Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Via Vela 6, Bellinzona, Switzerland.
Biochimie. 2010 Mar;92(3):317-20. doi: 10.1016/j.biochi.2009.12.012. Epub 2010 Jan 1.
Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells.
三聚体形成寡核苷酸（TFOs）是一种在转录水平上控制基因表达的有吸引力的工具。这种反基因方法已被证明在各种实验条件下是成功的。然而，导致细胞中转录抑制的机制尚未得到充分研究。在这里，我们使用染色质免疫沉淀分析研究了三聚体 DNA 形成对转录激活因子、共激活因子和 RNA 聚合酶 II 与 ets2 基因启动子结合的影响。三聚体靶序列位于转录起始位点（TSS）上游约 40 个碱基处，与 ets2 转录相关的 Sp1 结合位点重叠。我们发现，ets2-TFO 阻止了 Sp1、TAF(II)130 和 TAF(II)250 与 ets2 启动子的结合，而 RNA 聚合酶 II 和 TBP 的结合不受影响。这种作用具有序列和靶标特异性，因为 TFO 对 c-myc 启动子和突变的 ets2 启动子构建体没有影响。因此，TSS 附近的三聚体 DNA 形成通过阻断转录激活因子和共激活因子分子的结合，导致无功能的起始前复合物（PIC）的形成。这是首次在细胞中直接证明寡核苷酸三聚体 DNA 形成干扰 TSS 处 PIC 组装。
