Batarseh Amani, Barlow Keith D, Martinez-Arguelles Daniel B, Papadopoulos Vassilios
The Research Institute of the McGill University Health Centre and the Department of Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4.
Biochim Biophys Acta. 2012 Jan;1819(1):38-56. doi: 10.1016/j.bbagrm.2011.09.001. Epub 2011 Sep 18.
The translocator protein (18kDa; TSPO) is a mitochondrial drug- and cholesterol-binding protein that has been implicated in several processes, including steroidogenesis, cell proliferation, and apoptosis. Expression of the human TSPO gene is elevated in several cancers. To understand the molecular mechanisms that regulate TSPO expression in human breast cancer cells, the TSPO promoter was identified, cloned, and functionally characterized in poor-in-TSPO hormone-dependent, non-aggressive MCF-7 cells and rich-in-TSPO hormone-independent, aggressive, and metastatic MDA-MB-231 breast cancer cells. RNA ligase-mediated 5'-rapid amplification of cDNA ends analysis indicated transcription initiated at multiple sites downstream of a GC-rich promoter that lacks functional TATA and CCAAT boxes. Deletion analysis indicated that the region from -121 to +66, which contains five putative regulatory sites known as GC boxes, was sufficient to induce reporter activity up to 24-fold in MCF-7 and nearly 120-fold in MDA-MB-231 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1, Sp3 and Sp4 bind to these GC boxes in vitro and to the endogenous TSPO promoter. Silencing of Sp1, Sp3 and Sp4 gene expression reduced TSPO levels. In addition, TSPO expression was epigenetically regulated at one or more of the identified GC boxes. Disruption of the sequence downstream of the main start site of TSPO differentially regulated TSPO promoter activity in MCF-7 and MDA-MB-231 cells, indicating that essential elements contribute to its differential expression in these cells. Taken together, these experiments constitute the first in-depth functional analysis of the human TSPO gene promoter and its transcriptional regulation.
转位蛋白（18 kDa；TSPO）是一种线粒体药物和胆固醇结合蛋白，参与了包括类固醇生成、细胞增殖和凋亡在内的多个过程。人类TSPO基因在多种癌症中表达升高。为了了解调节人类乳腺癌细胞中TSPO表达的分子机制，在TSPO表达水平低的激素依赖性、非侵袭性MCF-7细胞以及TSPO表达水平高的激素非依赖性、侵袭性和转移性MDA-MB-231乳腺癌细胞中鉴定、克隆并对TSPO启动子进行了功能表征。RNA连接酶介导的cDNA末端5'快速扩增分析表明，转录起始于富含GC的启动子下游的多个位点，该启动子缺乏功能性TATA盒和CCAAT盒。缺失分析表明，从-121到+66的区域，其中包含五个被称为GC盒的假定调控位点，足以在MCF-7细胞中诱导报告基因活性高达24倍，在MDA-MB-231细胞中诱导近120倍。电泳迁移率变动分析和染色质免疫沉淀分析表明，Sp1、Sp3和Sp4在体外与这些GC盒结合，并与内源性TSPO启动子结合。Sp1、Sp3和Sp4基因表达的沉默降低了TSPO水平。此外，TSPO表达在一个或多个已鉴定的GC盒处受到表观遗传调控。TSPO主要起始位点下游序列的破坏在MCF-7和MDA-MB-231细胞中对TSPO启动子活性有不同的调节作用，表明关键元件有助于其在这些细胞中的差异表达。综上所述，这些实验构成了对人类TSPO基因启动子及其转录调控的首次深入功能分析。
