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首页> 外文期刊>Bioscience Reports >The human receptor tyrosine kinase Axl gene -promoter characterization and regulation of constitutive expression by Spl, Sp3 and CpG methylation
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The human receptor tyrosine kinase Axl gene -promoter characterization and regulation of constitutive expression by Spl, Sp3 and CpG methylation

机译：人受体酪氨酸激酶Axl基因启动子的表达及Spl，Sp3和CpG甲基化的组成型表达调控

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Axl is a receptor tyrosine kinase which promotes anti-apoptosis, mitogenesis, invasion, angiogenesis and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, c/s-acting elements and promoter methylation driving expression of Axl, The 2.4 kb sequence upstream of the translational start site, and sequential 5'-deletions were cloned and revealed a minimal GC-rich region (-556 to +7) to be sufficient for basal Axl promoter activity in Rko, HCT116 and HeLa cells. Within this minimal region, five Sp (specificity protein)-binding sites were identified. Two sites (Sp a and Sp b) proximal to the translation start site were indispensable for Axl promoter activity, whereas mutation of three additional upstream motifs (Sp c, Sp d and Sp e) was of additional relevance. Gel-shift assays and chromatin immunoprecipitation identified that Spl and Sp3 bound to all five motifs, and mutation of all motifs abolished binding. Mithramycin, which inhibits binding of Sp factors to GC-rich sites, dramatically reduced Axl promoter activity and Axl, Spl and Sp3 expression. In Drosophila Schneider SL2-cells, exogenous expression of Spl/Sp3 increased Axl promoter activity. Use of Spl/Sp3 siRNAs (small interfering RNAs) significantly reduced Axl promoter activity and protein levels in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG sites within three Sp motifs (Sp a, Sp b and Sp c) and GC-rich flanking sequences, and demethylation by 5-aza-2'-deoxycytidine up-regulated Axl and Sp3 expression in low-Axl-expressing Colo206f/WiDr cells, but not in high-Axl-expressing Rko cells. The results of the present study suggest that Axl gene expression in cancer cells is (1) constitutively driven by Spl/Sp3 bound to five core promoter motifs, and (2) restricted by methylation within/around Sp-binding sites This might enhance the understanding and treatment of essential mechanisms associated with cancer and other diseases.
机译：Axl是一种受体酪氨酸激酶，可促进抗凋亡，有丝分裂，侵袭，血管生成和转移，并在癌症中高度表达。然而，该重要基因的转录调控从未被表征过。启动本研究以表征Axl的启动子，c / s作用元件和启动子甲基化驱动表达，翻译起始位点上游的2.4 kb序列和顺序的5'缺失被克隆，并揭示了最小的GC富集区域（-556至+7）足以使Rko，HCT116和HeLa细胞中的基础Axl启动子活性。在该最小区域内，鉴定出五个Sp（特异性蛋白）结合位点。对于Axl启动子活性而言，最接近翻译起始位点的两个位点（Sp a和Sp b）是必不可少的，而另外三个上游基序（Sp c，Sp d和Sp e）的突变则具有其他意义。凝胶移位测定法和染色质免疫沉淀法鉴定出Spl和Sp3与所有五个基序结合，并且所有基序的突变消除了结合。抑制Sp因子与富含GC的位点结合的光神霉素，大大降低了Axl启动子活性以及Axl，Spl和Sp3表达。在果蝇施耐德SL2细胞中，Spl / Sp3的外源表达增加了Axl启动子的活性。 Spl / Sp3 siRNA（小干扰RNA）的使用显着降低了Rko和HeLa细胞中的Axl启动子活性和蛋白质水平。甲基化-亚硫酸氢盐测序检测到三个Sp基序（Sp a，Sp b和Sp c）和富含GC的侧翼序列中的甲基化CpG位点，并通过5-氮杂2'-脱氧胞苷上调了Axl和Sp3的表达，从而在低水平表达Axl的Colo206f / WiDr细胞，但不在表达高Axl的Rko细胞中。本研究的结果表明，癌细胞中Axl基因的表达是（1）由与五个核心启动子基序结合的Spl / Sp3组成性驱动的，并且（2）受Sp结合位点内/周围的甲基化限制。与癌症和其他疾病相关的基本机制的治疗。

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