Chromosome inheritance ensures transmission of genetic and genomic information. Abnormal chromosome number (aneuploidy) and altered chromosome structure cause birth defects, reproductive abnormalities, and cancer. The centromere is the locus required for chromosome segregation and genome stability. Normal chromosomes typically have one centromere, but genome rearrangements associated with birth defects and cancer produce chromosomes in which two centromeres are physically linked. These dicentrics are not naturally tolerated in most model organisms, as originally illustrated in maize by Barbara McClintock nearly 75 years ago. In humans, dicentric chromosomes occur non-randomly and can be extremely stable during cell division. Such stability has been attributed to centromere inactivation, the poorly understood process by which one centromere is functionally suppressed. Our goal is to define molecular pathways responsible for dicentric formation and long-term stability. We hypothesize that (A) formation of common dicentrics is linked to genomic features of acrocentric chromosomes, and (B) centromere inactivation routinely occurs by either genomic or epigenetic mechanisms. A major impediment in studying centromere inactivation in humans has been the absence of experimental systems. To circumvent this long-standing problem, we have developed assays to engineer dicentric human chromosomes that molecularly mirror those that occur naturally. Engineered dicentrics represented a model system to study normal centromere function, centromere repression and genome stability. Using these experimental models, we propose three specific aims: 1) To identify sequence-dependent mechanisms of dicentric formation, 2) To define the molecular pathways by which dicentric chromosomes are stabilized, including genomic, epigenetic and temporal changes associated with centromere inactivation, and 3) To experimentally test models of centromere inactivation using protein tethering and engineered genomic deletions. Collectively, these studies will define the mechanistic basis for non-random participation of acrocentric chromosomes in naturally occurring and experimentally produced chromosome fusions. Our studies will also be critical for evaluating current models of centromere function, and provide new insights into mechanisms that specify and maintain centromeres on human chromosomes.