Joshi Bharat, Ordonez-Ercan Dalia, Dasgupta Piyali, Chellappan Srikumar
Department of Interdisciplinary Oncology, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.
Oncogene. 2005 Mar 24;24(13):2204-17. doi: 10.1038/sj.onc.1208206.
The E2F transcription factors induce the expression of many genes in response to specific extracellular stimuli. Here, we show that human metallothionein 1G (hMT1G) promoter is upregulated by E2F1 upon VEGF stimulation of human aortic endothelial cells. Analysis of the hMT1G promoter showed the presence of many potential E2F-binding sites flanked by potential SP1 sites and metal response elements (MREs). hMT1G promoter could be induced by E2F1 in transient transfections; further, deletion analysis suggested that the region spanning the E2F-binding sites was necessary for VEGF-mediated induction. E2Fs 1-5 could bind to the hMT1G promoter in a chromatin immunoprecipitation assay. VEGF stimulation led to an increased binding of E2Fs 1-3 to the endogenous hMT1G promoter; at the same time, the binding of Rb, p107 and p130 to the promoter was abolished. VEGF stimulation also led to the increased acetylation E2F1 as well as the histones in the hMT1G promoter region. Stimulation with metals or VEGF led to dissociation of histone deacetylase 1 (HDAC1) from the promoter, leading to acetylation of histones. Induction of the hMT1G promoter upon exposure to heavy metals such as Zn and Cd is mediated by the MRE. Interestingly, mutation of MRE affected the metal response, but not the VEGF response of the hMT1G promoter. In contrast, deletion of the E2F-binding sites did not affect the metal response. Based on these findings, we conclude that induction of the hMT1G promoter by VEGF and heavy metals occurs through the utilization of different transcription factors.
E2F转录因子可响应特定的细胞外刺激诱导许多基因的表达。在此，我们表明，在血管内皮生长因子（VEGF）刺激人主动脉内皮细胞时，人金属硫蛋白1G（hMT1G）启动子会被E2F1上调。对hMT1G启动子的分析显示，其存在许多潜在的E2F结合位点，两侧为潜在的SP1位点和金属反应元件（MREs）。在瞬时转染中，hMT1G启动子可被E2F1诱导；此外，缺失分析表明，跨越E2F结合位点的区域对于VEGF介导的诱导是必需的。在染色质免疫沉淀试验中，E2F1 - 5可与hMT1G启动子结合。VEGF刺激导致E2F1 - 3与内源性hMT1G启动子的结合增加；同时，Rb、p107和p130与该启动子的结合被消除。VEGF刺激还导致hMT1G启动子区域中E2F1以及组蛋白的乙酰化增加。金属或VEGF刺激导致组蛋白去乙酰化酶1（HDAC1）从启动子上解离，从而导致组蛋白乙酰化。暴露于锌和镉等重金属时，hMT1G启动子的诱导由MRE介导。有趣的是，MRE的突变影响金属反应，但不影响hMT1G启动子的VEGF反应。相反，E2F结合位点的缺失不影响金属反应。基于这些发现，我们得出结论，VEGF和重金属对hMT1G启动子的诱导是通过利用不同的转录因子发生的。
