Fig 2: Immunoprecipitation and GST-pulldown experiments confirmed the binding of MOF and YY1. (A,B) Endogenous MOF and YY1 were immunoprecipitated by overexpressed Flag-YY1 and Flag-MOF in HCT116 cells. Bound proteins were measured by Western blot analysis. GAPDH was used as an internal control. (C) Co-transfection and Flag IP confirmed the interaction between YY1 and MOF. HCT116 cells were co-transfected with Flag-YY1 and untagged MOF, and bound MOF was measured after a Flag IP. (D) The colocalization analysis of YY1 and MOF. Endogenous YY1 (green) and MOF (red) in SW480 cells were visualized by IF staining. DAPI staining showed the nuclei. Scale bar indicates 200 µm. (E) Different lengths of YY1. (F) A GST-pulldown assay was performed by mixing whole cell lysates of Flag-MOF overexpressing 293T cells and the GST-tagged deletion mutants of YY1 proteins. (G) The lack of the 146–270 region in YY1 decreased its binding to MOF. (H) The 146–270 region of YY1 competed for binding to MOF. HCT116 cells were transiently transfected with YY1 and MOF, as indicated; 48 h later, the MOF protein level was analyzed after a Flag IP. IB, immunoblot.

Fig 3: The YY1K183 site regulated the acetylation-mediated ubiquitin degradation of YY1. (A) There are three predicted potential ubiquitination sites (K183, K208, and K258 indicated by red color) in the 146–270 region of YY1. (B) Protein expression of the mutant plasmids. (C) The effect of mutations of lysine K183, K208, and K258 to arginine on YY1 ubiquitination. (D) Lysine K183 and K258 mutations suppressed YY1 ubiquitin degradation. (E) The effect of acetylation on ubiquitination of YY1 and mutants. (F) Protein stability of the YY1K183R mutant. (G) The effects of MOF-mediated acetylation on the ubiquitin-mediated degradation of the YY1K183R mutant.

Fig 4: Acetylation of YY1 by the MOF/MSL complex negatively regulated its protein stability in HCT116 cells. (A) YY1 may be modified by MOF. Endogenous YY1 was detected by a Western blot analysis after overexpressing MOFwt or the MOFG327E mutant in HCT116 cells. The red arrow indicates YY1 that may be modified. (B) Acetylation of YY1 by MOF facilitated its degradation. (C) Degradation of YY1, caused by MOF, was inhibited by the MOF enzyme activity inhibitor MG149. (D) The inactivated MOFG327E mutant inhibited the degradation of YY1. (E) The knockdown of MOF reduced YY1 degradation. (F) Elevated MOF decreased the endogenous YY1 protein level. Scale bar indicates 200 µm. (G,H) The MOF/MSL complex promoted YY1 degradation. HCT116 cells were transfected with YY1 alone or co-transfected with MSL2 or MSL1, and 48 h later, YY1 degradation was measured by Western blot using an anti-HA antibody. The red arrow in (G) indicates MSL2 protein. GAPDH was used as an internal control.

Fig 5: The YY1/146–270 region and the YY1K183 site play important roles in p53RE-mediated downstream target gene transactivation in HCT116 cells. (A) The schematic diagram of target genes up- or down-regulated by YY1. (B) The schematic diagram of a p53RE-Luc plasmid. Multiple response elements of TP53 were inserted into the pp53-TA-Luc vector. (C,D) The effects of YY1 and its truncated mutants on p53RE-Luc luciferase activity and related protein levels. * p < 0.05 or # p < 0.05, compared to the p53RT-Luc group. (E) P53 and its downstream protein levels in YY1 knockdown HCT116 cells. (F) Effects of YY1 and its point mutants on p21 protein levels. Increasing amounts of YY1wt, YY1K183R, and YY1K258R were transfected into HCT116 cells, and p21 protein levels were analyzed by a Western blot using the anti-p21 antibody. (G) The effects of YY1 and MOF on p53RE-Luc luciferase activity. * p < 0.05 or ** p < 0.01 compared to the p53RT-Luc group; ## p < 0.01 or ### p < 0.001 compared to the p53RT-Luc+MOF groups.