Lee K D, Baek S J, Shen R F
Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, MD USA.
Biochem J. 1996 Nov 1;319 ( Pt 3)(Pt 3):783-91. doi: 10.1042/bj3190783.
Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.
人血栓素合酶（TS）基因5.5 kb启动子的特征分析揭示了一个近端正调控序列（PPRS，-90至-25 bp）和几个远端抑制元件。发现最大启动子活性存在于前285 bp内，其中约75%由PPRS贡献。-365至-665 bp之间的序列对报告基因表达具有强烈的抑制作用（约55%），且与方向和位置无关，这与沉默子预期的特性一致。-665 bp至-5.5 kb上游的序列主要包含抑制元件，可使启动子活性进一步降低30%。65 bp的PPRS以方向独立但位置依赖的方式起作用，可进一步分为两个独立元件，PPRS1（-90至-50 bp）和PPRS2（-50至-25 bp）。虽然来自不同细胞类型的相似核因子与PPRS2相互作用，但与PPRS1相互作用的那些核因子表现出细胞特异性。内部序列缺失和寡核苷酸竞争表明，PPRS1中NF-E2的结合序列（-60 tgctgattcat -50）对于增强HL-60细胞中TS启动子活性很重要。通过cDNA的逆转录PCR扩增和Northern印迹分析证明了HL-60细胞中存在NF-E2 mRNA。当NF-E2 cDNA与TS启动子/luc构建体共表达时，检测到荧光素酶（luc）报告基因表达有9倍的反式激活。尽管NF-E2和顺式元件可以改变TS转录的效率，但它们不足以限制细胞特异性TS表达。对几种人类细胞系中TS启动子甲基化状态的分析揭示了可能与TS表达相关的细胞特异性甲基化模式。综上所述，这些结果表明，人TS基因的表达受多种因素调节，包括顺式元件、反式激活因子，可能还有基因组甲基化。
