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

The human centromere comprises large arrays of repetitive a-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 a-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of a-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 a-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 chromo-somes follows a rounded pattern similar to that of a-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Collec-tively, our data provide a 3D reconstruction of a-satellite DNA along with CENP-A clusters that outlines the overall architecture of the mitotic centromere.