Functional analysis of the endothelin-1 gene promoter. Evidence for an endothelial cell-specific cis-acting sequence.
Endothelin-1 (ET-1) is a peptide synthesized by endothelial cells both in culture and in vivo. ET-1 induces contraction of smooth muscle cells and stimulates growth in a variety of mesenchymal cell types. We have previously characterized the genomic organization of the ET-1 gene and described its chromosomal localization and promoter region sequence. In this report, we describe the use of fusion plasmids containing ET-1 5'-flanking sequence and the chloramphenicol acetyltransferase gene to identify cis-acting sequences that direct transcription of the ET-1 gene. When transfected into bovine aortic endothelial cells, constructs containing 143 base pairs of ET-1 5'-flanking sequence allowed maximal transcription, whereas constructs containing 129 base pairs of sequence had 40-fold lower rates of transcription. A synthetic DNA fragment encoding the region delineated by these deletion mutants was found to have a positive effect on transcription when placed in either orientation upstream of short inactive ET-1 promoter constructs. However, this increase in transcription was noted only when a second region containing an AP1 consensus sequence was also included in the constructs. In experiments with a heterologous promoter and a 119-base pair DNA fragment containing these two functional regions, this 119-base pair sequence acted in a positive and endothelial cell-specific fashion. Taken together, these data localize cis-acting sequences important in determining the rate and tissue specificity of ET-1 gene transcription and should allow the study of protein-DNA interactions which mediate transcription of this gene in endothelial cells.
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
M E
ME
Lee
K D
KD
Bloch
J A
JA
Clifford
T
T
Quertermous
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D016232:Endothelins; D004730:Endothelium, Vascular / Q000378:metabolism*; D005796:Genes; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D016232:Endothelins; D015345:Oligonucleotide Probes; D010455:Peptides