Characterization of the human EDF-1 minimal promoter: involvement of NFY and Sp1 in the regulation of basal transcription.
Endothelial Differentiation Factor (EDF)-1 is a calmodulin binding protein involved in the repression of endothelial cell differentiation, a crucial, late step in angiogenesis. Its expression is cell cycle regulated, although its transcriptional regulation is yet to be determined. To map the promoter region and to understand its regulation, we cloned and fused 2300 bp upstream of EDF-1 translational start site to a luciferase reporter gene. After transient transfection in HeLa cells, this fragment was shown to possess a promoter activity. Deletion constructs of the 5' flanking region of EDF-1 lead to the identification of the minimal promoter region which was highly homologous to the mouse sequence. No TATA box was detected, whereas three consensus sequences--two GC boxes and a CAAT box--were identified. EMSA supershift and chromatin immunoprecipitation demonstrated that these sequences were binding sites for Sp1/Sp3 and NFY, respectively. Deletion of Sp1/Sp3 and NF-Y consensus sequences resulted in the total loss of EDF-1 promoter activity. Our studies indicate that Sp1 and NFY binding is essential for EDF-1 promoter activity.
University of Milan, Dipartimento di Scienze Precliniche-LITA Vialba, Via GB Grassi 74, Milan, Italy.
Fabrizio
F
Bolognese
Marià
M
Pitarque-Martì
Viviana
V
Lo Cicero
Roberto
R
Mantovani
Jeanette A M
JA
Maier
D024506:5' Flanking Region; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D023081:CCAAT-Binding Factor; D002148:Calmodulin-Binding Proteins; C115911:EDF1 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C402244:nuclear factor Y; D008156:Luciferases