Jin H M, Brady M L, Fahl W E
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7563-7. doi: 10.1073/pnas.90.16.7563.
The SIS/PDGFB gene, encoding the B polypeptide of platelet-derived growth factor (PDGF-B), is transcriptionally activated (> 50 fold) in human K562 erythroleukemia cells when they are induced to differentiate into megakaryocytic cells by treatment with phorbol 12-myristate 13-acetate ("tetradecanoylphorbol acetate," TPA). A 250-bp PDGF-B gene promoter attached to a reporter gene was shown to reproduce this TPA-induced activation. In a series of mutants that we constructed, a 10-bp linker sequence was systematically moved across the 250-bp PDGF-B promoter sequence, and the effect upon luciferase reporter activity was measured to identify a site through which this TPA-induced transcriptional activation occurred. We identified a site, which we named the SIS proximal element (SPE), at positions -58 to -39 relative to the PDGF-B mRNA initiation site that was essential for the TPA-induced activation. The SPE site contains two repeated sequences (TCTC and CACC) arranged in an ABBA configuration. The SPE sequence was not found in the existing list of consensus sequences for transcription factor binding sites. Gel mobility-shift assays using an SPE oligonucleotide and K562 cell nuclear extracts showed three shifted complexes, one of which was formed only following TPA treatment of K562 cells. In a time-course study, TPA induction of the endogenous PDGF-B mRNA and formation of the TPA-inducible complex occurred over the same time frame, and both events were specifically blocked by the addition of cycloheximide. The 20-bp SPE sequence was highly conserved (19/20) in both the cat and the mouse PDGF-B promoter, and conserved portions of the SPE sequence were also found at two sites within the human PDGF-A promoter. The role of the SPE in regulating the concurrent expression of the PDGF-B and PDGF-A genes in megakaryocytes, as well as various human tumor cells, is considered.
SIS/PDGFB基因编码血小板衍生生长因子(PDGF - B)的B多肽,当人K562红白血病细胞用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯(“十四酰佛波醇乙酸酯”,TPA)处理诱导分化为巨核细胞时,该基因转录激活(>50倍)。连接到报告基因的250 bp PDGF - B基因启动子显示可重现这种TPA诱导的激活。在我们构建的一系列突变体中,一个10 bp的接头序列在250 bp PDGF - B启动子序列上系统地移动,并测量对荧光素酶报告活性的影响,以确定该TPA诱导的转录激活发生的位点。我们确定了一个位点,相对于PDGF - B mRNA起始位点,位于-58至-39位,我们将其命名为SIS近端元件(SPE),它对于TPA诱导的激活至关重要。SPE位点包含以ABBA构型排列的两个重复序列(TCTC和CACC)。在现有的转录因子结合位点共有序列列表中未发现SPE序列。使用SPE寡核苷酸和K562细胞核提取物进行的凝胶迁移率变动分析显示有三个迁移复合物,其中一个仅在K562细胞用TPA处理后形成。在一项时间进程研究中,内源性PDGF - B mRNA的TPA诱导和TPA诱导复合物的形成发生在相同的时间范围内,并且这两个事件都通过添加放线菌酮被特异性阻断。20 bp的SPE序列在猫和小鼠的PDGF - B启动子中高度保守(19/20),并且在人PDGF - A启动子内的两个位点也发现了SPE序列的保守部分。考虑了SPE在调节巨核细胞以及各种人类肿瘤细胞中PDGF - B和PDGF - A基因同时表达中的作用。