We have used a large reference set of chromosome transmission fidelity mutants, the ctf mutants of S. cerevisiaein a colony color assay for chromosome loss. The first gene we characterized was S. cerevisiae SPT gene. We have established that Spt4p is a component of centromeric and heterochromatic chromatin with roles in kinetochore function and gene silencing. A human homolog of SPT4, HsSPT4, is able to functionally complement the phenotypes of S. cerevisiae spt4mutants. These studies represent one of the first examples of the silencing defects of a yeast mutant (spt4)being complemented by a human gene (HsSPT4) and the in vivoassociation of a human protein to the kinetochores of budding yeast. We also determined that spt4 mutants mis-localize the centromeric histone variant Cse4p to non-centromeric regions. Mis-localization of CENP-A, the human homolog of Cse4p has been reported in colorectal cancers. We have recently established that dosage of histone H3 and Cse4p affect chromosome transmission fidelity. Our preliminary studies show that in addition to kinetochore proteins, the state of centromeric chromatin is crucial for chromosome transmission fidelity. Future studies will establish the molecular role of Spt4p and its interacting partners Spt5p and Spt6p as well as histones in chromatin structure, chromosome segregation and gene silencing in both yeast and humans. To demonstrate the functional relevance of our findings in S. cerevisiae, we plan to extend our research to higher eukaryotes. To this end we are collaborating with the laboratory of Dr. Natasha Caplen in RNAi studies to investigate the role of human Spt4p/Spt5p/Spt6p in chromosome segregation and function of CENP-A. Studies of the second kinetochore mutant showed that the nucleoporin gene NUP170 is required for chromosome transmission fidelity in S. cerevisiae. We established an important physical and functional link between members of the Nup170p protein complex and mitotic spindle checkpoint proteins Mad1p and Mad2p. Spindle checkpoint proteins (Mad1p, Mad2p, Mad3p, Bub1p, Bub3p and Mps1p) monitor the interaction of the spindle apparatus with the kinetochores and halt anaphase if the microtubule attachment of even a single chromosome is altered. Ongoing studies are beginning to shed light led on the distinct sub-domains within Mad1p that are required for nucleopore or checkpoint/chromosome segregation functions. Studies with a third spindle checkpoint protein, Bub3p, have demonstrated for the first time that this spindle checkpoint protein can associate in vivo with a single defective kinetochore. Our experimental system will allow us to establish the order of assembly of checkpoint protein complexes and help elucidate the domains of checkpoint proteins required for chromosome segregation and checkpoint and nucleopore functions in both yeast and humans. In addition to chromosome segregation, the DNA damage and replication checkpoint pathways ensure genome stability by halting the cell cycle in response to genotoxic stress. We have recently established a functional relationship between oxidative stress genes SOD1 and LYS7and the MEC1 mediated checkpoint pathway for DNA damage and replication arrest. We will continue our studies with Sod1p and Lys7p to unravel molecular mechanisms studies that correlate oxidative stress, redox state and checkpoint pathways in S. cerevisiaethat may be applicable to other systems. Our research on the molecular determinants of faithful chromosome transmission in S. cerevisiaewill help us understand analogous processes in humans and their implications in human disease. Our laboratory is uniquely poised to utilize the conventional genetic, biochemical, and cell biology approaches, as well as high-throughput genomic analysis for our research projects. We use an array of gene-deletion strains and a colony picking robot for the identification of possible cancer drug targets and also for genetic screens by Synthetic Genome (SGA) analysis, developed in the laboratory of Charlie Boone (Univ. of Toronto)
| Deng, Yi; Guo, Yan; Watson, Hadiya et al. (2009) Gga2 mediates sequential ubiquitin-independent and ubiquitin-dependent steps in the trafficking of ARN1 from the trans-Golgi network to the vacuole. J Biol Chem 284:23830-41 |
