Functional analysis and genetic mapping of mouse centromere protein genes

This study focuses on the functional analysis and genetic mapping of a number of the centromere-associated protein genes. The mammalian centromere is the pinched region on metaphase chromosomes and is made up of an inner heterochromatin core coated with a protein complex known as the kinetochore. Numerous constitutive and transient proteins assemble at the centromere-kinetochore complex during mitosis and meiosis facilitating microtubule attachment and accurate separation of sister chromatids. Aberrations in cell division may lead to chromosome missegregation and a range of pathologies including cancer and miscarriage. The function of many centromere-associated proteins has been studied in yeasts, fungi, invertebrates and mammalian cell culture systems however their roles are undefined in whole animals. To determine the requirement for six centromere-associated proteins during mitosis, meiosis and development, gene targeting experiments in mice were undertaken. Heterozygous mice carrying disrupted Cenpa, Cenpb, Cenpc, Incenp, survivin (Api4) and Bub3 genes were healthy and fertile however, with the exception of Cenpb, their null offspring were embryonic lethal. The Cenpc-, Incenp- and Api4-deficient embryos failed to hatch from their zona pellucida and implant, whereas the Cenpa and Bub3 knockout embryos were able to hatch and form trophectoderm and inner cell mass outgrowths before mitosis halted at day-5.5 to -6.5 post conceptus. Morphological examination revealed affected embryos with severe mitotic problems including formation of micro- and macronuclei, nuclear bridging and lagging chromosomes. Surprisingly the Incenp- and Api4- disrupted embryos displayed bundling of microtubules and giant nuclei suggesting that these proteins act in concert in the regulation of microtubule dynamics and/or cell cleavage. In contrast, Cenpc appears to be essential at the metaphase stage of mitosis; and in an absence of Cenpa, immunofluorescence stainings demonstrated that Cenpb and Cenpc DNA-binding proteins were unable to form functional kinetochores. Finally Bub3- disrupted embryos failed to arrest following treatment with the microtubule-poison nocadazole indicating a vital role in the mitotic spindle checkpoint pathway. In comparison, assessment of Cenpb-null mice demonstrated that Cenpb is not required for mitosis and meiosis. However Cenpb-null mice are smaller in size and display significantly smaller testis (>14 %) and uteri (>30%) in adulthood. The observed body-weight reduction is dependent on the genetic background (R1, W9.5 and C57) as well as the sex of the mice. Likewise Cenpb-deficient female mice exhibit age-related uterine dysfunction that is more severe in the C57 background. Histological analysis of the uterus points to defective luminal and glandular epithelium as the likely primary cause. In a complementary study the chromosomal position of six of the centromere-associated protein genes was determined with a view to identifying possible candidate mouse mutants in the mouse database. The genetic mapping of the Cenpa, Cenph, Cenpe, Cenpf, Incenp and Bub3 loci to chromosomes 5, 13, 6, 1, 19 and 7 respectively, was achieved by linkage analysis using specific probes to analyse sets of recombinant inbred mouse strains and Mus spretus-based interspecific backcross panels. The mapping of these loci failed to reveal any pre-existing mouse mutants for further study however the mapping of Cenpa and Cenpe did extend or identify novel regions of mouse-human homology. Overall, the discoveries made in these studies have provided new insight into the functional intricacy of the centromere-kinetochore complex. Furthermore the observed phenotypes in mice bearing disrupted centromere protein genes provide fresh genetic clues to understanding the pathogenesis of mammalian pregnancy disorders such as miscarriage and pyometra.