A nonerythroid GATA-binding protein is required for function of the human preproendothelin-1 promoter in endothelial cells

Autor: D B Wilson, D M Dorfman, S H Orkin
Rok vydání: 1990
Předmět:
Zdroj: Molecular and Cellular Biology. 10:4854-4862
ISSN: 1098-5549
0270-7306
DOI: 10.1128/mcb.10.9.4854
Popis: 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.
Databáze: OpenAIRE
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