Journal of Leukocyte Biology eBioscience full spectrum cell analysis
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Originally published online as doi:10.1189/jlb.0803384 on November 21, 2003

Published online before print November 21, 2003
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(Journal of Leukocyte Biology. 2004;75:332-341.)
© 2004 by Society for Leukocyte Biology

{alpha}-Defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes

Cindy M. Yamamoto, Niaz Banaiee, Nannette Y. Yount, Bindi Patel and Michael E. Selsted1

Departments of Pathology & Microbiology & Molecular Genetics, College of Medicine, University of California, Irvine

1 Correspondence: Department of Pathology, College of Medicine, University of California, D440 Med. Sci. I, Irvine, CA 92697-4800. E-mail: meselste{at}uci.edu

{alpha}-Defensins are antimicrobial peptides that contribute to innate-immune functions of neutrophils and intestinal Paneth cells. Transcription of {alpha}-defensin genes occurs early in neutrophilic myelopoeisis. To examine the mechanisms that regulate {alpha}-defensin gene expression, we analyzed transcription of rat neutrophil {alpha}-defensin NP-3 in D4 cells, a subclone of the promyelocytic cell line IPC-81. Northern blot analysis showed that D4 cells express fivefold higher levels of {alpha}-defensin mRNA than the parental cell line in a manner relatively independent of passage number. Increased levels of steady-state mRNA in D4 cells correlated with markedly elevated peptide levels detected by immunocytochemical staining. To identify the cis-acting DNA elements involved in tissue-specific expression, D4 cells were transfected with luciferase reporter constructs containing NP-3 gene 5'-flanking sequences. Analyses of transfected D4 cells demonstrated that the proximal 87 base pair (bp) sequence contained cis-acting DNA elements necessary for optimal promoter activity. Mutational analyses within the 87-bp region suggested the involvement of the CAAT box and a putative polyoma enhancer-binding protein 2/core-binding factor (PEBP2/CBF) site in defensin gene transcription. Transient transfection analyses using tandem repeats of oligonucleotides containing these sequences demonstrated that proximity of the CAAT box and PEBP2/CBF site was important for defensin promoter activity. Electrophoretic mobility shift assays indicated that PEBP2/CBF or a PEBP2/CBF-related protein was involved in a specific protein-DNA interaction occurring within a DNA fragment containing the CAAT and PEBP2/CBF sequences. These data identify functional trans- and cis-elements that regulate rat defensin gene expression in high defensin-expressing promyelocytic cells.

Key Words: neutrophils • mRNA • cellular differentiation • gene regulation






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