Functional analysis of the SIS proximal element and its activating factors: regulated transcription of the c-SIS/PDGF-B gene in human erythroleukemia cells.
The SIS proximal element (SPE) is essential for the basal transcription of the c-sis/PDGF-B gene as well as the lineage-specific, activated transcription of this gene seen in megakaryocytes. In gel mobility shift analyses, the SPE element forms three gel-shift complexes; the t(op) and b(ottom) complexes were detected in nuclear extracts from both untreated and phorbol 12-myristate 13-acetate ('tetradecanoylphorbol acetate', TPA) treated K562 cells, whereas the m(iddle) complex was detected only in nuclear extracts from TPA-treated K562 cells. Site-directed mutagenesis of the SPE revealed a CCACCC motif that was essential for promoter activity as well as the formation of all three SPE gel-shift complexes. Nested-deletion analyses showed that the SPE was required for TPA-inducibility of c-sis/PDGF-B transcription. Antibody supershift analyses demonstrated that the t gel-shift complex contained both Sp1 and Sp3, and that the b complex contained only Sp3. In vitro transcription assays demonstrated that both Sp1 and Sp3 could support c-sis/PDGF-B transcription independent of each other in untreated K562 cells. However, overexpression of Sp1/Sp3 failed to significantly increase the c-sis/PDGF-B transcription in K562 cells.
Liang
Y
Y
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
Robinson
D F
DF
Kujoth
G C
GC
Fahl
W E
WE
D001483:Base Sequence; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D013755:Tetradecanoylphorbol Acetate