Figure 7
From: SUMOylation of ZFP282 potentiates its positive effect on estrogen signaling in breast tumorigenesis
SUMOylation of ZFP282 positively regulates its interaction with CoCoA and ERα. (a) 293T cells were transfected with expression vectors as indicated. Cell extracts were prepared in the presence of N-ethylmaleimide (NEM) and immunoprecipitated with anti-ZFP282 antibody. The immunoprecipitates and inputs were analyzed by immunoblot using antibodies as indicated. (b) MCF-7 cell extracts were prepared in the presence or absence of N-ethylmaleimide as indicated. Equal amounts of extracts were immunoprecipitated with anti-CoCoA antibody, followed by immunoblot using the indicated antibodies. The arrowhead and arrow correspond to the SUMOylated and unmodified ZFP282, respectively. The intensity of SUMOylated and unmodified ZFP282 bands was quantified by densitometry, and the ratio of SUMOylated to unmodified ZFP282 band is indicated. (c, d) Pull-down assays. SUMOylated GST-ZFP282 proteins were incubated with immobilized recombinant His-CoCoA (c) or His-ERα-LBD (d). Bound ZFP282 proteins were analyzed by immunoblot with anti-ZFP282 antibody. The arrowhead and arrow correspond to the SUMOylated and unmodified ZFP282, respectively. The intensity of SUMOylated and unmodified ZFP282 bands was quantified by densitometry, and the ratio of SUMOylated to unmodified ZFP282 band is indicated. (e) 293T cells were transfected with expression vectors as indicated. Cell extracts were prepared in the presence of N-ethylmaleimide and immunoprecipitated with anti-ERα antibody. The immunoprecipitates and inputs were analyzed by immunoblot with the indicated antibodies. (f) SUMOylation of ZFP282 facilitates recruitment of ZFP282 and CoCoA to the pS2 promoter. MCF-7 cells were transfected with pFLAG.CMV4-ZFP282 WT or 3KR and treated with E2. ChIP assays using the indicated antibodies and quantitative PCR (qPCR) analyses were performed as in Figure 2d. Data are means±s.d. (n=3). Protein levels were monitored by immunoblot using the indicated antibodies. (g) The proposed model for SUMOylation-dependent regulation of ERα-ZFP282-CoCoA complex assembly. SUMOylation of ZFP282 increases its binding affinity for CoCoA and ERα, thereby contributing to stabilization of ERα-ZFP282–CoCoA complex formation on ERα target promoters. Bold arrows represent strong interactions, and dashed arrows represent weak interactions.
