Stamatiou Konstantinos, Huguet Florentin, Serapinas Lukas V, Spanos Christos, Rappsilber Juri, Vagnarelli Paola
College of Health, Medicine and Life Science, Brunel University London, London, UB8 3PH, UK.
Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, EH16 4SB, UK.
Genome Biol. 2024 Apr 22;25(1):105. doi: 10.1186/s13059-024-03243-5.
The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle.
Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested.
We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.
增殖抗原Ki-67多年来一直在临床环境中广泛用于癌症分期，但对其生物学功能的研究却滞后了。最近，已证明Ki-67可调节染色体外周的组成和有丝分裂中的染色体行为，并在异染色质组织和基因转录中发挥作用。然而，Ki-67在整个细胞周期中的不同作用是如何被调节和协调的，仍知之甚少。由于缺乏可用于耗尽该蛋白的工具，再加上其在细胞周期中的丰度和波动，对Ki-67功能的理解进展有限。
在这里，我们使用强力霉素诱导的E3连接酶与生长素诱导的降解标签，在HCT116细胞中实现Ki-67的快速、急性和均匀降解。该系统与APEX2蛋白质组学和磷酸蛋白质组学方法相结合，使我们能够证明Ki-67在DNA复制过程中发挥作用。在其缺失的情况下，DNA复制会严重延迟，复制机制被卸载，导致DNA损伤，而这种损伤无法被经典途径感知，且依赖于HUWE1连接酶。这会导致复制和姐妹染色单体粘连出现缺陷，但也会在所有测试的细胞系中触发由cGAS/STING途径介导的干扰素反应。
我们揭示了Ki-67在DNA复制和基因组维持中的新功能，该功能独立于其先前已知的在有丝分裂和基因调控中的作用。
