ISOLATION AND FUNCTIONAL-CHARACTERIZATION OF THE HUMAN GENE ENCODING THE MYELOID ZINC-FINGER PROTEIN MZF-1

The expression of the human myeloid zinc finger gene (MZF-1) by human bone marrow cells is necessary for granulopoiesis. We have analyzed th e structure and function of the human MZF-1 gene by diagnostic polymer ase chain reaction, genomic cloning, and promoter analysis. Comparison of human promyelocytic HL-60 cell cDNA with isolated MZF-1 genomic cl ones indicated that the human MZF-1 gene is without introns and spans approximately 3 kb. Restriction enzyme mapping and Southern analysis i ndicated further that the human MZF-1 gene is a single-copy gene, Prim er extension studies identified the major transcription start site as a thymidine residue located 1102 bp upstream of the ATG translation st art codon, A putative TATA box sequence (TAAAAA) was found at -66 bp a nd a CCAAT box at -130 bp relative to the transcription initiation sit e. In HL-60 cells, MZF-1 mRNA levels are increased by granulopoietic i nducers including retinoic acid and GM-CSF. DNA upstream of the transc ription start site contains tandem-repeated consensus retinoic acid re sponse elements at -666 through -696 bp and paired putative GM-CSF-res ponsive sequences centered at -50 and -100 bp. CAT reporter gene const ructs containing these DNA regions promoted transcription and conferre d transcriptional responsiveness to retinoic acid and GM-CSF when tran sfected into HL-60 cells. Additional putative regulatory binding sites included conserved MZF-1 zinc finger binding sequences, the importanc e of which was suggested by the enhanced expression of the endogenous MZF-1 gene following vector-driven expression of MZF-1 constructs in K 562 myeloblastic leukemia cells. These findings provide a clearer basi s for understanding the role of MZF-1 gene expression in myeloid cell growth and differentiation.