The myeloid zinc finger gene, MZF-1, regulates the CD34 promoter in vitro

Abstract

MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.