Mitotic centromeres organize in repeat-subunits with specialized histone landscape and multi-domain architecture as revealed by expansion microscopy

Centromeres ensure faithful chromosome segregation during cell division, yet their underlying architecture and spatial arrangement of centromeric proteins remain incompletely resolved. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to human diploid hTERT RPE-1 cells to visualize mitotic centromeres at nanoscale resolution. We find that the centromeric histone variant CENP-A and the kinetochore protein KNL1 form discontinuous, repeating substructures. We further resolve centromeric chromatin into four spatially distinct domains: an inner kinetochore domain marked by CENP-A, a mantle domain marked by RAD21, an outer core domain marked by CENP-B, and a distinct, RAD21-associated inner centromere domain. Core and pericentromeric regions share a specialized chromatin landscape, extending the concept of centrochromatin, with enrichment of H3S10ph and H3K9me3, depletion of H3K27me3, and low-level enrichment of the euchromatic marks H3K4me3 and H3K36me3. Together, we uncover a discontinuous repeat-subunit organization and multidomain architecture of the human centromere that combines euchromatic and heterochromatic modifications.