v-Jun downregulates the SPARC target gene by binding to the proximal promoter indirectly through Sp1/3.
Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a downregulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Repression of SPARC contributes to the oncogenic process by facilitating tumor development in vivo. A proximal promoter fragment, designated -124/+16, is responsible for high constitutive activity of the SPARC gene and is the target of repression by v-Jun. In this paper, using electrophoretic mobility shift and pull-down assays in vitro, and transient transfections and chromatin immunoprecipitation assays in Sp1/3-deficient Drosophila SL2 cells and in chick embryo fibroblasts, we show that (i) Sp1 and/or Sp3 is required for constitutive activation of SPARC transcription, by binding directly to the GGA-rich -92/-57 fragment; and (ii) v-Jun does not bind -124/+16 directly, but binds to the GGA-rich fragment indirectly, most likely through a physical interaction with Sp1/3. Moreover, a transactivation-proficient v-Jun derivative, designated v-Jun/cebp/glz, which cannot bind Jun DNA motifs anymore and cannot heterodimerize, is still capable of downregulating SPARC efficiently. Taken together, these data strongly suggest that v-Jun downregulates SPARC through the formation of a DNA-Sp1/3-v-Jun, chromatin-associated complex.
Chamboredon
Sandrine
S
Unité de Virologie Humaine, INSERM-U412, Ecole Normale Supérieure, 46 allée d'ltalie, 69364 Lyon cedex 07, France.
Briggs
Joseph
J
Vial
Emmanuel
E
Hurault
Julien
J
Galvagni
Federico
F
Oliviero
Salvatore
S
Bos
Timothy
T
Castellazzi
Marc
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004330:Drosophila; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016759:Oncogene Protein p65(gag-jun) / Q000378:metabolism*; D015676:Osteonectin / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D016759:Oncogene Protein p65(gag-jun); D015676:Osteonectin; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase