FIG. 3.

Set2 from Schizosaccharomyces pombe is a robust nucleosome-selective K36-specific methyltransferase. (A) Bacterial lysates containing recombinantly expressed SpSet2 (or vector-only control) were incubated with recombinant H3 (rH3), chicken core histones, or oligonucleosomes (Nuc.) and [³H]SAM. ³H incorporation was analyzed by the filter-binding assay and monitored by scintillation counting. (B) Reaction products from HMT assays using oligonucleosomes and SpSet2 were resolved on a 15% SDS-PAGE gel and examined by Coomassie staining (lower panel) and fluorography (upper panel). The asterisk indicates the H3 breakdown product that is typically observed. (C) Oligonucleosomes or recombinant H3 was incubated with SpSet2 and unlabeled SAM in an HMT assay followed by immunoblotting with the anti-Me₂K36 antibody (upper panel). Parallel reactions were performed and examined by Coomassie staining to monitor loading (lower panel). (D) Filter-binding assays were performed as in A using bacterial lysates with or without SpSet2 and H3 peptides either unmodified (H3, 1-20 or H3, 27-45) or trimethylated at H3 K36 (H3, 27-45 K36 tri-Me) in the presence of [³H]SAM.