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. Author manuscript; available in PMC: 2014 Mar 28.
Published in final edited form as: Mol Cell. 2013 Feb 28;49(6):1121–1133. doi: 10.1016/j.molcel.2013.01.034

Figure 1. Establishment of In Vitro H3K4 Methylation Assays with Purified Yeast Set1 Complex and Ubiquitylated H2B-Containing Recombinant Chromatin.

Figure 1

(A) SDS-PAGE/Coomassie Blue staining and immunoblot analyses of the purified, baculovirus-reconstituted ySet1C. Band identities were confirmed by LC/MS analysis and by immunoblot. The dot indicates an N-terminal degradation product of FLAG-tagged Set1.

(B, C and D) Ubiquitylated H2B directly stimulates H3K4 methylation. Free H3 or H3K4R (B), chromatin templates assembled with indicated pre-modified histones (C) and octamers with indicated pre-modified histones (D) were subjected to in vitro HMT assays with purified ySet1C. H3 methylation status was monitored by immunoblotting with indicated antibodies or by fluorography (in this and subsequent figures). The asterisk in (C) indicates an uncharacterized methylation signal from a component in the chromatin assembly factor or ySet1C preparations.

Note: unmod, unmodified histones; H2Bub, lysine(K)120-ubiquitylated H2B; H3K4R, H3 containing an arginine(R) substitution at lysine(K)4; H3K4me, lysine(K)4-methylated H3; H3K4me1, lysine(K)4-mono-methylated H3; H3K4me2, lysine(K)4-di-methylated H3; H3K4me3, lysine(K)4-tri-methylated H3.

See also Figure S1.