Cloning and characterization of the human lung endothelial-cell-specific molecule-1 promoter.
Endothelial-cell-specific molecule-1 (ESM-1) is a cysteine-rich protein that is expressed primarily in endothelial cells of the lung, kidney and gut. In the present study, we have cloned and sequenced 3,888 bp of the 5' flanking region of the human ESM-1 gene. The full-length promoter directed high-level expression of the luciferase reporter gene in bovine lung microvascular endothelial cells and bovine aortic endothelial cells, but not in nonendothelial cell types. In 5' deletion analyses, a region spanning -81 to +58 was shown to contain information for endothelial-cell-specific expression. Mutational analysis in transient transfection assays uncovered an important role for an Ets-binding motif located between -77 and -74 and a cAMP-response-element (CRE)-like motif located between -68 and -62 in mediating high-level expression in endothelial cells. A second Ets site (-63 to -60) as well as a novel 6-bp palindromic sequence (-58 to -53) were found to inhibit expression. In DNase footprint analyses, both the Ets-binding motifs were protected specifically in endothelial cells, while the CRE-like element and palindrome were protected in endothelial and nonendothelial cells alike. Taken together, these results provide an important foundation for studying endothelial-cell-subtype-specific gene regulation.
Tsai
Jo C
JC
Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Mass 02215, USA.
Zhang
Jie
J
Minami
Takashi
T
Voland
Carole
C
Zhao
Shuping
S
Yi
Xianjin
X
Lassalle
Philippe
P
Oettgen
Peter
P
Aird
William C
WC
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003331:Coronary Vessels / Q000737:chemistry; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018983:DNA Footprinting; D004730:Endothelium, Vascular / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008168:Lung / Q000098:blood supply / Q000737:chemistry*; D008833:Microcirculation / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000235:genetics* / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D011651:Pulmonary Artery / Q000737:chemistry; D012333:RNA, Messenger; D017422:Sequence Analysis, DNA; D012867:Skin / Q000098:blood supply; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins / Q000737:chemistry
D017362:Cyclic AMP Response Element-Binding Protein; C101134:ESM1 protein, human; D009363:Neoplasm Proteins; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D014157:Transcription Factors