Figure 4: ZNF764 and GR physically interact through the former’s portion spanning amino acids 1-96 and the latter’s LBD.

(A) ZNF764 is co-immunoprecipitated with GR in HeLa cells. HeLa cells were treated with 10⁻⁶ M of dexamethasone and immunoprecipitation was performed using anti-GR antibody or control IgG. Coprecipitated ZNF764 and ZNF764/GR in input samples were detected with Western blots. (B) ZNF764 and GR physically interact through the former’s portion spanning amino acids 1-96 and the latter’s LBD in GST pull-down assays. ³⁵S-labelled GR or ZNF764 was incubated with indicated proteins fused with GST. NRB: nuclear receptor box (C) ZNF764 fragments containing amino acids 1-96 are co-precipitated with GR in HCT116 cells. HeLa cells were transfected with indicated His-ZNF764 fragment-expressing plasmid, and immunoprecipitation was carried out with anti-His antibody. Co-precipitated GR, and GR/ZNF764 fragments expressed in input samples were detected with Western blots. (D) ZNF764 and GR interact through the former’s amino acids 41-96 and the latter’s LBD outside the AF-2 surface in a mammalian two-hybrid assay. HCT116 cells were transfected with the plasmid expressing indicated VP16-AD-fused ZNF764 fragment and that expressing GAL4 DBD-GRα LBD (left panel) or -GRα full-length (FL) (right panel) together with GLA4 DBD-responsive p17mer-TK-Luc and pGL4.73[hRluc/SV40]. Bars represent mean ± S.E. values of the corrected firefly luciferase activity. In the right panel, numbers above the 2 bars indicate mean ± S.E. values of the fold association of the 2 molecules shown. **p < 0.01, n.s.: not significant, compared to the the two conditions indicated, or the condition in the presence of GAL4 DBD-GRα FL wild type (WT) and dexamethasone but in the absence of VP16 AD-ZNF764 (#). (E) GR-binding ZNF764 fragment (1-175) suppresses wild type ZNF764-induced enhancement of GR transcriptional activity in HCT116 cells. HCT116 cells were transfected with the plasmid expressing the indicated ZNF764-related molecules together with pRShGRα, pMMTV-Luc and pGL4.73[hRluc/SV40]. Bars represent mean ± S.E. values of the corrected firefly luciferase activity. **p < 0.01, compared to the the condition transfected with wild type (WT) ZNF764, but not its (1-175) fragment and in the presence of dexamethasone, or to the 2 conditions indicated.