Figure 8.

Recombinant Set4 does not methylate histones in vitro. A, autoradiograph and Coomassie Blue staining of SDS-PAGE with in vitro methylation assays using purified His₆-Set4 and histone substrates, including recombinant H3 (rH3), rH4, rH2A, rH2B, calf thymus histones (CTH), and nucleosomes purified from Hela cells (Nuc). A reaction with GST-Set5 and CTH as substrate was run on the same gel as a positive control. Coomassie Blue of KMTs shows both His₆-Set4 and GST-Set5 used in the reactions. B, autoradiograph and Coomassie Blue staining of SDS-PAGE with in vitro methylation assays using purified GST-Set2 and GST-Set5 on multiple substrates. C, 10-fold serial dilutions of WT yeast (yEG001) carrying WT and mutant SET4 under the control of the GAL1 promoter spotted on either SC-LEU with 2% glucose (GLU) or 2% galactose (GAL) and grown at 30 °C for 2–3 days. D, serial dilutions of WT strains (yEG315) with either WT or Y375A mutant pMN3-SET4 spotted on SC-URA or SC-URA with 100 μm β-estradiol and grown at 30 °C for 3 days.