Identification and characterization of an essential, activating regulatory element of the human SIS/PDGFB promoter in human megakaryocytes.
Identification and characterization of an essential, activating regulatory element of the human SIS/PDGFB promoter in human megakaryocytes.
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人巨核细胞中人 SIS/PDGFB 启动子的必需激活调节元件的鉴定和表征。
DOI：
10.1073/pnas.90.16.7563
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发表时间：
1993
影响因子：
11.1
通讯作者：
Fahl,WE
中科院分区：
文献类型：
--
作者：
Jin,HM;Brady,ML;Fahl,WE
编码血小板衍生生长因子（PDGF-B）的B多肽的SIS/PDGFB基因在人K562红白血病细胞中被转录激活（> 50倍），当它们通过用佛波醇12-肉豆蔻酸酯13-乙酸酯（“十四烷酰基佛波醇乙酸酯”TPA）处理而被诱导分化成巨核细胞时。一个250 bp的PDGF-B基因启动子连接到一个报告基因，重现TPA诱导的激活。在我们构建的一系列突变体中，将10-bp的接头序列系统地移过250-bp的PDGF-B启动子序列，并测量对荧光素酶报告基因活性的影响，以确定TPA诱导的转录激活发生的位点。我们确定了一个位点，我们命名为SIS近端元件（SPE），在位置-58至-39相对于PDGF-B mRNA的起始位点，这是必需的TPA诱导的激活。SPE位点含有两个以ABBA构型排列的重复序列（TCTC和CACC）。在转录因子结合位点的现有共有序列列表中未发现SPE序列。使用SPE寡核苷酸和K562细胞核提取物的凝胶迁移率变化测定显示了三种变化的复合物，其中之一仅在TPA处理K562细胞后形成。在一项时程研究中，TPA诱导的内源性PDGF-B mRNA和TPA诱导复合物的形成发生在相同的时间范围内，这两个事件都被特异性阻断放线菌酮的加入。20 bp的SPE序列在猫和小鼠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SPE序列的保守部分。SPE在调节巨核细胞以及各种人类肿瘤细胞中PDGF-B和PDGF-A基因的同时表达中的作用被考虑。
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.