Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.
Sci Adv. 2019 Jan 9;5(1):eaau1249. doi: 10.1126/sciadv.aau1249. eCollection 2019 Jan.
How DNA double-strand breaks (DSBs) affect ongoing transcription remains elusive due to the lack of single-molecule resolution tools directly measuring transcription dynamics upon DNA damage. Here, we established new reporter systems that allow the visualization of individual nascent RNAs with high temporal and spatial resolution upon the controlled induction of a single DSB at two distinct chromatin locations: a promoter-proximal (PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of a DSB resulted in a rapid suppression of preexisting transcription initiation regardless of the genomic location. However, while transcription was irreversibly suppressed upon a PROP DSB, damage at the EX2 region drove the formation of promoter-like nucleosome-depleted regions and transcription recovery. Two-color labeling of transcripts at sequences flanking the EX2 lesion revealed bidirectional break-induced transcription initiation. Transcriptome analysis further showed pervasive bidirectional transcription at endogenous intragenic DSBs. Our data provide a novel framework for interpreting the reciprocal interactions between transcription and DNA damage at distinct chromatin regions.
由于缺乏直接测量 DNA 损伤时转录动力学的单分子分辨率工具，DNA 双链断裂 (DSB) 如何影响正在进行的转录仍然难以捉摸。在这里，我们建立了新的报告系统，允许在两个不同的染色质位置（转录起始位点下游的启动子近端 (PROP) 区域和内含子内的区域 (EX2)）在受控诱导单个 DSB 时，以高时间和空间分辨率可视化单个新生 RNA。DSB 的诱导导致预先存在的转录起始迅速抑制，而与基因组位置无关。然而，虽然 PROP DSB 会导致转录不可逆地受到抑制，但在 EX2 区域发生的损伤会导致类似启动子的核小体缺失区域的形成和转录恢复。在 EX2 病变侧翼序列上的转录本的双色标记揭示了双向断裂诱导的转录起始。转录组分析进一步显示，在内源性基因内 DSB 处存在普遍的双向转录。我们的数据为解释不同染色质区域转录和 DNA 损伤之间的相互作用提供了一个新的框架。
