Synthetic lethal screens identify gene silencing processes in yeast and implicate the acetylated amino terminus of Sir3 in recognition of the nucleosome core

Abstract

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.

FIG. 1.

Synthetic lethal screens by SGA analysis reveal synthetic genetic interactions of dot1Δ. (A) A genome-wide SGA screen using the dot1Δ query strain was carried out in duplicate and was repeated with a query strain harboring the catalytically inactive dot1-G401V allele (dot1V). First-round candidates were retested first by SGA protocols. Two reproducible genetic interactions were identified. Strains carrying dot1Δ and dot1V were functionally indistinguishable. Final selection steps of synthetic lethal analysis by SGA in quadruplicate showed that the sir1Δ dot1Δ/V and pol32Δ dot1Δ/V double mutants (selected on medium containing G418 and CloNat [KAN+NAT]) grew more slowly than the pol32Δ and sir1Δ single mutants (selected on medium containing G418 [KAN]) or other dot1Δ/V double mutants (e.g., ada5Δ or apg17Δ). (B) Growth of haploid spores obtained by tetrad analysis of three heterozygous sir1Δ dot1V and pol32Δdot1V diploid strains. Dotted squares indicate double mutants.

FIG. 2.

The synthetic lethal interaction between SIR1 and DOT1 in SGA analysis is caused by the loss of HMLα silencing. (A) Schematic outline of the transcribed MATa locus and the silent mating type loci HMRa and HMLα on chromosome III in MATa cells. MATa cells express α1 and activate MATa-specific and haploid-specific genes (arrow labeled ASG). The final steps of SGA involve selection for MATa haploids through selection expression of HIS3 (or the S. pombe HIS5) gene which is driven by a MATa-specific promoter (MFA1 or STE2). Diploid cells or haploid MATa cells with a desilenced HMLα locus (pseudodiploids) express both a and α information and repress MATa-specific and haploid-specific genes. (B) Random spore analysis of haploid spores from the dot1V sir1Δ heterozygous diploids obtained during SGA analysis. Cells were plated on haploid selection media to select for all haploids, on medium containing G418 (+KAN) to select for the sir1Δ single mutants (either DOT1 or dot1V), or medium containing G418 and CloNat (+KAN+NAT) to select for the sir1Δ dot1V double mutants. The top panel shows medium lacking histidine to select for MATa haploids only. The bottom panel shows medium containing histidine to select for all haploids, without selection for mating type identity (MATa, MATα, and MATa pseudodiploids). (C) Spores obtained by tetrad dissection of the sir1Δ dot1V heterozygous diploids were mated with tester strains. Dotted squares indicate MATa (black) and MATα (white) sir1Δ dot1V double mutants. Lack of growth on diploid selection medium (bottom panels) indicates a mating defect. YPD, yeast-peptone-dextrose. (D) Silencing at HMLα was measured in strains carrying a URA3 reporter gene inserted at HMLα. When URA3 is silenced, cells are resistant to 5-FOA. When URA3 is expressed, cells are sensitive to 5-FOA, which is converted into a toxic compound by the URA3 gene product. Cells were pregrown in nonselective medium and spotted in a 10-fold dilution series on complete medium (complete) or medium containing 5-FOA (+FOA). Pictures were taken after 3 or 4 days of growth. Strains were UCC7268 and derivatives thereof (Table 1).

FIG. 3.

POL32 is involved in silencing at HMLα and telomeres and collaborates with SIR1 and DOT1. (A) Analysis of DNA content by fluorescence-activated cell sorting of the WT strain UCC7366 and the pol32Δ, dot1Δ, and pol32Δ dot1Δ derivatives thereof (Table 1). (B) Random spore analysis of haploid spores from the dot1V pol32Δ heterozygous diploids obtained during SGA analysis. Cells were plated as shown in Fig. 2B on medium to select for all haploids, the pol32Δ single mutants (either DOT1 or dot1V), or the pol32Δ dot1Δ double mutants. The top panel shows MATa haploids only; the bottom panel shows all haploids without selection for mating type identity. (C) Silencing at HMLα was measured in strains carrying a URA3 reporter gene inserted at HMLα (strain UCC7268 and derivatives thereof [Table 1]) as described in the legend to Fig. 2D. (D) Telomeric silencing was measured in a strain in which URA3 was integrated at telomere VII-L (the same strains as described above for panel A). Spot tests were performed as described in the legend to Fig. 2D. Silencing is stronger at higher temperatures. Cells were grown at 30°C (normal temperature) as well as 37°C (high temperature) to suppress silencing defects of the single mutants and thereby allow detection of phenotypic enhancement of double mutants.

