Live Cell Visualization of Chromatin Structure and Dynamics

Abstract

Recent studies have suggested that chromosomes are hierarchically organized into topologically associated domains. Direct visualization of the genomic elements in living cells is required to explore the relationship between such structural organization and its functional roles. To study the structural dynamics of genomic elements, we have developed an improved CRISPR-based imaging system for visualization of arbitrary non-repetitive gene loci in living cells. Use of split-fluorescent proteins nearly eliminated background fluorescence and signals from non-specific aggregation, allowing reliable long-term tracking of genomic loci. High resolution imaging of pericentromeric C9-1 region in S phase revealed highly extended fiber-like structures of chromatin. Deep-learning-based analysis of the chromatin extension revealed that they move toward nuclear membranes or nucleoli. Fusion of 53BP1 to CRISPR eliminated the chromatin extension. CRISPR imaging of centromeres revealed same behaviors. The chromatin extension is possibly induced by homology-assisted repair of damaged genome, which is suppressed by 53BP1 that forces non-homologous end joining. Our approaches provide novel tools to study the mechanism of reorganizing chromosomes and its relevance in genome replication, repair, and the regulation of gene expression.