Collaboration between the essential Esa1 acetyltransferase and the Rpd3 deacetylase is mediated by H4K12 histone acetylation in Saccharomyces cerevisiae

Abstract

Histone modifications that regulate chromatin-dependent processes are catalyzed by multisubunit complexes. These can function in both targeting activities to specific genes and in regulating genomewide levels of modifications. In Saccharomyces cerevisiae, Esa1 and Rpd3 have opposing enzymatic activities and are catalytic subunits of multiple chromatin modifying complexes with key roles in processes such as transcriptional regulation and DNA repair. Esa1 is an essential histone acetyltransferase that belongs to the highly conserved MYST family. This study presents evidence that the yeast histone deacetylase gene, RPD3, when deleted, suppressed esa1 conditional mutant phenotypes. Deletion of RPD3 reversed rDNA and telomeric silencing defects and restored global H4 acetylation levels, in addition to rescuing the growth defect of a temperature-sensitive esa1 mutant. This functional genetic interaction between ESA1 and RPD3 was mediated through the Rpd3L complex. The suppression of esa1's growth defect by disruption of Rpd3L was dependent on lysine 12 of histone H4. We propose a model whereby Esa1 and Rpd3L act coordinately to control the acetylation of H4 lysine 12 to regulate transcription, thereby emphasizing the importance of dynamic acetylation and deacetylation of this particular histone residue in maintaining cell viability.

Figure 1.—

Deletion of the histone deacetylase gene RPD3 suppresses the growth defect of esa1. Deletion of RPD3 suppressed the temperature sensitivity of the esa1-414 allele, whereas deletion of other genes encoding histone deacetylases did not. Top panel: serial dilutions of wild type (LPY5), esa1 (LPY4774), esa1 rpd3 (LPY12156), rpd3 (LPY12154), esa1 sir2 (LPY11160), and sir2 (LPY11) are shown on SC at the restrictive temperature for esa1 (35°), compared to growth at the permissive temperature (30°). Bottom panel: serial dilutions of wild type (LPY5), esa1 (LPY4774), esa1 hos1 (LPY13712), esa1 hos2 (LPY13585), esa1 hda1 (LPY13478), hos1 (LPY13706), hos2 (LPY13583), and hda1 (LPY13472) on SC at restrictive and permissive temperatures. Note that some of these interactions overlap with published results from a genomewide study (Lin et al. 2008), yet others are distinct. The differences may be due to strain background- or allele-specific effects (Y. Lin and J. Boeke, personal communication).

Figure 2.—

Rpd3L is the Rpd3-containing complex responsible for suppression of the growth defect in the esa1 mutant. (A) Cartoon highlighting the unique and shared members of the Rpd3S and Rpd3L complexes. (B) Deletion of RCO1, specific to Rpd3S, did not suppress esa1's growth defect at restrictive temperature. Deletion of PHO23 and SDS3, both specific to Rpd3L, mimicked the suppression seen in esa1 rpd3. Serial dilutions of wild type (LPY5), esa1 (LPY4774), esa1 rco1 (LPY12652), rco1 (LPY12645), esa1 sds3 (LPY12956), sds3 (LPY12958), esa1 pho23 (LPY12729), and pho23 (LPY12732), were plated on SC at permissive (30°) and restrictive temperatures (35°). Cartoon of complexes is modified from Roguev and Krogan (2007).

Figure 3.—

Disruption of Rpd3L suppresses esa1 silencing phenotypes. (A) Disruption of Rpd3L suppressed the rDNA silencing defect of an esa1 mutant. Wild type (LPY4909), esa1 (LPY4911), esa1 rpd3 (LPY12147), rpd3 (LPY12145), esa1 sds3 (LPY13517), sds3 (LPY13513), esa1 pho23 (LPY13859), pho23 (LPY13854), esa1 rco1 (LPY13505), and rco1 (LPY13501) all have the rDNA∷ADE2-CAN1 reporter to test for rDNA silencing on plates containing canavanine. Decreased growth on canavanine indicates a defect in rDNA silencing. (B) Disruption of Rpd3L suppressed the telomeric silencing defect of an esa1 mutant. Wild type (LPY4917), esa1 (LPY4919), esa1 rpd3 (LPY12211), rpd3 (LPY12093), esa1 sds3 (LPY13540), sds3 (LPY13536), esa1 pho23 (LPY13769), pho23 (LPY13765), esa1 rco1 (LPY13528), and rco1 (LPY13524) all have the TELVR∷URA3 silencing marker to test for telomeric silencing on plates containing 5-FOA. Decreased growth on 5-FOA indicates a defect in telomeric silencing.

