Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
J Cell Sci. 2013 Feb 15;126(Pt 4):889-903. doi: 10.1242/jcs.109413. Epub 2012 Dec 21.
Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.
电离辐射（IR）在天然染色质中诱导的 DNA 双链断裂（DSBs）引发 RNF8/RNF168 依赖性泛素化反应，从而触发各种修复因子的募集。然而，这种反应在染色质背景下是如何被调控的，在很大程度上仍未被探索。在这里，我们发现，SWI/SNF 染色质重塑复合物的催化亚基 SMARCA5/SNF2H，以依赖多聚（ADP-核糖）聚合酶 1（PARP1）的方式被募集到 DSB 上。值得注意的是，PARP 活性虽然对 γH2AX 有效地扩散到受损染色质中不是必需的，但选择性地促进 SMARCA5、E3 泛素连接酶 RNF168、泛素缀合物以及泛素结合因子 RAD18 和 RAP80-BRCA1 复合物在 DSB 侧翼染色质中的扩散。这表明 PARP 调节 RNF168 驱动的泛素对 DNA 损伤的空间组织。支持这一观点的是，我们表明 SMARCA5 和 RNF168 以 DNA 损伤和 PARP 依赖性的方式相互作用。在 DNA 损伤后，RNF168 发生多聚（ADP-核糖基）化，而 RNF168 和多聚（ADP-核糖）链是体内 SMARCA5 结合所必需的，这解释了 SMARCA5 如何与 RNF168 泛素级联反应相关联。此外，发现 SMARCA5 通过促进 RNF168 在 DSB 处的积累来调节泛素反应，从而随后促进有效的泛素缀合和 BRCA1 组装。强调这些发现的重要性，我们表明 SMARCA5 的耗竭使细胞对 IR 敏感，并导致 DSB 修复缺陷。我们的研究揭示了 DNA 损伤诱导的多聚（ADP-核糖基）化、SMARCA5 介导的染色质重塑以及 RNF168 依赖性信号转导和 DSB 修复之间的功能联系。
