Figure 5: The KRAB domain and the zinc finger domain are both necessary for the regulation of GR transcriptional activity by ZNF764.

(A) The transdominant negative fragment ZNF764 (1-175) competes with differential actions of endogenous ZNF764 on DUSP1 and GPR153, but not on BEST2. HeLa cells were transfected with control or ZNF764 (1-175)-expressing plasmid in the presence or absence of control or ZNF764 siRNA, and were cultured in the presence or absence of 10⁻⁶ M of dexamethasone. mRNA expression of DUSP1, BEST, GPR153 and RPLP0 was determined with the SYBR Green real-time PCR using their specific primers. Bars represent mean ± S.E. values of fold DUSP1 (left panel), BEST2 (middle panel) or GPR153 (right panel) mRNA expression normalized for RPLP0 mRNA expression. Broken lines indicate the level of fold expression as “1”. *p < 0.05, **p < 0.01, n.s., not significant, compared to the 2 conditions indicated. (B) Both ZNF764 (1-175) and (176-407) fragments are attracted to DUSP and GPR153 GREs, but not to BEST2 GREs in HeLa cells. HeLa cells were transfected with the indicated His-tagged ZNF764 fragment-expressing plasmid, and were cultured in the presence or absence of 10⁻⁶ M of dexamethasone. Association of His-tagged ZNF764 fragments to GREs of DUSP1, BEST or GPR153 was determined in the ChIP assays performed using the anti-His antibody and subsequent SYBR Green real-time PCR using specific primer pairs for these GREs. Obtained ChIP signals were normalized for those with control IgG, and fold association was further calculated as the condition transfected with ZNF764 (1-407) and in the absence of dexamethasone as “1”. Bars represent mean ± S.E. values of the fold association of the indicated ZNF764-related molecules to GREs of these genes. *p < 0.05, **p < 0.01, n.s., not significant, compared to the conditions indicated. Broken lines indicate the level of fold expression as “1”.