DOT4 links silencing and cell growth in Saccharomyces cerevisiae

Abstract

Transcriptional silencing in Saccharomyces cerevisiae occurs 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.

FIG. 1

Dot4p mutant map and Cys box sequence. (A) Dot4p contains sequence homology to the active site Cys box of other yeast deubiquitinating enzymes. (B) Schematic map of the Dot4 protein and various engineered alleles.

FIG. 2

Overexpression of the amino terminus of Dot4p disrupts silencing. (A) A wild-type DOT4 strain (UCC3505) containing URA3 near a telomere was transformed with a high-copy TRP1 plasmid carrying various alleles of DOT4 under the control of the DOT4 promoter. Telomeric silencing was measured by testing for growth on media lacking uracil in a serial dilution plating assay. In the case of dot4-2, control of transcription occurred through the GAL1 promoter, and silencing was assayed on medium containing galactose as the sole carbon source. (B) The extent of dot4-5 overexpression in panel A was determined by comparison with wild-type DOT4 overexpression, using Western analysis of total yeast protein extracts. Proteins were tagged with a six-Myc epitope, and blots were probed with anti-Myc antibodies (α-myc). Prestaining of the blot with India ink showed equivalent sample loading.

FIG. 3

Dot4p interacts with Sir4p in a two-hybrid assay. (A) A two-hybrid assay was used to test the interaction between a Dot4p bait and Sir4p C-terminal preys. GAL4BD-DOT4 fusions (bait) on a TRP1 plasmid and GAL4AD-SIR4 fusions (prey) on a LEU2 plasmid were transformed into PJ69-4a, which contains HIS3 and ADE2 under the control of synthetic GAL promoters. The ability of cells to grow in the absence of histidine or adenine was tested in a serial dilution plating assay. Activation of HIS3 and ADE2 indicates a positive interaction between the two protein fusions. WT, wild type. (B) Dot4p-Sir4p two-hybrid interaction was tested in a PJ69 background in which SIR2 (UCC4773), SIR3 (UCC4774), or SIR4 (UCC4775) was deleted.

FIG. 4

Dot4p is localized primarily to the nucleus. Dot4p localization was determined by using a DOT4-GFP-S65T gene fusion integrated at the DOT4 genomic locus of a diploid yeast strain (UCC4606). The strain (UCC4818) is heterozygous for the fusion gene. By fluorescence microscopy, the GFP signal was compared to a DAPI signal in cells that were fixed during exponential growth phase in 3.7% formaldehyde. (A to C) Control experiment using parental strain UCC4606 which lacks DOT4-GFP fusion; (D to F) experiment in which strain UCC4818 was stained with DAPI and tested for localization of Dot4-GFP.

FIG. 5

Dot4p and its deubiquitinating activity are important for transcriptional silencing. (A) Silencing was measured in DOT4 (UCC4877) and dot4Δ (UCC4879) strains grown on complete, uracil-lacking, and 5-FOA-supplemented media. These strains are fully prototrophic except that the URA3 gene was placed near a telomere and PPR1 was deleted. Growth on medium lacking uracil indicates a disruption of telomeric silencing. (B) Northern analysis of URA3 expression at a telomeric (Tel; UCC4877 [DOT4] and UCC4879 [dot4Δ::KanMX]) or internal (Int; BY4712 [DOT4] and UCC4884 [dot4Δ::KanMX]) locus. Northern analysis of PDA1 mRNA was used as a loading control in a parallel loading experiment. (C) A strain containing ADE2 near a telomere (UCC4825) was used to test the effects of two DOT4 alleles on silencing. DOT4 was deleted (UCC4857) or replaced in the genome with the dot4-1 (UCC4870) or dot4-5 (UCC4896) alleles, and telomeric silencing of ADE2 was observed following growth on rich medium. ADE2 expression results in a white colony color, whereas ADE2 repression results in a red colony color.

