Figure 3. ZNF764 differentially regulates DNA-binding and transcriptional activity of GR.

(A) Venn diagram for the genes differentially bound and regulated by GR in the presence or absence of ZNF764 knockdown (KD). Numbers of the GR-binding genes in the presence (right circle) or absence (left circle) of ZNF764 siRNA are shown. Group1: genes for which GR-binding sites are found only in the presence of ZNF764 (control KD), Group 2: genes for which GR-binding sites are found independently to ZNF764, Group 3: genes for which GR-binding sites are found only in the absence of ZNF764 (ZNF764 KD). (B) GR-binding profiles for the 3 representative genes. GR-binding profiles around the 3 representative genes bound and regulated by GR in the presence of (top: DUSP1), independent to (middle: BEST2) or in the absence of (bottom: GPR153) ZNF764 are shown. Statistically significant (p < 0.05) GR-binding sites are indicated with the colored boxes positioned under the corresponding peaks. (C) ZNF764 differentially regulates mRNA expression of the 3 representative genes and changes the attraction of GR and itself to GREs of these genes. HeLa cells were transfected with 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 (top panels). Association of GR or ZNF764 to GREs of DUSP1, BEST or GPR153 was determined using the ChIP assays performed using the anti-GR or -ZNF764 antibody and with the subsequent SYBR Green real-time PCR. Obtained ChIP signals were normalized for those with control IgG, and fold association was further calculated as the baseline with control siRNA in the absence of dexamethasone as “1” (middle and bottom panels). 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 or fold association of GR or ZNF764 to GREs of these genes. **p < 0.01, n.s., not significant, compared to the conditions indicated. Broken lines indicate the level of fold expression as “1”.