Figure 1

SET7/9 methylates YY2 in vitro. (a, b) In vitro methylation assay was performed by mixing purified bacterially expressed His-tagged YY2 protein with histone lysine methyltransferases (KMTs), either FL or truncations with enzymatic domain, from bacterial cells (a) or HEK293T cells with overexpression (b) as indicated, followed by autoradiogram. Stars indicate auto-methylation (auto-me) of KMTs. Black arrows indicate methylation of YY2 (YY2(me)). Wild type, wt; enzymatically dead mutant, m. (c) In vitro methylation assay was performed by mixing purified bacterially expressed His-tagged SET7/9 with YY2 amino- (1–226) or carboxyl terminus (227–372), followed by autoradiogram. (d) The expression of YY2 (1–226) and YY2 (227–372) in c was examined with the help of coomassie blue staining (C.B.S.). (e) In vitro methylation assay was performed by mixing purified bacterially expressed His-tagged SET7/9 with YY2 (1–226) wt or YY2 (1–226) with substitution of lysine 139 to arginine (K139R), followed by autoradiogram. (f) The expression of YY2 (1–226; wt) and YY2 (1–226; K139R) in e was examined by C.B.S. (g) In vitro methylation assay was performed by mixing purified bacterially expressed His-tagged SET7/9 with YY2 (227–372) wt or YY2 (227–372) with substitution of lysine 247 or 369 to arginine (K247R or K369R), followed by autoradiogram (top panel) or immunoblotting (IB) with anti-YY2K247me1 antibody (bottom panel). (h) The expression of YY2 (227–372; wt), YY2 (227–372; K247R) and YY2 (227–372; K369R) in g was examined by C.B.S. (i) In vitro methylation assay was performed by mixing synthetic short peptides from YY2 containing unmodified (K247) or monomethylated K247 (K247me1) with or without purified bacterially expressed SET7/9 proteins, followed by dot blot assay and autoradiogram as indicated.