FIG. 3.

Intrinsically enhanced in vitro histone H3K4 methyltransferase activity of Set1(Y1052F) proteins within COMPASS. (A) The levels of Set1 and TAP-tagged Cps60 proteins in purified COMPASS were determined. Increasing concentrations of TAP-tagged Cps60-purified COMPASS, containing either wild-type or mutant Set1, were analyzed by Western blotting using anti-Set1 and anti-calmodulin binding peptide (CaM) antibodies. Each panel shows the results for aliquots of identical samples applied for Western blotting using different antibodies. Comparison of histone methyltransferase (HMTase) activity of wild-type and mutant Set1 within COMPASS toward histone H3K4 mono-, di-, and trimethylation (H3K4me1, -2, and -3 and H3) was performed with a histone methyltransferase assay mixture consisting of recombinant histone H3 substrate, the cofactor S-adenosylmethionine, and equal volumes of each COMPASS. Histone methyltransferase reaction products were subjected to Western analysis with the indicated antibody. Anti-H3 was used as a loading control. α, anti. (B) Subunit composition of COMPASS purified from either wild-type or Set1(Y1052F) was determined via MudPIT analysis. Error bars show standard deviations. NSAF, normalized spectral abundance factor; PRS315, plasmid pRS315; WT, wild-type Set1.