Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

Abstract

Background: Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.

Result: We determined that loss of H3K79 methylation results in a partial silencing defect that could be bypassed by conditions that promote targeting of Sir proteins to heterochromatin. Furthermore, the silencing defect in strains lacking Dot1 was dependent on methylation of H3K4 by Set1 and histone acetylation by Gcn5, Elp3, and Sas2 in euchromatin. Our study shows that multiple histone modifications associated with euchromatin positively modulate the function of heterochromatin by distinct mechanisms. Genetic interactions between Set1 and Set2 suggested that the H3K36 methyltransferase Set2, unlike most other euchromatic modifiers, negatively affects gene silencing.

Conclusion: Our genetic dissection of Dot1's role in silencing in budding yeast showed that heterochromatin formation is modulated by multiple euchromatic histone modifiers that act by non-overlapping mechanisms. We discuss how euchromatic histone modifiers can make negative as well as positive contributions to gene silencing by competing with heterochromatin proteins within heterochromatin, within euchromatin, and at the boundary between euchromatin and heterochromatin.

Figure 1

Suppression of the silencing defects of dot1Δ strains. (A) Reporter genes used for telomeric silencing. Cells in which the ADE2 gene is silenced accumulate a red pigment whereas cells that express ADE2 are white. Cells in which URA3 is silenced are resistant to 5-FOA, whereas cells in which URA3 is expressed convert 5-FOA into a toxic product and are sensitive to 5-FOA. (B) Wild-type (WT) and dot1Δ strains were transformed with empty vector (p) or a Sir3 overexpression plasmid (pSir3) and were spotted in 10-fold dilution series on media (YC) with and without 5-FOA. (C) Immunoblot analysis of Sir3 expression in sir3Δ and WT cells, and cells containing the Sir3 overexpression plasmid. Ctrl indicates a non-specific band recognized by the Sir3 antibody that was used as a loading control. (D) Telomeric silencing in WT and dot1Δ strains lacking RIF1 or RPD3; sir2Δ and sir3Δ strains are shown as no-silencing controls (E) mRNA expression levels of ADE2 and URA3 relative to ACT1 were determined by RT-qPCR. mRNA was isolated and quantified in duplicate with the difference as the standard error. (F) Sir3 binding at ADE2-TEL-VR, URA3-TEL-VIIL and 3500 bp from telomere VIR (VIR3500) relative to binding at control locus ACT1 was determined by ChIP combined with real-time qPCR. Each clone was analyzed in duplicate with the difference as the standard error. (G) Silencing in strains lacking DEP1 (Rpd3L complex) or RCO1 (RPD3S complex).

Figure 2

Multiple euchromatic histone modifiers promote gene silencing by redundant mechanisms. (A) Analysis of telomeric silencing at 23, 30, and 37°C. (B-G) Single, double and triple mutants of the indicated genes involved in chromatin modification were analyzed as in Figure 1. dot1Δ strains have a partial silencing defect and are 5-FOA sensitive at 30°C. Cells were spotted at 37°C to partially restore silencing and identify mutants that enhance the partial silencing defect of dot1Δ. Each section represents a different experimental panel.

Figure 3

Histone lysine methyltransferases Dot1, Set1 and Set2 affect silencing by different mechanisms. Telomeric silencing in strains lacking DOT1, SET1, and/or SET2 was analyzed by growth on media with or without 5-FOA, at 30 and 37°C as described in Figure 1.

Figure 4

Silencing and viability of strains lacking Set1 is modulated by histone acetylation. (A) Deletion of RPD3 or DEP1 (Rpd3L complex) suppressed the silencing defects of strains lacking SET1, whereas deletion of RCO1 (Rpd3S complex) had no effect. (B) A diploid strain homozygous for the ADE2 and URA3 silencing reporters and heterozygous for SET1/set1::NatMX, GCN5/gcn5::HphMX, ELP3/elp3::KanMX, and SIR3/sir3::HIS3 was sporulated and spore viability was analyzed by tetrad analysis. Each row indicates the four-spore progeny of one diploid cell. Genotypes and mating type of the individual colonies were determined by replica-plating. Only those tetrads are shown of which the genotype of all four spores could be determined or deduced. The genotype of each colony is indicated by the position of the squares, where white indicates the WT allele and black indicates the mutant allele. (C) Combined deletion of SET1, GCN5, and ELP3 affected cell viability. Colony sizes of panel B (large, small, very small/no colony) were scored for each genotype indicated in SIR3 and sir3Δ backgrounds. (D) Wild type and set1Δ strains were transformed with an empty multi-copy plasmid (p) or a multi-copy plasmid carrying a genomic copy of Dot1 (pDot1) to examine the effect of intermediate levels of Dot1 overexpression on telomeric silencing. (E) Protein levels of Dot1 expressed from its endogenous locus and from the multi-copy plasmid were examined by immunoblot analysis. Pgk1 was used as a loading control and a dot1Δ strain was used as a negative control.

Figure 5

Expression of Sir2 and Sir3 in strains with altered silencing properties. Immunoblot analysis of whole-cell protein extracts using antibodies against Sir2 and Sir3. A Pgk1 antibody was used as a loading control. The specificity of the Sir2 antibody is shown in the left panel. The specificity of the Sir3 antibody was shown previously [33] and is shown in Figure 1.

Figure 6

Summary of genetic relationships identified in this study. Genetic interactions between Dot1 and other histone modifiers. Grey nodes indicate positive regulators of silencing and black nodes indicate negative regulators of silencing. Grey lines indicate phenotypic enhancement; black lines indicate phenotypic suppression; arrows indicate the directions of the interactions. No silencing phenotypes were observed for SAS3, HTZ1, and RCO1.

Figure 7

A competition model for positive and negative roles of euchromatic histone modifications in heterochromatin formation. Euchromatic histone modifications can have positive roles (arrows) and negative roles (blunt arrows) in heterochromatin formation. Competition between euchromatic histone modifiers and heterochromatin proteins for interactions with nucleosomes can occur at three locations and can have different outcomes (see text). 1) Competition within heterochromatin regions creates a semi-stable epigenetic state. 2) Competition at the interface between euchromatin and heterochromatin prevents local spreading of the Sir complex, thereby on the one hand avoiding ectopic silencing of regions adjacent to heterochromatin and on the other hand ensuring availability of limiting silencing proteins for the endogenous heterochromatic regions. 3) Competition throughout euchromatin prevents non-specific binding of the Sir2/3/4 complex to bulk chromatin, thereby enhancing targeting of Sir proteins to endogenous heterochromatic regions to ensure sufficient spreading of the Sir complex. By these mechanisms, the function of a euchromatic histone modification in gene silencing depends on the relative contribution that it makes to each of these mechanisms and to what extend the negative and positive functions counteract each other.