FIG. 6

Sir4p levels decrease in a dot4Δ strain. (A) To detect Sir2p, Western analysis was performed on total protein extracts from strains carrying wild-type DOT4 (UCC4786) or dot4Δ (UCC4794). A SIR2 deletion strain (UCC4888) is included as a negative control. Sir2p was detected with an anti-Sir2p antibody (α-Sir2p). A parallel loading experiment in which proteins were stained with Coomassie blue shows equivalent loading of proteins. (B) Anti-Sir3p antibody was used for Western analysis of protein extracts from strains UCC4825 (DOT4 SIR3), UCC4857 (dot4Δ SIR3), and UCC4889 (DOT4 sir3Δ). The presence of a cross-reacting band (asterisk) serves as a control for protein loading. (C) As for panel A except that anti-HA antibody was used to detect Sir4p in extracts of UCC4776 (DOT4 HA-SIR4), UCC4799 (dot4Δ HA-SIR4) and BJ5459 (DOT4 SIR4). The cross-reacting band (asterisk) serves as a control. (D) Northern analysis was used to test the effect of deleting DOT4 on SIR4 expression. In parallel loading experiments, SIR4 and PDA1 expression was analyzed by using RNA from exponentially growing DOT4 and dot4Δ cells. EtBr, ethidium bromide. (E) As for panel A except that anti-Rap1p antibody was used on extracts from UCC4786 (DOT4) and UCC4794 (dot4Δ). The nitrocellulose blot was stained with India ink to control for loading.

FIG. 7

Deleting DOT4 in auxotrophic strains causes a slow-growth defect that is dependent on 26S proteasome activity. (A) DOT4 wild-type and mutant strain pairs containing few (prototroph +) or multiple (prototroph −) auxotrophic markers were assayed for growth on nutritionally complete medium. From the top down, the strain pairs tested were UCC4786 plus UCC4794 (no markers), UCC4711 plus UCC4687 (ura3-52 his3-Δ200 trp1-Δ63 lys2-801 ade2-101), UCC4825 plus UCC4857 (ura3-Δ0), and BY4705 plus UCC4881 (ade2Δ::hisG his3-Δ200 leu2-Δ0 lys2-Δ0 met15-Δ0 trp1-Δ63 ura3-Δ0). In all cases, DOT4 was deleted without altering the auxotrophy of the strain. + in the prototroph column represents relative prototrophy. (B) Growth of strains carrying DOT4, dot4-1, or dot4Δ. Strain UCC4599 (dot4::HIS3 ura3-52 lys2-801 amber ade2-101ochre trp1-Δ63 his3-Δ200 leu2-Δ1 adh4::URA3-TEL ADE2-TEL ppr1::LYS2) carried a single-copy TRP1/CEN plasmid containing wild-type DOT4, the enzymatically inactive dot4-1, or empty vector. Multiple independent transformants were grown on medium lacking tryptophan but containing all other required nutritional supplements. (C) A deletion of DOT4 was combined with a mutation in DOA3, encoding a proteasome subunit, and growth was tested on the same plate at the permissive temperature (23°C) on nutritionally complete medium. Strain pairs, containing a DOT4 and dot4Δ::KanMX allele, respectively, were (from the top down) MHY784 plus UCC4875 (his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 doa3-Δ1::HIS3 YCp50DOA3::URA3) and MHY792 plus UCC4876 (his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 doa3-Δ1::HIS3 YCplac22doa3-1::TRP1). (D) Levels of free ubiquitin were compared in DOT4 wild-type and mutant strains. Ub1, monoubiquitin; Ub2, diubiquitin; Ub3, triubiquitin. Antiubiquitin antibodies were used on extracts from UCC4786 (DOT4) and UCC4794 (dot4Δ). In a parallel loading experiment, proteins were stained with Coomassie blue to control for differences in sample loading.

FIG. 8

The growth defect of dot4Δ mutants requires functional SIR genes. (A) SIR2, SIR3, or SIR4 was deleted in combination with a deletion in DOT4, and growth was assayed on nutritionally complete growth medium. The strains assayed were, from the top to bottom, UCC4887, UCC4891, UCC4892, UCC4893, and UCC4894. (B) The growth rates of strains from panel A were quantified. Doubling times were calculated by regression analysis for exponential equations. The bars represent the mean of three independent experiments, and the error bars represent standard deviations. (C) FACS analysis was used to determine the cell cycle profiles of strains from panel A. The proportions of cells in G₁ and G₂ phases of the cell cycle are presented.