epigenetic inheritance, we will develop a novel microfluidic device that allows us to track changes in chromatin 3D structures across individual cell lineages. Finally, to test our quantitative understanding, we will go back and forth between these single-cell data and theoretical modelling of chromatin dynamics. This research plan will greatly advance our understanding of chromatin dynamics and its functional role in transcription regulation, while at the same time contributing a whole new set of novel imaging technologies and engineered cell lines that will serve as a jumping board for the 4D Nucleome and broader scientific community.

项目总结