Spatial Distributions of Early and Late Replicating Chromatin in Interphase Chromosome Territories

Abstract

The surface area of chromosome territories has been suggested as a preferred site for genes, specific RNAs, and accumulations of splicing factors. Here, we investigated the localization of sites of replication within individual chromosome territories.In vivoreplication labeling with thymidine analogues IdUrd and CldUrd was combined with chromosome painting by fluorescentin situhybridization on three-dimensionally preserved human fibroblast nuclei. Spatial distributions of replication labels over the chromosome territory, as well as the territory volume and shape, were determined by 3D image analysis. During late S-phase a previously observed shape difference between the active and inactive X-chromosome in female cells was maintained, while the volumes of the two territories did not differ significantly. Domains containing early or mid to late replicating chromatin were distributed throughout territories of chromome 8 and the active X. In the inactive X-chromosome early replicating chromatin was observed preferentially near the territory surface. Most important, we established that the process of replication takes place in foci throughout the entire chromosome territory volume, in early as well as in late S-phase. This demonstrates that activity of macromolecular enzyme complexes takes place throughout chromosome territories and is not confined to the territory surface as suggested previously.