Project Summary/Abstract Defects in the equal partitioning of chromosomes at cell division causes aneuploidy, a genetic catastrophe that results in spontaneous abortion or birth defects if it arises in the gametes and that is a major contributor to gene dosage imbalances in almost all human cancers. The centromere is the locus on each chromosome that directs accurate chromosome segregation at cell division in healthy cells, but a paradox exists in the field because the DNA sequences typically found at the loci are neither necessary nor sufficient for centromere function. As our major area of contribution to science, thus far, we have made major headway during the past decade in elucidating the molecular basis for centromere identity, the epigenetic pathway that propagates centromeric chromatin in perpetuity, the relationship between epigenetic and genetic information in driving centromere evolution in eukaryotes, and key steps in the quality control pathway that ensures proper chromosome segregation at cell division. In the next five years, we are poised to make quantum leaps in our molecular understanding of centromeres in three areas. The first area is with a new type of human artificial chromosome (HAC) that we have recently developed. We will gain new insight regarding the relationship between DNA sequence and centromere formation and expand the utility of HACs in experimental and applied settings. The second area is with mouse models and biochemical reconstitution to expand our understanding of the balance of epigenetics and genetics at the centromere. We will build on our success with gaining the first molecular evidence in mammals of an evolutionary process known as “centromere drive” to now define the relationship of this process to the expansion of centromeric satellite DNA sequences. In addition, we will investigate the role of the centromere repeats typically found at human centromeres on the physical properties of centromeric chromatin using purified components. The third area is with a combination of biophysical, cell biological, and epigenomic approaches to extend our understanding of centromere regulation at mitosis. We will focus on the chromatin at the “inner centromere” (i.e. between the pair of sister centromeres on a mitotic chromosome) that plays a key role in the quality control step known as mitotic error correction. Altogether, our progress in these three areas will constitute a major advance in our understanding of the molecular mechanisms underlying the specification and regulation of centromeres.