CRISPR-based multi-locus real-time tracking reveals single chromosome dynamics and compaction

Abstract

In eukaryotic nuclei, individual chromosomes occupy discrete three-dimensional spaces with little overlap. Dynamic chromatin organization instantly influences DNA accessibility through modulating local macromolecular density and interactions, driving changes in transcription activities. Chromatin anchoring to nuclear landmarks often leads to massive reorganization and motion changes. Chromatin dynamics has been reported to be locally confined but contributes to the large-scale coherent chromatin motion across the entire nucleus. However, the dynamics and compaction of chromosomal sub-regions along a single chromosome are not well-understood. In this study, we combined quantitative real-time single-molecule fluorescence microscopy, CRISPR-based genomic labeling, biophysical analysis and polymer models to characterize the dynamics of specific genomic loci and chromatin-nuclear landmark interactions on human chromosome 19 in living cells. Precise genomic labeling allows us to dissect loci motions and chromatin elasticity on a single chromosome basis. We found that the dynamics of genomic loci were all subdiffusive but varied at different regions along the chromosome. The mobility of genomic loci was similar among interior chromosomal loci but deviated for the loci at pericentromeric and near-telomeric regions. Tighter compaction on chromosome 19 long arm, compared to the short arm was observed, which may correlate to more active genes on the short arm than the long arm, shown by our RNA-seq analysis. The strong tethering interaction was found for loci at the pericentromeric region, suggesting a higher degree of local condensation, perhaps through stronger interactions or association between pericentromeric regions and their microenvironments, such as chromatin-nuclear body association through sequence-specific domains on DNA.