A nonerythroid GATA-binding protein is required for function of the human preproendothelin-1 promoter in endothelial cells.
Endothelin-1 (ET-1) is a 21-amino-acid peptide synthesized by endothelial cells that has potent vasoconstrictor activity. Human ET-1 is derived from a 212-amino-acid prepropeptide, termed preproendothelin-1 (PPET-1). To identify cis-acting sequences essential for PPET-1 gene transcription, bovine aortic endothelial (BAE) cells were transfected with plasmids containing 5'-flanking sequences of the human PPET-1 gene fused to the human growth hormone gene as a reporter. Deletional analysis of these fusion plasmids showed that the sequence spanning positions -141 to -127 of the human PPET-1 promoter is required for full transcription activity. Introduction of clustered point mutations into this region of the promoter reduced transcription activity. Gel shift analysis, methylation interference, protein-DNA cross-linking, and oligonucleotide competition studies revealed that BAE cell nuclear extract contains a 47-kilodalton DNA-binding protein recognizing the core motif TATC (GATA) located at positions -135 to -132 of the PPET-1 promoter. The size and specificity of this DNA-binding protein resemble GF-1, a previously described transcription factor of erythroid cells that binds to the same core motif. Gel shift analysis indicated that GF-1 and the DNA-binding protein interacting with the PPET-1 promoter have different tissue distributions; the former is restricted to a subset of hematopoietic cells, and the latter is found in various cell types, including BAE, NIH 3T3, and HeLa cells. By using an antiserum to the C-terminal region of GF-1, the two proteins were also found to be antigenically distinct. When a growth hormone fusion plasmid containing the proximal 141 nucleotides of the PPET-1 promoter was transfected into a variety of cell types, these was preferential expression in cells of endothelial origin. We conclude that a nuclear factor with binding specificity for a GATA motif similar to that of the transcriptional activator GF-1 is necessary for the efficient and cell-specific expression of the human PPET-1 gene.
Wilson
D B
DB
Division of Hematology-Oncology, Children's Hospital, Boston, Massachusetts 02115.
Dorfman
D M
DM
Orkin
S H
SH
D000818:Animals; D001483:Base Sequence; D001973:Bromodeoxyuridine; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019332:Endothelin-1; D004730:Endothelium, Vascular / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D014162:Transfection
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D019332:Endothelin-1; D010455:Peptides; D011498:Protein Precursors; D001973:Bromodeoxyuridine