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Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z - PubMed

  • ️Mon Jan 01 2007

Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z

Shivkumar Venkatasubrahmanyam et al. Proc Natl Acad Sci U S A. 2007.

Abstract

In Saccharomyces cerevisiae, several nonessential mechanisms including histone variant H2A.Z deposition and transcription-associated histone H3 methylation antagonize the local spread of Sir-dependent silent chromatin into adjacent euchromatic regions. However, it is unclear how and where these factors cooperate. To probe this question, we performed systematic genetic array screens for gene deletions that cause a synthetic growth defect in an htz1Delta mutant but not in an htz1Delta sir3Delta double mutant. Of the four genes identified, three, SET1, SWD1, and SWD3, encode components of the Set1 complex, which catalyzes the methylation of histone H3 on lysine 4 (H3-K4), a highly conserved modification that occurs in the coding sequences of transcribed genes. Using microarray-based transcriptional profiling, we find that H2A.Z and Set1 cooperate to prevent Sir-dependent repression of a large number of genes located across the genome, rather than the local effects reported previously for the individual mechanisms. This global, redundant function appears to be direct: using a DamID chromatin profiling method, we demonstrate ectopic association of Sir3 and Sir4 in htz1Delta set1Delta mutants at loci distant from silent chromatin domains. Antisilencing mechanisms may therefore cooperate to play a considerably broader role in regulating genome-wide transcription than previously thought.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Identification of the Set1 complex in a screen for antisilencing factors that cooperate with H2A.Z. (A) Results of an SGA screen for factors that act redundantly with H2A.Z (Htz1) to antagonize Sir3 activity. Each node represents a gene, and each line represents a synthetic genetic interaction between two genes. Forty-five gene deletions have a synthetic growth defect with a deletion of HTZ1. These genes are grouped according to membership in a complex or common function. The nodes are colored according to whether suppression of the corresponding genetic interaction by deletion of SIR3 was observed (red), not observed (black), or not tested (gray). Of the four interactions that are SIR3-dependent, three represent members of the Set1 complex (circled in red). (B) Deletion of SIR2, SIR3, or SIR4 rescues the slow-growth phenotype of a set1Δ htz1Δ mutant as seen by growth on plates (Upper) and in liquid cultures (graph, Lower).

Fig. 2.
Fig. 2.

Genes up-regulated in set1Δ and H3K4A cells are enriched near telomeres. Histograms, showing the fraction of genes in 5-kb intervals that are up-regulated in set1Δ and H3K4A mutants, are plotted as a function of their distance to the nearest telomere. The P values were calculated by using a χ2 test.

Fig. 3.
Fig. 3.

Transcriptional profiling of set1Δ htz1Δ and set1Δ htz1Δ sir2Δ mutants. (A) Telomere-proximal genes are preferentially up-regulated in a set1Δ htz1Δ mutant. Histogram of the fraction of genes up-regulated in a set1Δ htz1Δ mutant as a function of distance to the nearest telomere. (B) Deletion of SIR2 suppresses the transcription defect of set1Δ htz1Δ cells. Shown is a color representation of the expression of 585 genes (≈10% of the genome) in set1Δ, htz1Δ, set1Δ htz1Δ, and set1Δ htz1Δ sir2Δ mutants relative to wild type. Each row indicates the change in expression of a single gene in each of the above mutants (columns). The colors indicate the average change in expression across four independent replicate experiments according to the scale shown. Hierarchical clustering of genes was performed by using Cluster 3.0, and the results were visualized by using Java Treeview software. These genes exhibit a reproducible expression defect in set1Δ htz1Δ cells but not in either set1Δ or htz1Δ cells. The expression of these genes is significantly higher in set1Δ htz1Δ sir2Δ cells compared with the set1Δ htz1Δ cells, indicating suppression of the set1Δ htz1Δ expression defect by deletion of SIR2.

Fig. 4.
Fig. 4.

DamID chromatin profiling of Sir3 and Sir4 in the set1Δ htz1Δ mutant. (A) Schematic showing the use of DamID to measure relative chromatin association of Sir3 in set1Δ htz1Δ cells compared with wild type. Expression of a functional fusion of Sir3 to E. coli dam results in local DNA methylation (red dots) at genes (rectangle), in a manner proportional to the extent of Sir3 binding to chromatin (gray). The percentage of cells methylated at a particular site in wild-type or mutant strains bearing Sir3-dam (or Sir4-dam) was normalized to that in corresponding strains bearing unfused dam to account for differences in chromatin accessibility. (B and C) Increased Sir3 and Sir4 binding at 11 telomere-distal euchromatic loci in the set1Δ htz1Δ mutant. The values on the y axis indicate the ratio of normalized DNA methylation in set1Δ htz1Δ cells to that in wild-type cells (average ± SEM, n = 3). Also shown is the relative chromatin association of Sir3 and Sir4 in set1Δ and htz1Δ cells.

Fig. 5.
Fig. 5.

Model of genome-wide antisilencing mediated redundantly by H2A.Z and Set1. In wild-type cells, nucleosomes containing H2A.Z (blue) and methylated on H3-K4 (green) by Set1 antagonize the binding of the Sir2–4 complex (red) across euchromatin. Disruption of both these antisilencing pathways in set1Δ htz1Δ cells results in redistribution of Sir proteins from telomeric heterochromatin to ectopic sites across the genome.

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