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. Author manuscript; available in PMC: 2022 Jul 4.
Published in final edited form as: Mol Cell. 2021 May 11;81(13):2793–2807.e8. doi: 10.1016/j.molcel.2021.04.021

Figure 2. Set2 and Set1 (COMPASS) methylate H3K37 in vitro.

Figure 2

(A) In vitro methyltransferase assay. Recombinant wild-type Set2p and Set2Y149Ap mutant proteins were incubated with wild-type (WT) H3.1 nucleosomes in the presence of SAM. Reactions were separated by SDS-PAGE in 16% acrylamide gels. Blots were probed with anti-H3K37me1 antibody and then re-probed sequentially with anti-H3K36me1 and anti-H3 antibodies. Finally, the membrane was blotted with anti-MBP antibody to monitor the amount of enzyme in each reaction. (B) In vitro methyltransferase assay. Equal amount of recombinant wild-type Set2p was incubated with H3.1WT or H3.1K37R nucleosomes in the presence of SAM. Reactions were separated by SDS-PAGE in 16% acrylamide gels. Blots were probed with anti-H3K37me1 antibody and then re-probed sequentially with anti-H3K36me1 and anti-H3 antibodies. (C) In vitro methyltransferase assay. Wild-type PtA-Set1p and PtA-set1ΔC92p yeast purified complexes were incubated with wild-type H3.1 nucleosomes in the presence of SAM. Reactions were separated by SDS-PAGE in 16% acrylamide. Blots were probed with anti-H3K37me1 antibody and sequentially with anti-H3K4me1 and anti-H3 antibodies in this order. * indicates Δ tail–H3. PtA-Set1p and PtA-set1ΔC92p in each reaction were detected using anti-PAP antibody. (D) In vitro methyltransferase assay. Equal amount of wild-type PtA-Set1p complex was incubated with H3.1WT or H3.1K37R nucleosomes in the presence of SAM. Reactions were separated by SDS-PAGE in 16% acrylamide gels. Blots were probed with anti-H3K37me1 antibody and sequentially with anti-H3K4me1 and anti-H3 antibodies in this order. * indicates Δ tail–H3.