Project Summary Cells must accurately segregate their chromosomes into daughter cells at each division in order for cells to reproduce and proliferate. Mistakes in chromosome segregation lead to imbalances in chromosome copy number termed aneuploidies that are hallmarks of cancer, that cause developmental diseases such as Down syndrome and that are a primary cause of miscarriage. This work is focused on understanding how cells maintain a normal chromosome copy number during the process of chromosome segregation. The key regulator of chromosome segregation is the chromosomal kinetochore. The kinetochore is the site on each chromosome that attaches to the microtubules of the mitotic spindle so that chromosome can move to daughter cells during cell division. Kinetochores also monitor proper chromosome alignment through the mitotic checkpoint to ensure that each daughter cell gets one copy of each chromosome. The foundation for kinetochore formation is a region of the chromosome termed the centromere. Centromeres are a chromatin domain that is uniquely determined by the presence of a variant histone termed centromere protein A (CENP-A). The formation of centromeres is epigenetically determined by the presence of CENP-A in chromatin and once a centromere is formed it can be stably maintained regardless of the DNA sequence on which it is built. CENP-A chromatin is essential for centromere and kinetochore formation and mutation or loss of CENP-A results in centromere loss and chromosome missegregation. We are studying the mechanisms that assemble and maintain CENP-A chromatin and how CENP-A chromatin is recognized to build the centromere and kinetochore. In our first specific Aim we dissect the biochemical mechanisms that build CENP-A nucleosomes at the right time and place in the chromosome. In particular, we focus on understanding how a key regulator of new CENP-A nucleosome formation, the Mis18 complex, is properly targeted to centromeres and how it functions in CENP-A assembly. In our second Aim we explore the function of DNA sequences in centromere formation. Although CENP-A is the primary epigenetic determinant of centromere function the sequence of the DNA at centromeres promotes efficient centromere formation and maintenance. We test whether specific DNA sequences stimulate CENP-A assembly and whether DNA or RNA molecules actively regulate new CENP-A nucleosome formation. Together our approach defines the basis for centromere formation and how the proteins of the centromere give rise to the properties required for chromosome segregation in mitosis.