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 transcriptio n of the c-sis/PDGF-B gene as well as the lineage-specific, activated transcription of this gene seen in megakaryocytes. In gel mobility shi ft analyses, the SPE element forms three gel-shift complexes; the t(op ) and b(ottom) complexes were detected in nuclear extracts from both u ntreated and phorbol 12-myristate 13-acetate ('tetradecanoylphorbol ac etate', TPA) treated K562 cells, whereas the m(iddle) complex was dete cted only in nuclear extracts from TPA-treated g562 cells. Site-direct ed mutagenesis of the SPE revealed 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 supershif t analyses demonstrated that the t gel-shift complex contained both Sp 1 and Sp3, and that the b complex contained only Sp3. In vitvo transcr iption assays demonstrated that both Sp1 and Sp3 could support c-sis/P DGF-B transcription independent of each other in untreated K562 cells. However, overexpression of Sp1/Sp3 failed to significantly increase t he c-sis/PDGF-B transcription in K562 cells.