Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

Abstract

Background: Histone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity.

Results: We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Deltaelp3 mutant. We examined genetic interactions between Deltaelp3 and two other HAT mutants, Deltamst2 and Deltagcn5 and used whole genome microarray analysis to analyze their effects on gene expression.

Conclusions: Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.

Figure 1

Characterization of mutant phenotypes . A: Δelp3 morphology. Wild-type (FY368) and Δelp3 (FY3851)cells were asynchronously grown at 32°C in YES. Cells were fixed and stained with DAPI. The scale bar represents 10 microns. B: Δelp3 mutants have a growth lag. Representative growth curves for wild type (triangle) (FY368) and Δelp3 (circle) (FY3851) cells in rich media (YES) at 30°C. The x-axis time scale indicates hours after inoculation that stared at a cell concentration of 4 × 10⁵ cells/ml. The y-axis indicates number of cells/ml. C: Comparison of growth rates on YES at different temperatures and with different levels of salt. Cells were grown to exponential phase, serially diluted 5× and compared for growth on rich media after 2 or 3 days at 30°C. and 36°C, or onYES supplemented with indicated salt and grown for 2 to 4 days at 30°C. Strain list: wild-type (FY368), Δelp3(FY3851), Δmst2 (FY1890), Δgcn5 Δmst2(Hu990), Δgcn5 Δelp3 (FY3847), Δmst2 Δelp3 (FY3850), Δgcn5 Δelp3 Δmst2 (3854) D: Comparison of drug sensitivity. Exponentially growing cells were diluted 5-fold on YES plates with the indicated drugs and grown 2-3 days at 32°C. Strain list: wild-type (FY261), Δswi6 (FY1584), Δrad3 (FY1104), Δelp3 (FY 3851), Δgcn5 (FY2943), Δmst2 (FY1890), Δgcn5 Δelp3 (FY3847), Δgcn5 Δmst2(FY3361), Δmst2 Δelp3 (FY3850), Δgcn5 Δelp3 Δmst2 (3854).

Figure 2

Overlapping expression in different mutations. A: Venn diagram of differentially down and up-regulated genes in double HAT mutants. (Additional File 5). B: Venn diagram of differentially down and up-regulated GO terms in the double HAT mutants using the affymetrix array. (Additional File 5) C: Venn diagram of differentially down and up-regulated GO terms of a double HAT mutant and the corresponding single HAT mutant. (Additional File 1). D: Localization of differentially regulated genes along chromosome 2 for the double mutant Δgcn5 Δmst2 (FY3361) and the corresponding single mutants, (Tables 1 and 2). Strains list: Δgcn5 Δelp3 (FY3847), Δgcn5 Δmst2(FY3361), Δmst2 Δelp3 (FY3850), Δgcn5 (FY2943) and Δmst2 (FY1890).

Figure 3

Validation of microarray results by qPCR. Genes that showed differential expression from microarray analysis were analyzed in different HAT mutants through qualitative PCR (qPCR). The log2 fold expression ratios from microarray and qPCR experiments for the mutants versus wild type were plotted. qPCR results are indicated by the blue bars, microarray by the purple bars. Strain list: Strain list: Δelp3 (FY 3851), Δgcn5 (FY2943), Δmst2 (FY1890), Δgcn5 Δelp3 (FY3847), Δgcn5 Δmst2(FY3361), Δmst2 Δelp3 (FY3850), Δgcn5 Δelp3 Δmst2 (3854).

Figure 4

Salt response is redundantly regulated by multiple HAT families. Venn diagram of differentially down and up-regulated genes after salt stress in Δgcn5, Δmst2 and Δgcn5 Δmst2 HAT mutants as determined by the Eurogenetech array platform (Additional File 3). Strains Δgcn5 (Hu799), Δmst2 (1890) Δgcn5 Δmst2(Hu990).

Figure 5

Histone H3 acetylation is differentially affected by different HAT mutants. Levels were determined by Western blots of whole cell lysates from the indicated strains with antibodies specific to H3K9ac, H3K14ac, H3K18ac, compared to levels of actin and total histone H3 (C-term). Strain list: wild-type (FY368), Δgcn5 (Hu799), Δelp3(FY3851), Δmst2 (FY1890), Δgcn5 Δmst2(Hu990), Δgcn5 Δelp3 (FY3847), Δmst2 Δelp3 (FY3850), Δgcn5 Δelp3 Δmst2 (3854)

Figure 6

Venn diagram of differentially down and up-regulated GO terms of mst1^ts mutant compared to single, double, and triple HAT mutants. (Additional File 1) Strain list: Strain list: wild-type (FY261), mst1^ts (FY2535) Δelp3 (FY 3851), Δgcn5 (FY2943), Δmst2 (FY1890), Δgcn5 Δelp3 (FY3847), Δgcn5 Δmst2(FY3361), Δmst2 Δelp3 (FY3850), Δgcn5 Δelp3 Δmst2 (3854).