RESUMO
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.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a Calmodulina/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Região 5'-Flanqueadora , Sequência de Bases , Sítios de Ligação , Fator de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Deleção de Sequência , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genética , Sítio de Iniciação de TranscriçãoRESUMO
Yondelis is a potent DNA-binding anticancer drug isolated from the tunicate Ecteinascidia turbinata currently undergoing phase III clinical trials. We and others have shown selective inhibition to the transcriptional induction of several genes. We tested the hypothesis that Yondelis specifically targets cell-cycle genes. Our analysis on endogenous and transfected reporter systems revealed complex patterns of transcriptional inhibition and, surprisingly, activation. Other inducible systems-the metallothionein and the CYP3A4 promoters-were little affected. We assayed whether interference of DNA binding of the common nuclear factor Y (NF-Y) activator was responsible for the observed inhibition: in vivo chromatin immunoprecipitation analysis in NIH3T3 and HCT116 cells indicates that NF-Y binding is little affected by Yondelis addition. Finally, histone acetylation was modestly affected only on Cdc2 and cyclin B2 but not on other repressed promoters. These data prove that Yondelis is not a general inhibitor of inducible genes, and its selective effects are exerted downstream from transcription factors binding and histone acetyl transferases recruitment.
