Abstract
BackgroundHuman eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported.ResultsIn this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors.ConclusionOur results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters.
Highlights
Human eosinophil-derived neurotoxin and eosinophil cationic protein are members of a subfamily of primate ribonuclease genes
The results showed that the ecp and edn promoters branched when the 34-nt segment disappeared in some species, in agreement with the notion that ecp and edn were the most rapidly evolving coding sequences of primate genes [4]
The results showed that the formation of the major band shift was completely abolished only by the Myc-associated zinc finger protein (MAZ) consensus oligonucleotide (Fig. 3B, lane 4), but not consensus SV-40 protein-1 (Sp1), liver factor A1 (LF-A1), hepatocyte nuclear factor-4 (HNF-4), and activator protein-2 (AP2) oligonucleotides, with serum responsive element (SRE) and c-myb oligonucleotides were used as negative controls
Summary
Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes They are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported. Human edn and ecp respectively encode eosinophilderived neurotoxin (EDN) and eosinophil cationic protein (ECP), two of the four major proteins found in granules of human eosinophilic leukocytes Their gene products belong to members of the human RNaseA superfamily, which comprises RNase1-13 [1,2,3,4,5,6]. The regulatory motifs in promoter regions of duplicated genes are generally conserved during duplication events [19,20,21]
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