Histone Density and Dynamics Shape Mitotic Chromatid Architecture in <i>Xenopus</i> Egg Extracts

Mitotic chromosomes, consisting of a pair of rod-shaped chromatids, emerge from a dramatic reorganization of nucleosome fibers. However, the mechanisms by which local chromatin organization influences large-scale mitotic chromatid architecture remain poorly understood. Here, we report a modified cell-free assay in which erythrocyte nuclei, instead of conventionally used sperm nuclei, are incubated in Xenopus mitotic egg extracts. This modification enables mitotic chromatids to assemble from substrates containing regularly spaced, dense nucleosome arrays, allowing chromatid formation to be experimentally separated from nucleosome assembly. In this system, depletion of the linker histone H1.8 results in thinner chromatids with enhanced individualization, whereas excess H1.8 loading promotes chromatid clustering through its C-terminal tail. Chromatid clustering is also observed upon depletion of the histone chaperone Nap1 or the chromatin remodeler ISWI, whose roles have been underappreciated in conventional assays. Together, our findings demonstrate that histone density and dynamics cooperate with condensins and topoisomerase IIα to shape mitotic chromatid architecture.