The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer
The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer
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DOI：
10.4049/jimmunol.171.4.1825
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发表时间：
2003-08-15
影响因子：
4.4
通讯作者：
Ross, R
中科院分区：
文献类型：
--
作者：
Bros, M;Ross, XL;Ross, R
树突状细胞（DC）被认为是免疫系统中最有效的APC，能够激活幼稚T细胞。因此，DC是免疫治疗的主要靶点。然而，对DC中的基因调控知之甚少，并且对于人DC的有效转录靶向，仍然缺少合适的启动子。最近，我们成功地使用小鼠肌动蛋白捆绑蛋白fascin的启动子，通过DNA疫苗接种小鼠，转录靶向DC。在这项研究中，我们报告分离的人肌成束蛋白启动子及其调控元件的特性。将活性表达的基因与保守的非活性基因组基因座区分开，亚克隆覆盖该基因的14 kb的连续区域和3 kb的5 '-侧翼序列，测序并分析调控元件。调控序列仅在5 '侧翼启动子区中发现。该启动子在DC和fascin阳性神经元细胞系中发挥了强大的活性，但在测试的fascin阴性细胞中没有。值得注意的是，DC中的启动子活性随着DC的成熟而显著增加。通过进行性5'缺失，我们鉴定了一个核心启动子区域，包含一个推定的GC盒，一个复合cAMP应答元件/AP-1结合位点和一个TATA盒。通过内部缺失，我们证明了任一调节元件的功能重要性。此外，我们确定了一个更远的阶段特异性增强子区域也含有沉默元件。总之，人肌成束蛋白启动子允许成熟DC的转录靶向，并且代表了用于免疫治疗的有前景的工具。据我们所知，本研究首次报道了人单核细胞来源的DC中的启动子活性。
Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5'-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5'-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5' deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.