The eosinophil-derived neurotoxin (EDN/RNS2) is a member of the mammalian ribonuclease gene family and is one of four proteins found in the large specific granules of human eosinophilic leukocytes. The gene encoding EDN consists of two exons, including a noncoding exon 1, separated by a single intron from the coding sequence in exon 2. We have identified a functional promoter of the EDN gene and shown that optimal expression depends on interaction between the promoter and one or more sequence elements found in the single intron. Cells of the clone 15 eosinophilic variant of the human promyelocytic HL-60 cell line were transfected with constructs that included the promoter region of the EDN gene alone, promoter with exon 1, and promoter with both exon 1 and the intron positioned 5` to the chloramphenicol acetyltransferase (CAT) reporter gene (constructs referred to as PrCAT, PrExCAT, and PrExInCAT, respectively). Although reporter gene activity from either PrCAT or PrExCAT was only 2-3 fold higher than baseline (CAT alone), inclusion of the single intron (PrExInCAT) resulted in a 28-fold increase in reporter gene activity in uninduced clone 15 cells, and an 80-fold in activity when clone 15 cells were induced to differentiate toward eosinophils with butyric acid. The intron-mediated enhancer activity was reproduced in other human hematopoietic cell lines (K562, Jurkat, U937, and HL-60), but was not found in human 293 kidney cells, suggesting that the function of the enhancer element(s) may be tissue-specific. A significant portion of the observed enhancer activity resides in the first 60 base pairs the the intron, which includes consensus binding sites for both AP-1 and NF-ATp transcription factors, and a 15-base pair segment that is identical to a sequence found in the promoter of the gene encoding the neutrophil granule protein, lactoferrin. The noncoding exon 1/single intron/coding exon 2 genomic structure is a common feature among the mammalian ribonucleases; this finding suggests the possibility of a conserved mechanism of regulation in this gene family.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Z. Qiu, K. D. Dyer, Z. Xie, M. Radinger, and H. F. Rosenberg GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene J. Biol. Chem., May 8, 2009; 284(19): 13099 - 13109. [Abstract] [Full Text] [PDF]

				K. D. Dyer and H. F. Rosenberg The mouse RNase 4 and RNase 5/ang 1 locus utilizes dual promoters for tissue-specific expression Nucleic Acids Res., February 18, 2005; 33(3): 1077 - 1086. [Abstract] [Full Text] [PDF]

				R. M. Phillips, V. E. L. Stubbs, M. R. Henson, T. J. Williams, J. E. Pease, and I. Sabroe Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter J. Immunol., June 15, 2003; 170(12): 6190 - 6201. [Abstract] [Full Text] [PDF]

				P.-C. Tang, H.-C. Huang, S.-C. Wang, J.-C. Jeng, and Y.-D. Liao Regulation of ribonuclease expression by estradiol in Rana catesbeiana (Bullfrog) Nucleic Acids Res., July 15, 2002; 30(14): 3286 - 3293. [Abstract] [Full Text] [PDF]

				J. Zhang and H. F. Rosenberg Sequence Variation at Two Eosinophil-Associated Ribonuclease Loci in Humans Genetics, December 1, 2000; 156(4): 1949 - 1958. [Abstract] [Full Text]

				M. A. Giembycz and M. A. Lindsay Pharmacology of the Eosinophil Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340. [Abstract] [Full Text] [PDF]

				T. J. O'Leary Reducing the Impact of Endogenous Ribonucleases on Reverse Transcription-PCR Assay Systems Clin. Chem., April 1, 1999; 45(4): 449 - 450. [Full Text] [PDF]

				Y. Yamaguchi, S. J. Ackerman, N. Minegishi, M. Takiguchi, M. Yamamoto, and T. Suda Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2, Transactivates the Promoter of the Eosinophil Granule Major Basic Protein Gene Blood, May 1, 1998; 91(9): 3447 - 3458. [Abstract] [Full Text] [PDF]

				T. B. van Dijk, E. Caldenhoven, J. A.M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, and R. P. de Groot The Role of Transcription Factor PU.I in the Activity of the Intronic Enhancer of the Eosinophil-Derived Neurotoxin (RNS2) Gene Blood, March 15, 1998; 91(6): 2126 - 2132. [Abstract] [Full Text] [PDF]

				J. Z. Zhang and C. R. Somerville Suspensor-derived polyembryony caused by altered expression of valyl-tRNA synthetase in the twn2 mutant of Arabidopsis PNAS, July 8, 1997; 94(14): 7349 - 7355. [Abstract] [Full Text] [PDF]

				J. S. Handen and H. F. Rosenberg Intronic Enhancer Activity of the Eosinophil-derived Neurotoxin (RNS2) and Eosinophil Cationic Protein (RNS3) Genes Is Mediated by an NFAT-1 Consensus Binding Sequence J. Biol. Chem., January 17, 1997; 272(3): 1665 - 1669. [Abstract] [Full Text] [PDF]