Labeling DNA Replication Foci to Visualize Chromosome Territories In Vivo

Abstract

While a detailed understanding of chromatin dynamics is needed to explain how higher-order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin dynamics by labeling DNA during S phase. Using these methods, we have found that foci labeled during early and mid/late S phase have significantly different dynamic behavior. Spatially constrained heterochromatic foci restrict long-range transformations of the chromosome territory (CT) structure while providing a structural framework on which highly mobile euchromatic foci undergo positional oscillations that drive local changes in the chromosome shape. Despite often dramatic mobility, we have demonstrated a preservation of structural integrity which ensures that DNA from neighboring CTs is not able to mix freely within the same nuclear space. Finally, other potential applications of the presented protocols are discussed. © 2017 by John Wiley & Sons, Inc.