Lok Chun-Nam, Lang Alexander J, Mirski Shelagh E L, Cole Susan P C
Cancer Research Laboratories and Department of Pharmacology & Toxicology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Biochem J. 2002 Dec 15;368(Pt 3):741-51. doi: 10.1042/BJ20020791.
Eukaryotic topoisomerase II (topo II) catalyses topological genomic changes essential for chromosome segregation, chromatin reorganization, DNA replication and transcription. Mammalian topo II exists as two isoforms, designated alpha and beta. Human topo IIalpha is an important cancer drug target, and an established determinant of drug sensitivity and resistance. Human topo IIbeta is also the target of anticancer drugs but its role in drug resistance is less clear. The two human topo II proteins are encoded by the TOP2A and TOP2B genes, respectively, which despite their highly conserved structural organization, are subject to distinctly different modes of regulation. In the present study, we have cloned and characterized the human TOP2B promoter containing a 1.3 kb fragment of the 5'-flanking and untranslated region (-1067 to +193). We found that the promoter activity of this TOP2B fragment was constant throughout the cell cycle, in contrast to the activity of the proximal promoter of TOP2A which was low in resting cells and enhanced during proliferation. Analyses of 5'-serially and internally deleted luciferase reporter constructs revealed that 80% of the TOP2B promoter activity could be attributed to the region between -533 and -481. Mutational analyses of putative regulatory elements indicated that two inverted CCAAT boxes (ICBs) within this region were essential for TOP2B promoter activity and gel mobility-shift assays indicated these sites bound the transcription factor nuclear factor-Y (NF-Y). Co-transfection experiments using a dominant-negative form of subunit A of NF-Y suggested that TOP2B promoter activity required direct interaction of NF-Y with the ICBs. In addition, a specificity protein-1 (Sp1)-binding GC box located just upstream of the ICBs was shown to contribute to TOP2B promoter activity in a synergistic manner with the ICBs. Our results suggest that the binding sites for NF-Y and Sp1 are critical for TOP2B transcription.
真核生物拓扑异构酶II（拓扑异构酶II）催化对染色体分离、染色质重组、DNA复制和转录至关重要的拓扑基因组变化。哺乳动物拓扑异构酶II以两种同工型存在，分别命名为α和β。人类拓扑异构酶IIα是一种重要的癌症药物靶点，也是药物敏感性和耐药性的既定决定因素。人类拓扑异构酶IIβ也是抗癌药物的靶点，但其在耐药性中的作用尚不清楚。两种人类拓扑异构酶II蛋白分别由TOP2A和TOP2B基因编码，尽管它们的结构组织高度保守，但受到截然不同的调控模式。在本研究中，我们克隆并鉴定了包含5'-侧翼和非翻译区1.3 kb片段（-1067至+193）的人类TOP2B启动子。我们发现，与TOP2A近端启动子的活性相反，该TOP2B片段的启动子活性在整个细胞周期中保持恒定，TOP2A近端启动子在静止细胞中活性较低，在增殖过程中增强。对5'-连续和内部缺失的荧光素酶报告基因构建体的分析表明，80%的TOP2B启动子活性可归因于-533至-481之间的区域。对假定调控元件的突变分析表明，该区域内的两个反向CCAAT框（ICB）对TOP2B启动子活性至关重要，凝胶迁移率变动分析表明这些位点结合转录因子核因子-Y（NF-Y）。使用NF-Y亚基A的显性负性形式进行的共转染实验表明，TOP2B启动子活性需要NF-Y与ICB直接相互作用。此外，位于ICB上游的一个特异性蛋白-1（Sp1）结合GC框被证明与ICB协同作用，有助于TOP2B启动子活性。我们的结果表明，NF-Y和Sp1的结合位点对TOP2B转录至关重要。
