Figure 2. ELK3 represses the transcriptional activity of the ID4 promoter. Expression of ELK3 and ID4 (a) mRNA and (b) protein in control, ELK3KD, and ELK3KD-231 & 578T cells is restored by introduction of an ELK3-expressing plasmid. (c) ELK3 binding motif on the ID4 promoter of humans, mice, rats, and zebrafish (cut off, p-value < 0.01). Protein-binding motif alignment was performed using EPD (Eukaryotic Promoter Database). (d) (left) Schematic showing construction of the WT ID4 promoter (−1098~+100 bp) and the Mut ID4 promoter for the luciferase reporter assay. The Mut ID4 promoter has point mutation in the ELK3 binding sequence, as described. (Right) Luciferase assay of the pGL3 reporter plasmid, including the WT ID4 promoter region (−1098~+100 bp) and the Mut ID4 promoter. The luciferase reporter plasmid (cloned into the ID4 promoter) was cotransfected with ELK3-expressing plasmids or control plasmids into ELK3KD MDA-MB-231 cells. (e) ChIP-qPCR analysis of ELK3 binding to the ID4 promoter region. A Flag-ELK3 expressing plasmid or a Flag-control plasmid was transfected into ELK3KD MDA-MB-231 cells, and Flag-immunoprecipitates were subjected to qPCR using primers specific for the ID4 promoter region (−28~+107 bp). All data used in statistical analyses were originated from at least three independent experiments. Data represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.