Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
J Biol Chem. 2021 Sep;297(3):101049. doi: 10.1016/j.jbc.2021.101049. Epub 2021 Aug 8.
Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis. FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of double-strand break-associated factors, including 53BP1 and BRCA1. FUS cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of prereplication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase-associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS complementary DNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.
融合肉瘤（FUS）编码一种 RNA 结合蛋白，在转录激活和 RNA 剪接中具有多种作用。虽然 FUS 和转录因子 DNA 结合域的致癌融合与软组织肉瘤有关，但 FUS 的显性突变可导致肌萎缩侧索硬化症。FUS 也与基因组维护有关。然而，其在基因组稳定性中的作用的潜在机制尚不清楚。在这里，我们应用基因编辑、功能重建以及整合蛋白质组学和转录组学来阐明 FUS 在 DNA 复制和修复中的作用。与 DNA 双链断裂修复的支持作用一致，FUS 缺陷细胞在双链断裂相关因子（包括 53BP1 和 BRCA1）的募集和保留方面表现出微妙的改变。FUS 细胞还表现出增殖潜力降低，这与复制叉推进速度降低、前复制复合物加载减少、微核形成增强以及 S 期相关基因的表达和剪接减弱相关。最后，FUS 缺陷细胞表现出 DNA 复制时间的全基因组改变，在重新表达 FUS cDNA 后得到逆转。我们还表明，依赖 FUS 的复制结构域富含转录活性染色质，并且 FUS 是转录活性 DNA 及时复制所必需的。这些发现表明，DNA 复制动力学和编程的改变导致了基因组不稳定和 FUS 缺陷细胞的功能缺陷。
