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.