Identification of histone demethylases in Saccharomyces cerevisiae

Abstract

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

FIGURE 1. The five JmjC domain-containing proteins in S. cerevisiae

A, domain structures. The diagrams were adapted from the SMART data base. Each protein name is followed by the open reading frame name of the gene. ZF represents a zinc finger domain. B, core β-sheet sequence alignment of the five yeast JmjC proteins. Predicted β-sheet sequences are indicated with a letter b and highlighted by bold italic letters. The predicted co-factor Fe²⁺ binding sites are shown in red and the predicted α-ketoglutarate binding sites in blue.

FIGURE 2. Global changes of histone H3 methylation in JmjC knock-out strains

A, typical LC-MS spectrum of WT H3. The highest peak has six methylation equivalents. B, bar plots showing “relative percentage change” for each peak in knock-out strains relative to WT. For a given peak (i), the following equation was used to calculate the relative percentage change: 100 × [percentage (mutant, i) − percentage (WT, i)]/percentage (WT, i).

FIGURE 3. Site-specific quantification of histone H3 methylation in knock-out strains

The relative levels of unmodified, mono-, di-, and trimethylated peptides are shown as percentages, with panels A, B, and C representing H3-K4, -K36, and -K79, respectively. *, p < 0.05; #, p < 0.1.

FIGURE 4. MS analyses of overexpression strains

A, bar plots showing “relative percentage change” of each peak in overexpression strains relative to WT. B, full-length histone H3 MS spectra for WT and the overexpression strains of RPH1 and JHD2. C, corresponding spectra from truncated H3 (residues 22–135).

FIGURE 5. Site-specific quantification of histone H3 methylation in overexpression strains

Quantification of H3-K4, -K36, and -K79 peptides is shown in A, B, and C, respectively, *, p < 0.05; #, p < 0.1.

FIGURE 6. In vitro enzyme assays of GST-Rph1-(1–373)

A, MALDI-TOF MS data on demethylation of the K36me2 peptide. B, MS data on demethylation of the K36me3 peptide. C, control experiments. Solid and open columns represent the percentages of conversion from K36me3 and K36me2 peptides, respectively. Columns: 1, normal assays; 2, control assays without the enzyme; 3, control assays without α-ketoglutarate (α-KG); 4, control assays without Fe²⁺; 5, control assays with excess EDTA; 6, assay with Rph1 H235A mutant.

FIGURE 7. Growth phenotypes of yeast strains with overexpression of histone demethylase genes

A, overexpression of RPH1 in synthetic medium SC-uracil has a growth defect. Cells grown in raffinose until A₆₀₀ reached 1.0 were diluted 10-fold and spotted on SC-uracil plates with 2% dextrose (left panel) or galactose (right panel). B, hypersensitivity of the RPH1 overexpression strain to UV irradiation. Cells were grown in rich medium (YP) with 2% glucose (DEX) or galactose (GAL). Cell viability was assayed by plating serial dilutions of cells on YP-galactose plates followed by exposure to UV irradiation (10 mJ/cm²). C, histone demethylase activity of Rph1 was associated with cell survival to DNA damage. The rph1 deletion strain (rph1Δ) was transformed with plasmids containing no insert (Vector) or GAL1 promoter-driven wild type (RPH1) and activity-deficient mutant (rph1^H235A), respectively. UV sensitivity assays were performed as described in B.