Identification of a small molecule inhibitor of Sir2p

Abstract

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.

Figure 1

(A) Chemical structure of splitomicin. (B) Activation of a TRP1 reporter at the silent HMR mating locus by splitomicin (S). Wild-type (SIR2) or sir2Δ cells with TRP1 integrated into HMR (19). Cells were replica-plated onto complete synthetic medium or medium lacking tryptophan (−trp) without or with the indicated concentrations of splitomicin. (C) Loss of responsiveness to α factor in the presence of splitomicin. Logarithmically growing MATa cells were imbedded into agar with synthetic medium containing 2.5 μM α factor. The paper discs with 5 μl of DMSO or 5 mM splitomicin were placed onto the agar, and the plate was incubated at 30°C for 2 days. The halo of cells indicates those able to grow. (D) Splitomicin increases recombination of an ADE2 reporter integrated within the ribosomal DNA array. Logarithmic-phase cells were exposed to splitomicin (15 μM) or DMSO for 6 h and plated onto rich medium. The recombination rate was calculated directly from the frequency of loss of the ADE2 gene in the first division after plating by counting half-sectored red colonies. Three independent determinations were performed for each experimental group.

Figure 2

Splitomicin-treated wild-type (wt) cells and sir2Δ cells display similar transcriptional changes relative to untreated wild-type cells. (A) Correlation of transcriptional changes between genetic and chemical inactivation of Sir2p. Transcriptional changes were determined by competitive hybridization to DNA microarrays containing >6,000 yeast ORFs. The correlation plot shows transcriptional changes in a sir2Δ mutant relative to wild type (sir2Δ/wt) on the vertical axis and changes in wild-type cells treated with splitomicin relative to untreated wild-type cells (15 μM splitomicin/no treatment) on the horizontal axis. (B) A Venn diagram comparing genes up-regulated (Left) and down-regulated (Right) more than 2-fold relative to wild-type or untreated cells and sir2Δ, hst1Δ, or splitomicin-treated wild-type cells. (C) Correlation of transcriptional changes in wild-type cells in response to splitomicin treatment with and without cycloheximide. The correlation plot shows transcriptional changes in splitomicin- and cycloheximide-treated wild-type cells relative to cells treated with cycloheximide alone (15 μM splitomicin CYH/CYH) on the vertical axis and changes in wild-type cells treated with splitomicin relative to untreated wild-type cells (15 μM splitomicin/no treatment) on the horizontal axis. (D) Venn diagrams comparing transcriptional changes (up- or down-regulation) in hst2Δ, hst3Δ, and hst4Δ cells and splitomicin-treated cells (split).

Figure 3

(A) Inhibition of NAD-dependent HDA of Sir2p by splitomicin. The effect of splitomicin on NAD⁺-dependent HDA in wild-type and drug-resistant Sir2p mutants is shown. Chemically [³H]-acetylated histone H4 peptide (40,000 cpm per reaction) was incubated with whole-cell protein extracts (50 μg) prepared from hst2Δ strain containing overexpressed wild-type SIR2 or two drug-resistant SIR2 alleles (SIR2-Y298N and SIR2-H286Q), NAD⁺, and splitomicin at 30°C for 16 h. The assays were done in triplicate. The NAD⁺-dependent activity in the extract without splitomicin was 1,776 ± 25 cpm for wild-type SIR2, 1,620 ± 44 cpm for SIR2-Y298N, 1,795 ± 36 cpm for SIR2-H286Q, and 28 ± 14 cpm for cells containing the empty vector. (B) Immunoblot of Sir2p in whole-cell lysates containing overexpressed wild-type or drug-resistant mutant SIR2. The whole-cell lysates (25 μg) prepared from hst2Δ strain containing empty vector or overexpressed wild-type SIR2 and two drug-resistant SIR2 alleles (SIR2-Y298N and SIR2-H286Q) were probed with an anti-Sir2p antibody (Santa Cruz Biotechnology). (C) Telomeric silencing in SIR2, sir2Δ, and drug-resistant SIR2 mutants. Cells from a sir2Δ strain with telomeric URA3 gene containing either empty plasmid (sir2Δ), a plasmid with wild-type SIR2, or drug-resistant alleles SIR2-H286Q, SIR2-L287M, and SIR2-Y298N were replica-plated onto selective medium lacking leucine (for plasmid selection), selective medium lacking uracil (−ura), or selective medium to which 5-flouroorotic acid was added (+5-FOA) with or without 10 μM splitomicin and incubated at 30°C for 2 days. Expression of the telomeric URA3 gene kills cells, because Ura3p converts 5-fluoroorotic acid into a toxic metabolite. (D) Sequence alignment between yeast Sir2p and Hst1–4p. The region displayed in the alignment contains the putative substrate-binding site. Arrows indicate the positions of residues that, when mutated in Sir2p, confer splitomicin resistance.

Figure 4

(A) Cell cycle analysis of α factor arrested MATa cells treated with splitomicin. Logarithmically growing MATa cells were treated first with α factor for 90 min. At time 0, splitomicin (20 μM) or DMSO was added to the culture. DNA content of the cells was determined by flow cytometry at several time points after the addition of splitomicin. (B) α2 mRNA expression from the silent HML locus in G₁-arrested cells treated with splitomicin. A strain containing the galactose-inducible CLN3 gene in which genomic G₁ cyclin genes were deleted [MATa cln1Δ, cln2Δ, cln3Δ, GAL-CLN3 (35)], was arrested in G₁ by exposure to glucose for 90 min. Splitomicin (20 μM) or DMSO was added to the culture of these G₁-arrested cells, and the expression of MATα from the silent HML locus was assessed at several time points. Both splitomicin- and DMSO-treated cells remained arrested in G₁ as judged by flow cytometry (data not shown). The RNA from MATα and MATa sir2Δ cells is included for comparison. The weak lower molecular weight band is caused by cross hybridization to a2 mRNA. The blot was stripped and reprobed for the PDA1 mRNA as a loading control.