Figure 2

Ik-1 and MZF1 interact with and bind the IGF-IR gene promoter. (A) IGF-IR gene promoter map including the transcription (AGT) and translation (ATG) start sites. The 3 PCR fragments used in the luciferase assay (F1: −682/-137, F2: −137/+583, F3: +530/+1137) and 5 sites that potentially bind with Ik-1 are shown. *: 2 sites confirmed by ChIP assay. (B) In addition to the 3 PCR fragments, 6 sites that potentially bind with MZF1 are depicted. *: 3 sites confirmed by the ChIP. (C) Luciferase assay performed in R⁻ cells demonstrates that transfection of Ik-1 or MZF1 decreased IGF-IR promoter activity with F1 and F2, but not with F3. Cotransfection of Ik-1 and MZF1 induced more pronounced inhibitory effects on F1 and F2 than one transcription factor alone (*: P < 0.05 vs. F1+EV and F2+EV; †: P < 0.01 vs. F1+Ik-1 and P < 0.0001 vs. F1+EV; ‡: P < 0.0001 vs. F1+EV and F1+Ik-1 and P < 0.01 vs. F1+MZF1; ¥: P < 0.001 vs. F2+EV; ¶: P < 0.001 vs. F2+EV and < 0.0001 vs. F2+Ik-1 and F2+MZF1). Ik-1 and MZF1 failed to induce similar effects when F1 and F2 were mutated (MT) at potential binding sites. The results are shown as means ± SE of 3 consistent experiments. (D) ChIP assay shows that Ik-1 binds with binding site 2 (BS2) and BS4, but not BS3, of the promoter. Controls, including EV, IgG, input, and H₂O, worked properly. The 2 binding sites are identified by an (*) in (A). (E) ChIP assay confirms that MZF1 binds with the promoter at BS2, BS3, and BS4, and not BS5, which are marked by an (*) in (B). Controls worked properly. Some of the panels shown in (D) and (E) have been slightly enhanced in their entirety to assist with the visualization of the weak bands, which are pertinent to the results.