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Characterization of the c‐specific promoter of the gene encoding human endothelin‐converting enzyme‐1 (ECE‐1)
Author(s) -
Funke-Kaiser H.,
Bolbrinker J.,
Theis S.,
Lemmer J.,
Richter C.-M.,
Paul M.,
Orzechowski H.-D.
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01086-3
Subject(s) - promoter , microbiology and biotechnology , gene isoform , biology , gene , start codon , transfection , reporter gene , rnase p , mutant , transcription (linguistics) , transcriptional regulation , messenger rna , gene expression , genetics , rna , linguistics , philosophy
Human ECE‐1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE‐1, we studied the regulatory region of ECE‐1c, the major ECE‐1 isoform. A genomic clone comprising the complete human ECE‐1 gene including the putative ECE‐1c‐specific promoter was obtained. Up to 968 bp upstream of the putative c‐specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE‐1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (−142/−240 and −240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5′‐RACE revealed multiple transcriptional start sites located at about −110, −140 and −350 bp. Site‐directed mutagenesis demonstrated a crucial role for the E2F cis‐element for basal ECE‐1c promoter activity. Additionally, we found a correlation between isoform‐specific ECE‐1 mRNA levels and corresponding ECE‐1a, 1b, 1c promoter activities.