Figure 1

YY1 is methylated by SET7/9 in vitro.

(A,B) In vitro methylation assay was performed by mixing purified bacterially-expressed His-tagged YY1 protein with various protein lysine methyltransferases (KMTs), either full length (FL) or truncations with enzymatic domain, from bacterial cells (A) or HEK293T cells with over-expression (B) as indicated, followed by autoradiogram. Wild-type: wt; Enzymatically dead mutant: m. White arrows indicate automethylation (auto-me) of KMTs; Black arrows indicate methylation of YY1 (YY1(me)). (C) Schematic representation of the domain architecture of YY1 protein. Amino acid information for the three linker regions, aa321–324, 347–352 and 377–382, between Zn fingers as well as the very carboxyl-terminus was depicted. Acidic Region (light green); His-cluster (yellow); GA-rich region (light blue); GK-rich region (blue); Spacer (dark blue); Zn finger (purple); Linker region (black); The very carboxyl-terminus (red). (D) In vitro methylation assay was performed by mixing purified bacterially-expressed His-tagged SET7/9 protein with YY1 amino-terminus (1-266) or carboxyl-terminus (267-414), followed by autoradiogram (top panel). The expression of YY1(1-266) and YY1(267-414) was examined by coomassie blue staining (C.B.S) (bottom panel). (E) In vitro methylation assay was performed by mixing purified bacterially-expressed His-tagged SET7/9 with YY1(1-266) wild type (wt) or its mutant form with substitution of lysine 173 to arginine (K173R), followed by autoradiogram (top panel). The expression of YY1(1-266)(wt) and K173R was examined by C.B.S (bottom panel). (F) In vitro methylation assay was performed by mixing purified bacterially-expressed His-tagged SET7/9 with YY1(267-414) wild type (wt) or its mutant form with substitution of lysine 288, 305, 339, 341 or 411 to arginine (K288R, K305R, K339R, K341R or K411R), followed by autoradiogram (top panel). The expression of YY1(267-414)(wt), K288R, K305R, K339R, K341R and K411R was examined by C.B.S (bottom panel). (G) In vitro methylation assay was performed by mixing short peptides containing unmodified (K173 or K411) or mono-methylated K173 or K411 (K173me1 or K411me1) with or without purified bacterially-expressed SET7/9 proteins. Increased amount of each reaction was taken for dot blot as indicated, followed by autoradiogram.