Fig. 4

EP300-ZNF384 fusion protein transactivates the promoter activity of IL3RA (A) EP300-ZNF384- or ZNF384-expressing vectors were co-transfected with the PGL3-IL3RA-promoter and pRL-TK into HEK-293T cells. After 24 h, dual luciferase assay was performed. Data represents the mean ± SD. **P < 0.01, ***P < 0.001. (B) Increasing doses of EP300-ZNF384-expressing vectors and the luciferase reporter vectors were co-transfected into HEK-293T cells. Luciferase analysis for the IL3RA promoter was performed. ***P < 0.001. (C) Diagram showing the putative binding sites of ZNF384 in IL3RA promoter (orange). (D) Truncated mutations of the IL3RA promoter are shown. Numbering is indicated with respect to the transcriptional start site. (E) Luciferase analysis for the truncated IL3RA promoters was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. (F) Schematic presentation of the IL3RA promoter mutations used in this study. The mutated IL3RA promoter reporter along with pRL-TK were co-transfected with the empty vector, EP300-ZNG384, or ZNF384-expressing vectors into HEK-293T cells for 24 h. Dual-luciferase assay was performed. ***P < 0.001, ns, no significance. (G) HEK-293T cells were transfected with flag-tagged EP300-ZNF384- and ZNF384- expressing vectors for 48 h. Chromatin fragments of these cells were immunoprecipitated with a flag or nonspecific antibody (ns-Ab). PCR was conducted with primers corresponding to the genomic IL3RA and MMP7 promoter sequences. MMP7 served as a positive control