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DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates approximately 90% of histone H3. In dot1delta cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.

Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for chromosome stability. Because of the nature of DNA replication, telomeres require a specialized mechanism to ensure their complete duplication. Telomeres are also capable of silencing the transcription of genes that are located near them. In order to identify genes in the budding yeast Saccharomyces cerevisiae that are important for telomere function, a screen was conducted for genes that, when expressed in high amounts, would suppress telomeric silencing. This screen lead to the identification of the gene TLC1 (telomerase component 1). TLC1 encodes the template RNA of telomerase, a ribonucleoprotein required for telomere replication in a variety of organisms. The discovery of TLC1 confirms the existence of telomerase in S. cerevisiae and may facilitate both the analysis of this enzyme and an understanding of telomere structure and function.

Mitochondria play a central role in ageing. They are considered to be both a target of the ageing process, as well as a contributor to it1. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes2, but how this occurs is poorly understood. Here, we identify a functional link between the lysosome-like vacuole and mitochondria in S. cerevisiae, and show that mitochondrial dysfunction in replicatively-aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and show that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather, by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient sensing pathways3. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential4. Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because functions of the vacuole are highly conserved throughout evolution5, we hypothesize that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.