Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by superresolution microscopy.

The human centromere comprises large arrays of repetitive α-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Because CENP-A is bound to only a fraction of the α-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of α-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the centromere chromosome orientation fluorescence in situ hybridization (Cen-CO-FISH) technique together with structured illumination microscopy. Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that α-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in the presence or absence of spindle's microtubules. Using expansion microscopy, we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of α-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Collectively, our data provide a 3D reconstruction of α-satellite DNA along with CENP-A clusters that outlines the overall architecture of the mitotic centromere.

Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization.

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.