Project Summary The process of cell division requires that each cell receive an identical complement of the genome. Errors in chromosome segregation give rise to chromosomal aneuploidies that are causative for human genetic disease such as Down syndrome and promote the progression of most human cancers. The work in this proposal is focused on understanding the functions of the centromere and kinetochore, the primary site on the chromosome that attaches each chromosome to the mitotic spindle for proper segregation during division. The centromere and kinetochore serve as the primary control center for chromosome segregation through their roles in microtubule attachment, force coupling for chromosome movement, and error correction when chromosome attachment or alignment on microtubules is perturbed. Underlying the centromere is a specialized chromatin domain that is delineated by replacement of histone H3 in nucleosomes with a centromere specific histone variant termed CENP-A. The position of CENP-A defines where the centromere and kinetochore will form and defects in CENP-A assembly or maintenance cause centromere and kinetochore loss which result in chromosome missegregation. In this proposal we study three different aspects of CENP-A chromatin. In our first Aim we work to understand how the assembly of CENP-A chromatin is controlled so that the centromere is replenished during each cell cycle and maintained through replication and cell division. We focus on three important factors for CENP-A assembly, CENP-C, HJURP and the Mis18 complex that are essential for building new CENP-A nucleosomes at centromeres. Our second aim studies the problem of how the presence of CENP-A in chromatin dictates the sites of new CENP-A assembly. We apply a new method we have developed called DiMeLo-seq that overcomes existing limitations in studying the complex repetitive centromeres of vertebrates. This approach uses directed methylation to mark the sites where proteins are bound to chromatin and then uses long read sequencing to map the binding sites through the repetitive centromere sequences. We use this approach to study the density, distribution, and sites of regeneration of CENP-A through the cell cycle. In our third aim we study how centromeres are condensed and organized by the activities of CENP-A nucleosome binding proteins. We discovered that two centromere proteins (CENP-C and CENP-N) can bridge CENP-A nucleosomes and condense centromeric chromatin. We use human cells to test the role of this condensation activity in centromere condensation, resistance to force, and chromosome segregation. Overall, our proposal will elucidate the regulatory mechanisms and structural organization of vertebrate centromeres and how those properties ensure accurate chromosome segregation.