FIG. 4.

Set2 is responsible for mediating global K36 methylation in S. pombe. (A) S. pombe nuclear extracts prepared from wild-type (WT) and set2Δ strains were probed with antibodies against H3 K36 mono-, di-, and trimethylation. An antibody specific for the C terminus of H3 was used as a loading control. Antibodies specific for H3 K9 acetylation and H3 K4 trimethylation were used as additional controls. The asterisk indicates H3 breakdown products that were observed. (B) A slow-growth phenotype develops in the absence of S. pombe set2⁺ under nutrient-deprived conditions. Wild-type or set2Δ cells were spotted at a serial dilution of 1:10 and grown at 30°C on rich medium (YEA) for 3 days or minimal medium (EMM) for 5 days before being photographed. (C) SpSet2 interacts with the hyperphosphorylated form of Pol II. Whole-cell extracts (WCE) prepared from wild-type or genomically tagged SpSet2 (SpSet2-3Flag) were immunoprecipitated with anti-Flag antibody followed by immunoblotting with antibodies directed against unmodified CTD (8WG16, anti-unmod CTD), Ser5-phosphorylated CTD (H14, anti-Ser5P), or Flag (anti-Flag). The locations of Pol II and SpSet2-3Flag are indicated. The input whole-cell extracts were also examined by immunoblot analysis to monitor the presence of Pol II and SpSet2-3Flag.