Liang Y, Robinson D F, Kujoth G C, Fahl W E
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
Oncogene. 1996 Aug 15;13(4):863-71.
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.
SIS近端元件（SPE）对于c-sis/PDGF-B基因的基础转录以及在巨核细胞中所见的该基因的谱系特异性激活转录至关重要。在凝胶迁移率变动分析中，SPE元件形成三种凝胶迁移复合物；在未处理的和经佛波醇12-肉豆蔻酸酯13-乙酸酯（“十四烷酰佛波醇乙酸酯”，TPA）处理的K562细胞的核提取物中均检测到顶部（t）和底部（b）复合物，而中间（m）复合物仅在经TPA处理的K562细胞的核提取物中检测到。对SPE进行定点诱变揭示了一个CCACCC基序，该基序对于启动子活性以及所有三种SPE凝胶迁移复合物的形成至关重要。嵌套缺失分析表明，SPE是c-sis/PDGF-B转录的TPA诱导性所必需的。抗体超迁移分析表明，t凝胶迁移复合物同时包含Sp1和Sp3，而b复合物仅包含Sp3。体外转录分析表明，在未处理的K562细胞中，Sp1和Sp3均可相互独立地支持c-sis/PDGF-B转录。然而，Sp1/Sp3的过表达未能显著增加K562细胞中c-sis/PDGF-B的转录。
