Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto, Japan.
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Nucleic Acids Res. 2018 Apr 6;46(6):2932-2944. doi: 10.1093/nar/gky058.
During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.
在低剂量阿菲迪霉素（APH）处理引起的轻度复制应激下，关键的范可尼贫血蛋白 FANCD2 积累在常见的脆弱部位，表现为姐妹焦点，并保护基因组稳定性。为了更深入地了解 FANCD2 的功能及其调控机制，我们通过 ChIP-seq 分析研究了在此环境下 FANCD2 在全基因组染色质上的定位。我们发现 FANCD2 主要积累在一组大型转录基因的中央区域，这些基因与已知的 CFS 广泛重叠。与先前的研究一致，我们发现这种 FANCD2 保留依赖于 R 环。然而，在耗尽 R 环的细胞中，FANCD2 单泛素化和 RPA 焦点形成仍被诱导。有趣的是，我们在 APH 处理后检测到 FANCD2 和 R 环之间的近端连接分析点增加，这一增加被转录抑制所抑制。总的来说，我们的数据表明，R 环是将 FANCD2 保留在大型基因中间内含子区域染色质中的必需条件，而导致 FA 途径激活的复制应激诱导的上游事件不是由 R 环触发的。