FIG. 4.

DOT1 and NAT1 and the N-terminal mutants of SIR3 genetically interact with SIR1 but not with each other. (A) The effect of combined deletions of SIR1, NAT1, and DOT1 on silencing of HMLα was assessed by analysis of the mating efficiency of MATa strains. Cells from the mating reaction were plated in a 10-fold dilution series on media selecting for MATa haploids and diploids (input) and on media selecting for diploids only (mated). Mating efficiency was calculated as the ratio between the mated and the input. All mating assays were carried out at least two times. Error bars indicate the spread of the data. Strains were UCC7366 and derivatives thereof (Table 1). (B) MATa strains lacking endogenous SIR3 (strain NKI2111 and derivatives thereof [Table 1]) were transformed with empty vector (p), or single-copy CEN plasmids carrying the WT SIR3 or sir3-A2G genes. The plasmid carrying a WT copy of SIR3 complemented the mating defect of the sir3Δ strain. Mating efficiency was determined as described above. Error bars indicate the spread of the data. (C) Endogenous SIR3 was replaced by WT SIR3 (SIR3i) or sir3-A2G (sir3-A2Gi) by integrating the respective constructs into the SIR3 genomic locus of the WT, sir1Δ, and nat1Δ strains. Mating efficiency was determined as described above. Error bars indicate the standard deviations of four mating reactions. (D) qRT-PCR analysis of HMLα1 mRNA normalized to ACT1 mRNA of strains are shown in panels A to C. The arbitrary units are plotted relative to the HMLα1 expression in the sir3Δ strain. Error bars represent standard deviations of measurements of at least three independent RNA isolations.

FIG. 5.

Sir3-A2G mutant protein shows reduced binding to telomeres but retains interactions with nucleosomes. (A) Immunoblotting analysis of the expression of reintegrated (i) or plasmid-encoded (p) WT Sir3 and Sir3-A2G in WCE. A WT strain with endogenous SIR3 (end.) and a sir3Δ strain transformed with an empty vector were used as controls. Yeast strains are described in the legend to Fig. 4. An antibody against Pgk1 was used as a loading control. An asterisk indicates a nonspecific band. (B) Binding of Sir3 to silent chromatin was analyzed by three independent ChIP experiments using the Sir3 antibody followed by multiplex PCR and quantitation. DNA of WCE (input) was used for normalization (Sir3 ChIP/input), and binding signals were plotted relative to Sir3 binding at HMLα in the WT. Genomic regions analyzed were ACT1, HMLα, YFR057W (TEL-VIR), and HMRa. (C) The overall binding of Sir3 to chromatin was determined by ChIP assay using anti-Sir3 antibodies, followed by immunoblotting analysis of the immunoprecipitated proteins (Sir3-IP) and the input using antibodies against the C terminus of H3. (D) Fractionation of a WCE into a soluble supernatant (soluble), which lacks chromatin, and a pellet fraction (chromatin), in which chromatin is highly enriched. The three fractions were analyzed by immunoblotting. Histone H3 and Pgk1 were used as chromatin-bound and nonchromatin controls, respectively. Sir3 copurified with the chromatin-containing pellet fraction.

FIG. 6.

A model for the interaction between SIR1, DOT1, and the acetylated N terminus of SIR3 in silencing. Sir1 recruits the Sir2/3/4 complex in cis to silencer elements at HM loci. Dot1 acts by methylation (me) of histone H3K79 in euchromatin, which prevents promiscuous binding of Sir proteins to euchromatin, which enhances targeting of Sir proteins to regions of silent chromatin in trans. The data described in this paper show that Dot1, the N-terminal acetyltransferase complex Nat1/Ard1, and the N terminus of Sir3 effect a common silencing process. The results are compatible with a model in which the N terminus of the BAH domain of Sir3 interacts with the core domain of the nucleosome encompassing histone H3K79, whereby the ability of Sir proteins to discriminate between methylated and unmethylated histone H3K79 depends on acetylation of the N-terminal alanine of Sir3 by the Nat1/Ard1 complex (left panel). In the absence of components of this pathway (right panels), Sir3 loses its specificity for nucleosomes in silent chromatin unmethylated at histone H3K79 and binds to euchromatic regions. This pathway most likely acts in parallel with pathways that affect interactions of the C-terminal domain of Sir3 with the tails of histones H3 and H4.