Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States of America.
PLoS Genet. 2020 Feb 7;16(2):e1008597. doi: 10.1371/journal.pgen.1008597. eCollection 2020 Feb.
Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
将组蛋白 H3 变体 CENP-A（酵母中的 Cse4、苍蝇中的 CID）的定位限制在着丝粒对于忠实的染色体分离至关重要。CENP-A 的定位错误会导致酵母、苍蝇和人类细胞中的染色体不稳定（CIN）。在许多癌症中观察到 CENP-A 的过表达和定位错误，这与侵袭性增加和预后不良相关。然而，在生理条件下调节 CENP-A 水平和定位的基因尚未确定。在这项研究中，我们使用全基因组遗传筛选来识别维持 Cse4 内稳态所必需的基因，以防止其定位错误，从而维持染色体稳定性。我们表明，两种 Skp、Cullin、F-box（SCF）泛素连接酶与进化上保守的 F-box 蛋白 Met30 和 Cdc4 相互作用并协同调节内源性 Cse4 的蛋白水解，防止其在生理条件下定位错误，从而忠实的染色体分离。Met30 与 Cdc4 的相互作用独立于 D 结构域，D 结构域对于它们的同源二聚化和其他底物的泛素化是必需的。Cdc4 和 Met30 对泛素化的需求都是针对 Cse4 的；Cdc4 和 Met30 的共同底物以前没有被描述过。Met30 是 Cdc4 与 Cse4 之间相互作用所必需的，这种相互作用的缺陷会导致 Cse4 的稳定和定位错误，从而导致 CIN。我们提供了 Cse4 定位错误与动粒结构缺陷之间的直接联系，并表明 SCF 介导的 Cse4 蛋白水解是防止 Cse4 在非着丝粒区域稳定维持的主要机制，从而确保忠实的染色体分离。总之，我们已经确定了调节细胞内源性 Cse4 水平的必要途径，并表明 SCF-Met30/Cdc4 对 Cse4 的蛋白水解防止了未受干扰细胞中的定位错误和 CIN。
