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. 2019 Jun 8;11(6):792. doi: 10.3390/cancers11060792

Figure 2.

Figure 2

SCAN zinc finger MZF1 directly trans-activates the CDC37 gene. (A) Schemes of the CDC37 promoter–reporter constructs. Truncated mutants of CDC37 promoter were connected with the luciferase (Luc) gene. Blue box, MZF1 binding site. Orange box, heat shock element (HSE). 5′UTR, 5′ untranslated region. (B) The secondary structures of native MZF1 and the SCANless construct (zinc finger domain alone). S, SCAN box. Z, zinc finger motif. DE, aspartic acid- and glutamic acid-rich region. GP, glycine- and proline-rich region. MZF1 was overexpressed with an N-terminal Flag-tag. The SCANless construct was overexpressed with a C-terminal V5-tag. (C) Luciferase activities from the truncated CDC37 promoters controlled by MZF1 and the SCANless construct. Plasmid constructs shown in panels A and B were co-transfected into DU-145 cells. Vec., empty vector control. n = 3, * p < 0.05, ** p < 0.01 (vs. Vec.). (D) Chromatin Western blotting of MZF1 overexpressed in DU-145. Crosslinked chromatin was prepared from DU-145 overexpressed with MZF1. (E) ChIP-qPCR analysis showing MZF1 occupancy of CDC37. Chromatin was prepared from DU-145 overexpressed with Flag-MZF1 or GFP (control) and immunoprecipitated using anti-Flag antibody beads or the control, IgG. Co-immunoprecipitated DNA was analyzed by qPCR for CDC37 (−1.8k or −0.4k regions) or control regions in GAPDH or ACTB. N = 3, ** p < 0.01 (vs. IgG control). Similar results were obtained from three independent experiments.