Histone Deacetylases with Antagonistic Roles in Saccharomyces cerevisiae Heterochromatin Formation

Abstract

As the only catalytic member of the Sir-protein gene-silencing complex, Sir2's catalytic activity is necessary for silencing. The only known role for Sir2's catalytic activity in Saccharomyces cerevisiae silencing is to deacetylate N-terminal tails of histones H3 and H4, creating high-affinity binding sites for the Sir-protein complex, resulting in association of Sir proteins across the silenced domain. This histone deacetylation model makes the simple prediction that preemptively removing Sir2's H3 and H4 acetyl substrates, by mutating these lysines to unacetylatable arginines, or removing the acetyl transferase responsible for their acetylation, should restore silencing in the Sir2 catalytic mutant. However, this was not the case. We conducted a genetic screen to explore what aspect of Sir2's catalytic activity has not been accounted for in silencing. Mutation of a nonsirtuin histone deacetylase, Rpd3, restored Sir-protein-based silencing in the absence of Sir2's catalytic activity. Moreover, this antagonism could be mediated by either the large or the small Rpd3-containing complex. Interestingly, this restoration of silencing appeared independent of any known histone H3 or H4 substrates of Rpd3 Investigation of Sir-protein association in the Rpd3 mutant revealed that the restoration of silencing was correlated with an increased association of Sir proteins at the silencers, suggesting that Rpd3 was an antagonist of Sir2's function in nucleation of Sir proteins to the silencer. Additionally, restoration of silencing by Rpd3 was dependent on another sirtuin family member, Hst3, indicating multiple antagonistic roles for deacetylases in S. cerevisiae silencing.

Keywords: H4K16; Hst3; Rpd3; Sir2; heterochromatin.

Figure 1

A genetic screen identified suppressors of Sir2’s catalytic activity. (A) Schematic of the screen to identify mutations that restored silencing in the absence of Sir2 catalytic activity. A sir2N345A sas2Δ strain had the URA3 reporter integrated in place of the a1 ORF at HMR. Expression of the URA3 gene resulted in cells that grow on complete synthetic media lacking uracil but are sensitive to 5-FOA. Expression of the α-genes from HML and a-genes from MAT result in a pseudodiploid cell identity that cannot mate. This strain was mutagenized with EMS and mutants were picked that reestablished silencing as assayed by resistance to 5-FOA and the ability to mate. (B) The phenotypes of a SIR2 wild-type strain (JRY10676) exhibiting complete silencing, the parent strain (JRY9097), and the four mutants that were isolated from the suppressor screen. Fivefold serial dilutions assayed for growth phenotypes on the indicated media. Growth in the patches on the right indicates successful matings as either an a- or α-cell identity.

Figure 2

Mutation of RPD3 and SIN3 restored silencing. (A) Fivefold serial dilutions of yeast were spotted on plates to determine their growth phenotype. Growth on 5-FOA indicates silencing of the hmra1Δ::URA3 reporter. All strains are in the sir2N345A sas2Δ background. The first row is the unmutagenized, RPD3 parent (JRY9097). The next two rows are replicates of the rpd3P264L point mutant (JRY9568). The fourth row is isogenic to the unmutagenized parent except for the opposite mating type, MATα, and ade2-1 (JRY9572). In the fifth row, RPD3 is deleted in the parent strain (JRY9573). In the last row, RPD3 is deleted in the MATα ade2-1 strain isogenic to the parent (JRY9574). (B) The parental strain (JRY9097) and the recovered rpd3P264L mutant (JRY9568), and the rpd3Δ (JRY9573), were crossed with the MATα mating tester (JRY2728) to test for silencing at HML. Growth on the media indicates mating and thus silencing. (C) The Rpd3 subunit gene indicated was deleted in JRY9097 to determine if deletion of that subunit could also restore silencing. Green labels indicate Rpd3L subunits and red labels indicate Rpd3S subunits. (D) The sin3Δ strain that exhibited silencing restoration was transformed with plasmids expressing full-length SIN3 or one of the four SIN3 PAH domain deletions (M1635–M1639). Silencing was assayed on media lacking histidine to select for the plasmid.

