Lichtenheld M G, Podack E R
Department of Microbiology and Immunology, University of Miami, School of Medicine, FL 33101.
J Immunol. 1992 Oct 15;149(8):2619-26.
Gene expression of the cytolytic protein perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine perforin promoter and upstream region. The murine perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in perforin-negative cell types the perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in perforin-negative cells, the transcriptional activity of the immediate proximal perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.
溶细胞蛋白穿孔素的基因表达局限于溶细胞淋巴细胞，并在其中受到严格调控。为了初步了解穿孔素基因转录的分子基础，我们克隆并分析了5.1 kb的小鼠穿孔素基因真实启动子及上游区域。由于5'非翻译序列中有一个内含子，小鼠穿孔素启动子位于基因组DNA中翻译起始密码子上游约2.1 kb处。尽管已测序的小鼠启动子及上游区域与人基因的该区域具有较高的同源性，但大多数种间保守序列与已知调控元件缺乏明显的一致性。然而，对该区域的功能分析表明，它含有可能决定这种杀伤蛋白细胞类型特异性表达的调控元件。将穿孔素启动子及上游区域瞬时转染到几种细胞系后，用于驱动氯霉素乙酰转移酶（CAT）报告基因的表达。在细胞毒性T淋巴细胞（CTL）中表达了高水平的CAT活性，超过无启动子报告基因构建体表达水平的110倍。相比之下，在穿孔素阴性的细胞类型中，穿孔素启动子及上游区域介导的CAT基因转录几乎检测不到。对穿孔素启动子紧邻的近端区域（-120至+2）的分析表明，它在所有测试细胞中均有普遍活性，其表达的CAT活性比无启动子报告基因构建体高20至50倍。然而，穿孔素启动子及上游区域的细胞类型限制转录活性受至少四个正负顺式作用上游区域的控制，这些区域分布在克隆DNA的整个5 kb区域，并在不同细胞中相互作用。因此，在穿孔素阴性细胞中，紧邻的近端穿孔素启动子的转录活性被几个上游负调控元件显著抑制，而在穿孔素阳性细胞中，启动子活性被几个上游调控元件增强了五倍以上。
