Structural and functional dynamics of chromatin by live-cell genome imaging

Abstract

The structural dynamics of chromatin provide access to nuclear machinery, thus play a central role in regulating mammalian genome during DNA replication, repair, and gene regulation. The structural organization and dynamics of chromatin exhibit in time and space. To explore this structure–function relationship, it is necessary to directly visualize genomic elements in living cells. These important and transient nuclear process are not elucidated by current chromatin conformation methods. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. Next, we use CRISPR imaging system to simultaneously induce loop like chromatin extrusions at the repeat-rich genomes and visualize the ultra-structures of endogenous chromatin by live-cell microscopy. Our results revealed ultrastructural dynamics of chromatin suggesting cell cycle-dependent structural changes in physical size and shape. Loop like chromatin extrusion fibers shows preferential and directional movement towards nucleolus during G1/S phase to late S phase and re-establishment of compact chromatin structure as cell progress to G2/M phase of cell cycle. We further demonstrate that recruitment of 53BP1 fused with dCas9 to the target sites condensed the chromatin and protects from higher-order structure formation. Our study provides an attractive approach to engineering the 3D genome to harness the regulatory function of genome for diagnosis and therapeutic purposes.