Figure 5. Locus-specific changes in H3K36me3 and inhibition of Gis1 and Rph1 by 2-HG.

(A) Locus-specific increase for H3K36me3 in the rph1Δ and gis1Δ strains shown for the YRO2 and GDB1 genes. Increased methylation at YRO2 is accompanied by changes in gene expression while GDB1 is repressed specifically in rph1Δ strain. (B) The number of DMGs (FDR < 0.05, log2FC > 0.3) for H3K36me3 in the rph1Δ, gis1Δ, and jhd2Δ strains is shown as in Fig 3B. (C, D, E) Correlation analysis between differentially methylated genes in different histone demethylase deletion strains and the dld3Δ strain. (C, D, E) Log₂-FCs in H3K36me3 in the dld3Δ strain are plotted on the x-axis, whereas log₂-FCs in the (C) rph1Δ, (D) gis1Δ, and (E) jhd2Δ mutant strains are plotted on the y-axis. Each dot represents a gene and red dots represent DMGs found to change significantly for H3K36me3 in both mutant strains. Pearson correlation is calculated for each set. The strongest correlation was observed between the dld3Δ and rph1Δ strains. (F) Inhibition of the Gis1 and Rph1 enzymatic activities by 2-HG. A demethylation assay using recombinant enzymes and H3K36me3 peptide as substrate was performed in vitro in the presence of increasing 2-HG concentrations. Enzymatic activities were determined based on initial velocities (in μM NADH.min⁻¹) and normalized to the value obtained in the absence of 2-HG (set to 100%). Data shown are means ± SDs from three independent experiments. Statistical significance was calculated using t test; * = P < 0.05, ** = P < 0.01, *** = P < 0.001.