Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells.
Activated transcription of the urokinase-type plasminogen activator (uPA) gene depends on the enhancer, located approximately 2 kb from the start of transcription. The proximal promoter, driving basal transcription, contains a GC-/GA-rich sequence immediately upstream of the TATA box. We have investigated the role played by this element in the transcription of the uPA gene in HeLa and PC3 cells, which do not express or constitutively express the gene, respectively. This region binds either Sp1 or Sp3, as monomers or multimers, but not a combination of the 2 proteins. The more efficient binding of Sp1 to the proximal promoter in PC3 cells is correlated to its phosphorylation state. Polymerase chain reaction (PCR)-coupled, chromatin immunoprecipitation experiments with anti-Sp1 antibodies indeed show an enrichment of proximal promoter sequences in PC3 cells and support the observed difference in transcription levels from proximal promoter constructs in HeLa versus PC3 cells. Furthermore, overexpression of Sp1 increases transcription from the reporter construct in HeLa cells, whereas in PC3 cells, overexpression of Sp3 does not reduce transcription from the same construct, indicating that the Sp1/Sp3 balance cannot be shifted. We conclude that the GC-/GA-rich element of the uPA regulatory region is an independent functional element, regulated by Sp family proteins. Phosphorylation of Sp1 determines the presence in vivo and the functionality of this element in PC3 cells. Thus, the cellular context determines the relevance of the GC-/GA-rich region in uPA gene transcription, which contributes to constitutive gene expression, related, in turn, to the invasive phenotype.
Ibañez-Tallon
Inés
I
Laboratory of Molecular Genetics, Department of Biological and Technological Research (DIBIT) and Università Vita-Salute, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Ferrai
Carmelo
C
Longobardi
Elena
E
Facetti
Ileana
I
Blasi
Francesco
F
Crippa
Massimo P
MP
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D017930:Genes, Reporter; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D005810:Multigene Family; D009361:Neoplasm Invasiveness / Q000235:genetics*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000473:pathology; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*
D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases; D003850:Deoxyribonuclease I; D014568:Urokinase-Type Plasminogen Activator