Figure 3

The rpd3 mutant restored Sir-based silencing. (A) α2, a1, and URA3 transcripts were measured from the RPD3 parent strain (JRY9097), the rpd3P264L point mutant (JRY9568), and the rpd3Δ (JRY9573) to determine expression levels at HML, MAT, and HMR, respectively. RNA was normalized to ACT1 for each sample and reported as relative to the RPD3 sample. Error bars are the standard error of four biological replicates. The asterisks indicate a P-value of <0.05 (Student’s t-test). (B) Fivefold serial dilutions of the indicated genotypes were spotted to determine the growth phenotype. All strains are sir2Δ, sas2Δ, hmra1Δ::URA3, and all but the bottom two rows also express sir2N345A (JRY9588 and JRY9589). SIR3 and SIR4 were deleted as indicated (JRY9580–JRY9587). The replicates represent individual transformants for the knockouts or individual segregants from the same tetrad dissection. (C) Restoration of silencing at HMR by rpd3Δ was tested in a SAS2 wild-type strain. Fivefold serial dilutions were plated on the indicated media to determine growth phenotype. All strains express the catalytically incactive SIR2 allele, sir2N345A, and have the hmra1Δ::URA3 reporter. Six individual segregants derived from a single tetrad dissection were tested for their ability to restore silencing depending on their SAS2 genotype (from top to bottom: JRY9598, JRY9599, JRY9600, JRY9601, JRY9602, and JRY9603). Strains with the same indicated genotype represent two different segregants from the tetrad dissection to test for reproducibility of the silencing phenotype.

Figure 4

Silencing by rpd3Δ was independent of known Rpd3 histone H4 targets. Endogenous copies of H3 and H4 were deleted and wild-type or mutant H3 and H4 were expressed from a CEN-ARS plasmid. Complete genotypes for all strains tested are in Table S1 (JRY9605, JRY9607–JRY9613, and JRY10644–JRY10655). Silencing was tested by mating to a MATa tester strain (JRY2726) and replica plated to minimal media. Growth indicates successful mating, which reflected extent of silencing.

Figure 5

Deletion of RPD3 increased Sir4 association with the silencers. Sir4-myc ChIP was performed in the MATa sas2Δ hmra1Δ::URA3 with the following SIR2 and RPD3 genotypes: SIR2 RPD3 (JRY10635), sir2N345A RPD3 (JRY10632), and sir2N345A rpd3Δ (JRY10633). No enrichment was observed in a strain not expressing the Sir4-myc (JRY10634). Enrichment is shown as the Immunoprecipitation (IP)/input values relative to the SEN1 IP/input values as a negative control. Error bars represent standard error of four biological replicates. The asterisks indicate a P-value of <0.05 (Student’s t-test).

Figure 6

Silencing by the rpd3 mutant was dependent on Hst3. (A) Fivefold serial dilutions were spotted on the indicated media to determine growth phenotype. All strains are sir2Δ, sir2N345A, sas2Δ, and have the hmra1Δ::URA3 reporter to measure silencing as growth on 5-FOA. RPD3 and HST genotypes are as indicated (JRY9590–JRY9597). (B) α2, a1, and URA3 transcripts were measured to determine expression levels at HML, MAT, and HMR, respectively in RPD3 (JRY9097), rpd3P264L (JRY9568), and rpd3P264L hst3Δ (JRY9596). RNA was normalized to ACT1 for each sample and reported as relative to the RPD3 sample. Error bars are the standard error of four biological replicates. The asterisks indicate a P-value of <0.05 (Student’s t-test). (C) Endogenous copies of H3 and H4 were deleted and mutant H3 was expressed from a CEN-ARS plasmid (JRY9604 and JRY9606). Silencing was tested by mating to a MATa tester strain (JRY2726) and replica plated to minimal media. Growth indicates successful mating and silencing. (D) Fivefold serial dilutions of the indicated RPD3, HST3, and RTT109 genotypes were spotted to determine the growth phenotype. The strains assayed are (from top to bottom) JRY9097, JRY9567, JRY9573, JRY10636, JRY10637, and JRY10638. All strains are sir2Δ, sas2Δ, hmra1Δ::URA3, and sir2N345A.