Characterization of upstream activation elements essential for the expression of germ cell alkaline phosphatase in human choriocarcinoma cells.
Characterization of upstream activation elements essential for the expression of germ cell alkaline phosphatase in human choriocarcinoma cells.
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人绒毛膜癌细胞中生殖细胞碱性磷酸酶表达所必需的上游激活元件的表征。
DOI：
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发表时间：
1993
影响因子：
4.8
通讯作者：
J. Chou
中科院分区：
文献类型：
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作者：
N. Wada;J. Chou
生殖细胞碱性磷酸酶的表达是一个高度调控的过程，与人类胎盘的恶性转化有关。人绒毛膜癌细胞（恶性滋养细胞）主要表达生殖细胞碱性磷酸酶基因，只有低水平或无法检测到的胎盘碱性磷酸酶通常在人胎盘中发现。在这里，我们发现相对于基因转录起始位点（+1）的核苷酸-156至-1区域含有顺式作用的DNA元件，这些元件在绒毛膜癌细胞中指导生殖细胞碱性磷酸酶的表达。在最小激活子区域内，至少有三个核蛋白结合位点，I (-63/-44)， II（-87/-67）和III（-136/-103）被dna酶I足迹分析鉴定出来。这三个站点都富含gc。位点I和II包含已知与转录因子AP-2结合的序列；位于II位点的AP-2位点与转录因子Sp1的一致基序重叠。凝胶阻滞实验表明，在JEG-3绒毛膜癌细胞中，类似的核蛋白因子(s)可以与这三个位点结合，其中对位点I和II的亲和力最高。位点定向诱变可阻止核蛋白与位点I或II或位点I和II结合，导致因子结合丧失和激活剂活性降低。生殖细胞碱性磷酸酶启动子含有一个完整的结合位点III，但位点I和II发生了改变，激活子活性很小，这表明蛋白质-蛋白质相互作用对生殖细胞碱性磷酸酶基因激活很重要。
Expression of the germ cell alkaline phosphatase is a highly regulated process tied to malignant transformation of the human placenta. Human choriocarcinoma cells (malignant trophoblasts) express primarily the germ cell alkaline phosphatase gene and only low or nondetectable levels of the placental alkaline phosphatase normally found in the human placenta. Here, we show that nucleotides -156 to -1 region relative to the gene transcription start site (+1) contain cis-acting DNA elements that direct germ cell alkaline phosphatase expression in choriocarcinoma cells. Within the minimal activator region, at least three nuclear protein-binding sites, I (-63/-44), II (-87/-67), and III (-136/-103), were identified by DNase I footprinting analysis. All three sites are GC-rich. Sites I and II contain a sequence known to bind the transcription factor AP-2; the AP-2 site in site II overlaps a consensus motif for the transcription factor Sp1. Gel retardation experiments showed that similar nuclear protein factor(s) in JEG-3 choriocarcinoma cells bind to all three sites, with highest affinity to sites I and II. Site-directed mutagenesis that prevents binding of nuclear proteins to either site I or II, or both sites I and II, resulted in the loss of factor binding and reduced activator activity. The germ cell alkaline phosphatase promoter that contains an intact binding site III but altered sites I and II had little activator activity, suggesting that protein-protein interaction is important for germ cell alkaline phosphatase gene activation.