Molecular and Cellular Biology Department, The Salk Institute for Biological Studies, La Jolla, California, United States of America.
PLoS One. 2011;6(6):e21407. doi: 10.1371/journal.pone.0021407. Epub 2011 Jun 23.
Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG) and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci). We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1), a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach.
端粒是核蛋白复合物，可防止线性染色体的末端被识别为 DNA 损伤，并为复制相关的缩短提供序列缓冲。在哺乳动物中，端粒由重复的 DNA 序列（TTAGGG）和相关蛋白组成。端粒的核心复合物称为 shelterin，由 TRF1、TRF2、POT1、TIN2、TPP1 和 RAP1 蛋白组成。端粒缩短过多或通过 shelterin 亚基的丢失而使端粒脱保护，会导致端粒被识别为 DNA 损伤，这可以在染色体末端看到称为 TIF（端粒功能障碍诱导焦点）的 DNA 损伤蛋白焦点。我们试图利用 TIF 表型作为端粒功能障碍的标志物，通过 siRNA 介导的一组 386 个候选基因的敲低来鉴定新的参与端粒保护的基因。在这里，我们报告了这种筛选的建立、特异性和可行性，以及测试基因的结果。在候选基因中，只有一个基因表现出与主要 shelterin 成分 TRF2 或 TRF1 抑制相当的独特 TIF 表型，该基因被鉴定为 TRF1 样假基因。我们还鉴定出剪接因子和转录共激活因子 SKIIP（SNW1）的弱 TIF 表型。然而，SKIIP 的敲低也诱导了一般而非端粒特异性的 DNA 损伤反应，这使得关于端粒作用的结论变得复杂。总之，本报告是端粒脱保护的基于细胞的筛选的可行性的技术论证，具有将其扩展为高通量方法的潜力。
