Synthetic lethality between the Rpd3(L) and the SAS-I complex. (A) Cells disrupted for subunits of the SAS-I complex are synthetically lethal with rpd3Δ. Tetrad dissection of crosses of sas2Δ, sas4Δ or sas5Δ with rpd3Δ isogenic W303 strains. The four spores from individual asci are aligned in vertical lines. Double mutants are marked with circles. (B) The lethality between rpd3Δ and sas2Δ is specific for the Rpd3(L) complex. sas2Δ cells with pURA3-SAS2 and additional deletions of RCO1, SDS3, or both were incubated on supplemented yeast minimal medium (YM) or 5-fluoroorotic acid (5-FOA) medium. (C) set2Δ does not cause synthetic lethality in sas2Δ. (D) sas2Δ cells are sensitive to trichostatin A (TSA). Filter discs with DMSO control (Upper row) or increasing amounts (16, 12 and 8 μg) of TSA (Lower row) were placed on lawns of 2 × 10⁵ cells of a WT or sas2Δ strain. Plates were incubated for 1 day at 30°C.

The synthetic lethality between the Rpd3(L) and the SAS-I complex is caused by inappropriate SIR spreading. (A) Deletions of subunits of the telomeric SIR complex suppress the sas2Δ rpd3Δ synthetic lethality. Derivatives of an sas2Δ rpd3Δ pURA3-SAS2 strain (AEY 3923) with deletions for SIR1, SIR2, SIR3, or SIR4 were grown on minimal plates (YM, growth assay) and on 5-FOA plates to select against pURA3-SAS2 for 2 days at 30°C. (B) Mutations within the histone H3 and H4 N-termini suppress the sas2Δ rpd3Δ synthetic lethality. Alleles of H3 and H4 were introduced into an rpd3Δ sas2Δ pLYS2-SAS2 strain by plasmid shuffle (AEY3945; SI Materials and Methods for details), and the ability of the derivatives to survive in the absence of the SAS2 plasmid was tested. “wt” refers to WT copies of H3 and H4 in AEY3945. “Control” refers to a WT strain. H4 K -> Q designates H4 K5, 8, 12, and 16 Q. H3 K -> Q designates H3 K4, 9, 14, 18, 23, and 27 Q.

Deletion of RPD3 causes mislocalization of Sir2 and Sir3, gene silencing, and changes in histone acetylation in subtelomeric regions. (A) Sir2 and Sir3 binding at the right telomere of chromosome VI is shown as enrichment in ChIP experiments relative to their enrichment at the control gene SPS2. The amount of enrichment is given as a function of the distance to the telomere end in kb in strains with the indicated genotype. ChIP was performed with antibodies against myc-Sir2 and HA-Sir3. Error bars give SD (SI Materials and Methods). (B) Telomeric ADE2 is repressed in rpd3Δ strains. WT and rpd3Δ strains carrying telomeric ADE2 were grown for 2 days at 30°C. (C) Subtelomeric genes are repressed in rpd3Δ cells in a SIR-dependent fashion that correlates with Sir2 association. The upper row of diagrams shows the amount of cDNA of selected subtelomeric genes in the indicated strains as X-fold expression level relative to SPS2. Error bars give standard deviations of at least three PCR analyses from at least two independent reverse transcriptase reactions. The lower row shows ChIP analysis of Sir2 at the respective genes and is represented as in (A). (D) rpd3Δ causes changes in subtelomeric histone acetylation levels. Acetylation was measured by ChIP using antibodies against the indicated residues. Values indicate enrichment of a modification relative to enrichment of H4 (SI Materials and Methods). (E) Sir2-independent changes in subtelomeric histone acetylation by rpd3Δ. Experiments were performed as described in (D). (F) Schematic representation of the telomere VI-R with ORFs and fragments amplified in the ChIP experiments. Genes drawn above the upper line are transcribed from left to right; those below that line are transcribed in the opposite direction.

Tethered Rpd3 and Hos2 create a boundary against heterochromatin spreading. (A) Cells with URA3 inserted at TEL VII-L and with (+UAS) or without a Gal4 binding site at the telomere-proximal side (−UAS) were transformed with plasmids carrying the RPD3 or HOS2 genes fused to the GAL4 DNA binding domain (GBD-HDAC) or with the vector control (GBD). Repression of URA3 was tested by growth on plates lacking uracil and on 5-FOA plates. Serial dilutions of cells were grown for 2 days at 30°C. (B) The targeted boundary function of Rpd3 depends on its catalytic activity. Cells with a Gal4 UAS between telomeric heterochromatin and a subtelomeric URA3 reporter (as in A) were disrupted for endogenous RPD3 and transformed with RPD3 or a catalytically dead rpd3 allele (rpd3-H150:151A, referred to as “rpd3-HDAC⁻”). (C) Tethered Rpd3 disrupts silencing at HML and has insulating activity at HMR. Cells with ADE2 and URA3 inserted at HML or HMR were provided with GBD or with GBD-Rpd3. (D) Other HDACs did not display boundary function. GBD-HDAC fusions were assayed for boundary function as in A.

The putative OAADPR binding domain of Sir3 is necessary for its function in silencing. (A) Model of the AAA+ ATPase-like domain of Sir3. For a computational modeling of Sir3, the primary sequence of Sir3 AAA⁺ domain and structures of other proteins of the AAA⁺ family were subjected to a sequence-structure alignment, using Modeler 9v2 (see SI Materials and Methods for details). The model was visualized using PyMOL. The position of the putative OAADPR binding domain is marked by OAADPR. The mutations of Sir3 used in this study are indicated in red and blue. (B) Mutation of the AAA⁺ domain of Sir3 abrogates its ability to spread at telomeres. rpd3Δ sas2Δ sir3Δ pURA3-SAS2 strains carrying the indicated sir3 alleles were tested for their ability to lose the SAS2 plasmid on 5-FOA. sir3-AAA designates mutation of residues 575–577 to alanine. (C and D) sir3-Δ575–577 and sir3-AAA were deficient in telomeric and HML silencing. (E) Mutation of the AAA⁺ domain of Sir3 (Sir3-Δ578–585, designated “Sir3*”) reduces its ability to interact with Sir4 and Sir3. Fusions of the indicated proteins to the Gal4 DNA-binding (BD) and activation domain (AD) were tested for activation of the HIS3 reporter gene on medium lacking histidine. (F) Sir3Δ578–585 and Sir3Δ575–577 show reduced binding to the telomere. ChIP analysis was performed with Sir3-HA.