Figure 4.—

Rpd3L disruption does not suppress esa1's camptothecin sensitivity. The same strains tested in Figure 1 and Figure 2B were plated on a control YPD plate containing DMSO and a plate containing 20 μg/ml of camptothecin in DMSO.

Figure 5.—

Deletion of RPD3 restores global acetylation levels of specific histone H4 residues in esa1 mutants. (A) Diagram of the histone H4 N-terminal tail highlighting sites of acetylation modifications. (B) Deletion of RPD3 restored global acetylation of H4K5 and H4K12, but not H4K8 and H4K16. Whole cell protein extracts from wild-type (LPY5), esa1 (LPY4774), esa1 rpd3 (LPY12156), and rpd3 (LPY12154) cells at both permissive (30°) and restrictive (34°) temperatures were immunoblotted with an antiserum specific to the C terminus of H3 to control for histone levels, and with H4 antisera to detect the amount of bulk histone acetylation at each lysine residue.

Figure 6.—

Suppression of esa1's growth defect by rpd3 is dependent on H4K12. Strains are deleted for all copies of H3 and H4 and carry a TRP1 plasmid with either wild-type H4 or H4 with one mutated lysine residue. Plasmid retention was required for survival. (A) Serial dilutions of the following strains were plated at permissive (30°) and restrictive temperatures (35°) on SC. Top panel: growth of H4K12A mutants in combination with esa1 rpd3. Strains are wild type (LPY12383), H4K12A (LPY12394), esa1 (LPY12384), esa1 H4K12A (LPY12071), esa1 rpd3 (LPY12707), esa1 rpd3 H4K12A (LPY12714), rpd3 (LPY12695), and rpd3 H4K12A (LPY12702). Bottom panel: growth of esa1 rpd3 mutants in combination with each lysine individually mutated to alanine. Strains are esa1 rpd3 (LPY12707), esa1 rpd3 H4K5A (LPY12708), esa1 rpd3 H4K8A (LPY12711), esa1 rpd3 H4K12A (LPY12714), and esa1 rpd3 H4K16A (LPY12717). (B) Suppression of esa1's growth defect through deletion of noncatalytic Rpd3L subunits was also dependent on H4K12. Top panel: twofold dilutions, starting at an A₆₀₀ of 0.1, were plated on SC−Trp for assaying growth of esa1 sds3 in combination with each lysine individually mutated to alanine. Strains are esa1 sds3 (LPY14175), esa1 sds3 H4K5A (LPY14176), esa1 sds3 H4K8A (LPY14177), esa1 sds3 H4K12A (LPY14178), and esa1 sds3 H4K16A (LPY14179). Bottom panel: as above except in esa1 pho23 mutant. Strains are esa1 pho23 (LPY14165), esa1 pho23 H4K5A (LPY14166), esa1 pho23 H4K8A (LPY14167), esa1 pho23 H4K12A (LPY14168), and esa1 pho23 H4K16A (LPY14169).

Figure 7.—

A model depicting a critical role for Esa1 and Rpd3L in coordinating the dynamic acetylation of H4K12. (A) Esa1 and Rpd3L control H4K12Ac for general transcriptional targets contributing to cell viability and growth. (B) Esa1 and Rpd3L contribute to rDNA and telomeric silencing. This relationship is not mediated specifically through H4K12 acetylation, but likely through a number of other targets. Sir2 deacetylation of H4K16 appears downstream of the role for Esa1 and Rpd3L. (C) Esa1 and Rpd3S, but not Rpd3L, may control acetylation at sites of DNA damage.