DOT4 Links Silencing and Cell Growth in Saccharomyces cerevisiae

Abstract

Transcriptional silencing in Saccharomyces cerevisiaeoccurs at specific loci and is mediated by a multiprotein complex that includes Rap1p and the Sir proteins. We studied the function of a recently identified gene, DOT4, that disrupts silencing when overexpressed. DOT4 encodes an ubiquitin processing protease (hydrolase) that is primarily located in the nucleus. By two-hybrid analysis, the amino-terminal third of Dot4p interacts with the silencing protein Sir4p. Cells lacking DOT4 exhibited reduced silencing and a corresponding decrease in the level of Sir4p. Together, these findings suggest that Dot4p regulates silencing by acting on Sir4p. In strains with several auxotrophic markers, loss of DOT4 ubiquitin hydrolase activity also results in a slow-growth defect. The defect can be partially suppressed by mutations in a subunit of the 26S proteasome, suggesting that Dot4p has the ability to prevent ubiquitin-mediated degradation. Furthermore, wild-type SIR2, SIR3, and SIR4 are required for full manifestation of the growth defect in a dot4 strain, indicating that the growth defect is caused in part by a silencing-related mechanism. We propose that Dot4p helps to restrict the location of silencing proteins to a limited set of genomic loci.

ACKNOWLEDGMENTS

We thank J. Berman, M. Hochstrasser, P. James, D. Moazed, P. Philippsen, M. Roth, and R. Sternglanz for generously providing plasmids, yeast strains, and/or antibodies, and we thank Liz Wayner for her expertise and help in generating antiubiquitin antibodies. We also thank our friends at the FHCRC and The University of Chicago for helpful discussions and/or critical reading of the manuscript.

A.K. gratefully acknowledges support from a Glenn Foundation/AFAR Award for Aging Research and Medical Scientist National Research Service Award 5T32 GM07281. This work was supported by a Pew Charitable Trust Biomedical Scholars Fellowship, a Cancer Research Foundation Fletcher Scholarship, and National Institutes of Health grant GM43893 (D.E.G.).