Dunoyer-Geindre Sylvie, Rivier-Cordey Anne-Sophie, Caetano Carlos, Fish Richard J, Kruithof Egbert K O
Division of Angiology and Hemostasis, University Medical Center, University of Geneva, Geneva, Switzerland.
Department of Genetic Medicine and Development, University Medical Center, University of Geneva, Geneva, Switzerland.
PLoS One. 2016 Dec 14;11(12):e0167588. doi: 10.1371/journal.pone.0167588. eCollection 2016.
Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.
组织型纤溶酶原激活剂基因（t-PA；基因名PLAT）的表达部分受表观遗传机制调控。我们研究了五种原代人类细胞类型和六种转化细胞系中PLAT甲基化与PLAT表达之间的关系。对PLAT基因近端启动子以及PLAT转录起始位点（TSS，-7.3 kb）上游-7.3千碱基对处的多激素反应增强子（MHRE）附近的CpG甲基化进行了分析。在鲍伊斯黑色素瘤细胞中，PLAT启动子和MHRE完全未甲基化，t-PA分泌极高。在其他细胞类型中，-647至-366区域完全甲基化，而-121至+94的一段未甲基化DNA片段是可检测到的t-PA mRNA和t-PA分泌所必需的，但并不充分。MHRE附近的DNA甲基化与t-PA分泌无关。插入萤火虫荧光素酶报告基因的PLAT启动子区域-151至+151的特异性甲基化消除了报告基因活性。-121至+94区域包含两个已充分描述的调控元件，一个位于-106附近的佛波酯反应元件（CRE）和一个位于+59附近包含Sp1结合位点的富含GC的区域。含有CRE或富含GC区域的双链DNA寡核苷酸的甲基化对转录因子结合几乎没有影响。甲基化的CpG可能会吸引含有组蛋白去乙酰化酶（HDAC）的共抑制复合物。然而，HDAC抑制剂曲古抑菌素并未恢复甲基化质粒的报告基因活性。总之，高效的PLAT基因表达需要近端启动子中一小段未甲基化的CpG位点。
