Silencing And Yeast Aging

Yeast cells can be of two "sexes"—referred to as MATa and MATa mating types. All yeast cells contain the genetic information for both mating types. Yeast cells are normally fertile because the two repositories of mating-type information, HML

and HMR, are maintained in a transcriptionally silenced state. Yeast are capable of switching between MATa and MATa mating types. To switch between the two mating types, a cell must transpose the opposite, silenced, information into the mating type locus where it is then expressed. The maintenance of silencing at HML and HMR requires the activity of many genes, including SIR2, SIR3 and SIR4 (17,18).

The first phenotype of old yeast cells to be explained on a molecular level was sterility. That sterility is caused by a loss of silencing at the silenced mating type loci HML and HMR was demonstrated when the a1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in old cells of MATa mating type (10). The a1 mRNA is not normally expressed in MATa strains; expression of both a and a mating type information leads to sterility in yeast. That sterility is caused by loss of silencing also has been demonstrated directly by the failure of old MATa cells to respond to a mating factor. The response of old MATa cells to a mating factor was restored by deletion of HMLa, indicating the derepression of the HM loci is the causal agent in the age-specific phenotype of sterility (10). In addition to the loss of silencing at the silenced mating type loci, a loss of silencing at telomeres also occurs in old cells (19).

The importance of silencing in yeast aging was independently indicated when a SIR protein was identified in a screen for long-lived mutants. Genetic screens for mutations that extend life span in S. cerevisiae have been limited by the technical obstacle that the life span can only be determined for individual cells and only by micromanipulation. The performance of an unbiased aging screen is impractical. To circumvent this limitation, Kennedy et al. (20) capitalized on the well-established correlation between stress resistance and longevity (reviewed in ref. 21). A genetic screen for starvation resistance mutations was performed, and each mutant was independently tested for increased longevity. Four complementation groups were obtained (UTH1-4) that increased the life span by 20-55%. Two of these, UTH2 and UTH4, were further characterized in detail. Both of these genes have been shown to play a role in transcriptional silencing.

To facilitate subsequent life span determinations, the strain used in the genetic screen for stress resistance was chosen for its short life span. The shorter life span was due to a frame-shift mutation in the UTH4 gene that truncated the gene product after 207 residues (22) (designated the uth4-14c allele). UTH2 was found to be an allele-specific suppressor of uth4-14c mutation and is an allele of S1R4. This mutation, S1R4-42, was created by the generation of a stop codon that removes 121 residues from the C-terminus of Sir4p. This region of Sir4p has been shown to bind to Rap1p (23,24), a protein that is present at telomeres (25,26) and at mating-type loci (27). Overexpression of the terminal 154 residues of Sir4p not only eliminated silencing at the silent mating type loci and at telomeres, but also extended the life span only in strains expressing wild-type S1R4 (20).

The S1R4-42 mutation behaved like a null allele with respect to silencing at the mating-type loci and telomeres; silencing was abolished. However, only the

S1R4-42 allele, and not S1R4 deletion, extended the life span. S1R4-42 was dominant to S1R4 for life span extension. A null mutation in S1R4 actually shortened the life span. The extension of the life span by the S1R4-42 allele required S1R2 and S1R3, indicating that this was a property of the SIR complex (20).

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