The SAGA histone deubiquitinase module controls yeast replicative lifespan via Sir2 interaction

Abstract

We have analyzed the yeast replicative lifespan of a large number of open reading frame (ORF) deletions. Here, we report that strains lacking genes SGF73, SGF11, and UBP8 encoding SAGA/SLIK complex histone deubiquitinase module (DUBm) components are exceptionally long lived. Strains lacking other SAGA/SALSA components, including the acetyltransferase encoded by GCN5, are not long lived; however, these genes are required for the lifespan extension observed in DUBm deletions. Moreover, the SIR2-encoded histone deacetylase is required, and we document both a genetic and physical interaction between DUBm and Sir2. A series of studies assessing Sir2-dependent functions lead us to propose that DUBm strains are exceptionally long lived because they promote multiple prolongevity events, including reduced rDNA recombination and altered silencing of telomere-proximal genes. Given that ataxin-7, the human Sgf73 ortholog, causes the neurodegenerative disease spinocerebellar ataxia type 7, our findings indicate that the genetic and epigenetic interactions between DUBm and SIR2 will be relevant to neurodegeneration and aging.

Figure 1

Deletion of SAGA DUBm components SGF73, UBP8, and SGF11 significantly increases yeast RLS, and mutation of H2B-K123 or deletion of its monoubiquitinating enzymes shortens RLS. (A) sgf73Δ; (B)ubp8Δ ; (C) sgf11Δ; (D) summary of remaining viable SAGA component deletions; (E) bre1Δ; (F) rad6Δ; (G) H2B-K123R. Legends show (mean RLS, number of mother cells scored). See also Supplemental Figure S1, S2, and S3.

Figure 2

RLS epistasis to determine the role of SAGA components and other nuclear transcription factors in sgf73Δ life extension. Genes encoding the SAGA component GCN5 (A) is necessary for the extended RLS of sgf73Δ, while those encoding transcription factors GCN4 (B) and HAP4 (C) are not. Legends show (mean RLS, number of mother cells scored).

Figure 3

RLS epistasis to determine the relative roles of SAGA and SLIK in sgf73Δ and ubp8Δ lifespan extension. SAGA-specific component SPT8 is necessary for the extended RLS of (A) sgf73Δ and (B) ubp8Δ while neither PEP4, the protease necessary for processing of the SLIK-specific form of Spt7 (C, D), nor SLIK– specific component RTG2 (E) are necessary. Legends show (mean RLS, number of mother cells scored). See also Supplemental Figure S4.

Figure 4

SIR2 is necessary for the extended RLS of sgf73Δ (A) and ubp8Δ(B), even in strains lacking FOB1 (C, D). E, SIR2OE ubp8Δ double shows extended lifespan comparable to sgf73Δ. Legends show (mean RLS, number of mother cells scored). See also Supplemental Figure S5.

Figure 5

Sgf73 influences silencing at rDNA and telomeres. (A) Sir2 protein levels are unchanged in sgf73Δ; (B) rDNA-dependent recombination of an ADE2 locus is dramatically reduced in sgf73Δ. At telomeres, sgf73Δshows increased silencing of URA3 marker on VIIL (C), but decreased silencing of endogenous ORF YFR057W on VIR (D). Error bars indicate standard error of the mean. See also Supplemental Figure S6.

Figure 6

Sgf73 interacts with Sir2. (A), Interaction of recombinant Sir2-GST and Sgf73-GFP in the presence and absence of Sir2. Immunoprecipitation was done with glutathione beads coupled to either GST tagged Sir2, GST tagged enzymatically dead Sir2R319K, or GST, and western blot was probed with anti-GFP. (B), Interaction of endogenous Sir2 and Sgf73-GFP. Immunoprecipitation was done using anti-GFP antibody and western blots were probed using anti-Sir2 antibody.