The three-dimensional structure of in vitro reconstituted Xenopus laevis chromosomes by EM tomography

Abstract

We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30-40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30-40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30-40-nm chromatin domains. These observations suggest that 30-40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation.