Increased amounts and stability of telomeric repeat-containing RNA (TERRA) following DNA damage induced by etoposide

Telomeric repeat-containing RNAs (TERRAs) are long noncoding RNAs transcribed from subtelomeres toward telomeric repeat tracts, which have been implicated in telomere protection and heterochromatin formation. Genotoxic stress leads to upregulation of TERRAs. However, the mechanism of DNA damage-mediated TERRA induction remains elusive. Here, we treated HeLa cells with etoposide, a DNA double-strand break-generating agent, for various times and monitored the levels of TERRAs. Etoposide treatment led to a gradual time-dependent increase in TERRAs. Etoposide-mediated induction was evident in many TERRAs arising from various chromosome loci, including 20q and XpYp. Chromatin immunoprecipitation assays revealed no significant changes in the occupancy of RNA polymerase II at telomeres upon etoposide treatment. Interestingly, TERRAs arising from 20q, XpYp, 10q, and 13q degraded at slower rates in cells treated with etoposide, while degradation rates of TERRAs from many loci tested were nearly identical in both etoposide- and mock-treated cells. Telomere damage occurred from early time points of etoposide treatment, but telomere lengths and abundance of telomeric repeat-binding factor 2 (TRF2) at telomeres remained unchanged. In summary, etoposide treatment led to telomere damage and TERRA accumulation, but telomere lengths and TRF2-mediated telomere integrity were maintained. Etoposide-mediated TERRA accumulation could be attributed partly to RNA stabilization. These findings may provide insight into the post-transcriptional regulation of TERRAs in response to DNA damage.含端粒重复序列的RNA（Telomeric repeat-containing RNAs，TERRAs）是一类从亚端粒区向端粒重复序列区域转录的长链非编码RNA，已被证实参与端粒保护与异染色质形成。基因毒性应激可导致TERRA表达上调，但DNA损伤介导的TERRA诱导机制仍不明晰。本研究使用DNA双链断裂诱导剂依托泊苷（etoposide）处理HeLa细胞，并设置不同处理时长以监测TERRA水平。结果显示，依托泊苷处理可使TERRA水平随处理时长逐渐升高，且该诱导效应在包括20q、XpYp在内的多个染色体位点产生的多种TERRA中均显著存在。染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）实验显示，依托泊苷处理后，端粒区域的RNA聚合酶II结合占据率无显著变化。有趣的是，由20q、XpYp、10q及13q位点产生的TERRA在依托泊苷处理的细胞中降解速率更慢；而在受试的众多染色体位点中，其余TERRA的降解速率在依托泊苷处理组与模拟处理对照组中几乎一致。依托泊苷处理早期即可出现端粒损伤，但端粒长度及端粒处端粒重复序列结合因子2（Telomeric repeat-binding factor 2，TRF2）的丰度未发生明显改变。综上，依托泊苷处理可引发端粒损伤与TERRA积累，但端粒长度及TRF2介导的端粒完整性得以维持。依托泊苷介导的TERRA积累可部分归因于RNA稳定性增强。本研究结果可为解析DNA损伤应答过程中TERRA的转录后调控机制提供新的见解。