Alpha-defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes.
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.
Yamamoto
Cindy M
CM
College of Medicine, University of California, D440 Med. Sci. I, Irvine, CA 92697-4800, USA.
Banaiee
Niaz
N
Yount
Nannette Y
NY
Patel
Bindi
B
Selsted
Michael E
ME
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D042381:Granulocyte Precursor Cells / Q000378:metabolism*; D009154:Mutation; D038042:Myelopoiesis; D009504:Neutrophils; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D023084:alpha-Defensins / Q000096:biosynthesis / Q000235:genetics*
D023084:alpha-Defensins; C482907:neutrophil peptide 3, rat