Figure 4.

GATA2 and MZF1 Are Transcription Factors of CDH2 Induction by EDN1

(A) Luciferase reporter assay to determine activity of the CDH2 promoter region (the −462- to −18-bp fragment from the ATG) and fragments of the promoter sequence (bases −335 to −18, −214 to −18, and −74 to −18). EDN1 significantly enhanced the promoter activity (−462- to −18-bp fragment; p < 0.05). Deletion of the sequence between −462 and −335 bp retarded this effect (−462 to −18 bp versus −335 to −18; p < 0.05). (B) The sequence from −462 to −335 bp contained one GATA2 and one MZF1 binding sequence. Putative GATA2 and MZF1 binding regions were identified using the TFSEARCH database v1.3, and point mutagenesis was carried out. (C) Luciferase reporter assay to determine the promoter activity of the −462- to −335-bp fragment with and without point mutations. The promoter activity of the −462- to −335-bp region was increased by EDN1 treatment (p < 0.05) but was obliterated by point mutations at either GATA2 or MZF1 site (p < 0.05). (D) Luciferase reporter assay to determine the promoter activity of the −462- to −335-bp fragment with and without point mutations in cells overexpressing GATA2 or MZF1 is shown. The promoter activity of the −462- to −335-bp region was increased by co-expression of GATA2 or MZF1 (p < 0.05). Reduced activity was observed in each point mutant (ΔGATA2-MZF1 or ΔMZF1-GATA2; no significant difference).