| The centromere is a complex locus characterized by heterochromatic DNA and associated proteins and is essential for the faithful distribution of a cell's genetic material to subsequent generations. Despite intense scrutiny, how the centromere DNA and associated kinetochore proteins come together to create a functional centromere is still unknown. We have developed a somatic cell hybrid model system, the Pushmi-Pullyu system, that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp)s, in culture. Cytogenetic analysis of these hybrids using fluorescence in-situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. These Pushmi-Pullyu isochromosomes were then used to study the formation and behavior of mono- and dicentric isochromosomes. Pulse field gel electrophoresis was used to analyze the centromere DNA of monocentric isochromosomes. These data suggest that mammalian isochromosomes can form as a result of centromere misdivision, as proposed by Darlington in 1939.;Analysis of dicentric Pushmi-Pullyu i(Xp) chromosomes uncovered three classes of dicentric chromosomes isolated in different cell lines: functionally monocentric chromosomes, in which one centromere is consistently inactivated, functionally dicentric chromosomes, in which both centromeres are consistently active, and dicentric chromosomes heterogeneous with respect to centromere activity. Characterization of all three classes of dicentric chromosomes with ∼<20 Mb of intercentromere distance revealed no influence on centromere activity and all classes were stably transmitted during anaphase. A study of serial single cell clones from heterogeneous cell lines revealed that while centromere activity is generally clonal, the centromere state (i.e. active or inactive) can switch within a growing population of cells. These experiments further defined the heterogeneous class of dicentric chromosomes into three types: stable functional monocentric chromosomes, stable functional dicentric chromosomes and dicentric chromosomes that are prone to switching their centromere state.;Switching of the centromere state appears to be influenced by epigenetic changes. Investigation of possible epigenetic mechanisms at work at the centromere revealed that histone acetylation may play a role in centromere function. This study provides evidence that the activity of mammalian centromeres, while largely stable, can be subject to dynamic change, most likely due to epigenetic modification. |
