A role for centromeric alpha satellite DNA in the formation and function of human centromeres is strongly implicated by chromosome fragmentation and re-introduction studies and by its association with CENtromere Protein-A (CENP-A) and CENP-C. However, neocentromeres, found in mitotically stable rearranged marker chromosomes, have no detectable alpha satellite DNA at inactive centromeres in stable dicentric chromosomes, and in artificially created ectopic alpha satellite DNA arrays, do not form functional centromeres and do not bind CENPs. Thus, these paradoxical findings suggest that centromere formation: 1) may not be strictly sequence dependent, 2) is reversible and plastic, and 3) requires the presence of certain CENPs. Thus, the following three objectives are designed to further examine the epigenetic chromosomal modifications involving centromeric DNA and associated proteins that are required to form and propagate centromeres. 1) Mini-chromosomes obtained by a novel in vivo strategy for the mitotic rescue of acentric chromosomal fragments will be characterized, and more efficient vectors will be developed. Mitotic rescue vectors containing alpha satellite DNA sequences with cloned telomeric DNA at one end and selectable markers on the other are designed to fragment chromosomes efficiently by Telomere Associated Chromosome Fragmentation (TACF). Acentric chromosomal fragments will become mitotically stable if the integrated DNA forms a centromere. At least one HeLa cell line is being characterized with small mitotically stable chromosomal fragments that contain the integrated DNA and kinetochore proteins. Modified mitotic rescue vectors will be made that incorporate a gene expressing Green Fluorescent Protein (GFP) in order to more efficiently obtain mini-chromosomes. The relative frequencies of mitotic rescue and other predicted integration and TACF events will allow quantitative assessment of the frequency and relative efficiency of minichromosome formation. 2) The ability of CENP-A to enhance the efficiency of de novo centromere formation will be investigated. Endogenous expression of human CENP-A is limited to late S/G2 phase of the cell cycle. The mitotic rescue vectors developed in objective 1 will be transfected into HeLa cells that contain an epitope-tagged (HA) human CENP-A gene under Tetracycline-regulated expression control. Thus, providing a pool of available CENP-A for incorporation into the nucleosomes of transfected DNA may allow formation of centromeric chromatin and an increased frequency of mini-chromosomes. 3) The ability of DNA-associated centromere proteins such as CENP-A and CENP-C to establish kinetochore structure will be investigated. DNA coding for these proteins will be fused to DNA coding for the alpha satellite DNA binding domain of CENP-B. Chimeric fusion proteins will be transiently expressed in mammalian cells containing ectopic alpha satellite DNA or dicentric chromosomes, thereby targeting the kinetochore proteins to inactive and ectopic alpha satellite DNA. The formation of kinetochores at these locations will be detected by aberrant chromosome behavior during mitosis, monitored by immunofluorescence to epitope tags in the chimeric proteins.
Investigation of centromere functions will provide information on the fundamental process of chromosome segregation and cell division. Since telomeres have been cloned and shown to be functional when reintroduced into cells, and origins of replication appear to be abundant and easily provided, the centromere remains the least characterized functional component of chromosomes. Only when the functional components of the centromere are defined will it be possible to readily construct artificial human chromosomes, which will prove invaluable to understanding chromosome dynamics during cell division. Furthermore, since chromosomes, by definition, contain all the elements necessary for their own maintenance during cell division, artificial chromosomes may be extremely valuable mitotically stable gene expression vectors for use in cultured cells or dividing tissues such as stem cells.
