Ali Abdullah Mahmood, Bajaj Vineeta, Gopinath K S, Kumar Arun
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Gene. 2009 Jan 15;429(1-2):37-43. doi: 10.1016/j.gene.2008.10.004. Epub 2008 Oct 22.
SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 bp to +78 bp is required for the core promoter activity. No consensus TATA or CAAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis.
溶质载体家族22成员18（SLC22A18）是一种多特异性有机阳离子转运体，在人类和小鼠中呈现父系印记。它在儿童和成体肿瘤中表现出杂合性缺失，而在肝癌和乳腺癌中则表现出印记增加。尽管该基因很重要，但其转录调控尚未得到研究，启动子也尚未得到表征。因此，我们着手鉴定包括该基因启动子在内的潜在顺式调控元件。在人类细胞中进行的荧光素酶报告基因检测表明，核心启动子活性需要-120 bp至+78 bp的区域。在该区域未发现共有TATA或CAAT框，但在人类、黑猩猩、小鼠和大鼠中两个Sp1结合位点是保守的。对这两个Sp1位点的突变分析表明它们对启动子活性是必需的。染色质免疫沉淀显示Sp1在体内与启动子区域结合。在果蝇Sp1缺失的SL2细胞中过表达Sp1表明Sp1是该启动子的反式激活因子。人类核心启动子在小鼠3T3细胞和猴COS7细胞中具有功能。我们发现了一个跨越核心启动子和外显子1的CpG岛。COBRA技术未在10个正常口腔组织、14个口腔肿瘤以及两种人类细胞系HuH7和A549中检测到启动子甲基化。本研究首次深入了解了控制这个印记肿瘤抑制基因表达的机制。已开发出一种基于COBRA的检测方法来寻找不同癌症中的启动子甲基化。目前的数据将有助于理解该基因的调控及其在肿瘤发生中的作用。
