The long-term goal of this research is to identify the mechanisms by which chromosomal structure contributes to the down regulation of gene expression and to characterize this regulation during the cell cycle by using S. cerevisiae as a model for other eukaryotes. S. cerevisiae has three loci, HMR, HML, and MAT, that encode alternative alleles, a or alpha, which determine the mating type of yeast. The allele at MAT is expressed. However, the alleles at HMR and HML are not transcribed, or silenced by the combined action of specific sequences on the chromosome called silencers, and several proteins, including SIR1, SIR2, SIR3, and SIR4, which do not bind DNA directly. The point in the cell cycle when the establishment of silencing occurs will be defined by monitoring mRNA expression of alpha1 from a modified HMR regulated by a synthetic silencer in the presence of hydroxyurea, a temperature-sensitive mutant of CDC9, or nocodazole to block cells in S, G2 or M phase, respectively. The role of DNA replication in establishing silencing will be assessed by using FLP1 recombinase to excise this modified HMR from its chromosomal context. To bypass the role of SIR1 in establishing silencing, SIR3 and SIR4 will be localized to a synthetic silencer containing Ga14 DNA binding sites via their association with the amino terminal tails of histones H3 and H4 fused to the Ga14 DNA binding domain.
| Rusche, Laura N; Kirchmaier, Ann L; Rine, Jasper (2002) Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. Mol Biol Cell 13:2207-22 |
| Kirchmaier, A L; Rine, J (2001) DNA replication-independent silencing in S. cerevisiae. Science 291:646-50 |
