專家點評EMBO J | 汪方煒實驗室揭示有絲分裂期基因組穩定性的重要保護機制

細胞來自於細胞，產生更多細胞的唯一途徑是已存在細胞的分裂。所有生物，包括從單細胞的細菌到多細胞的哺乳動物，都是上溯至30億年前生命起源後連續不斷的細胞生長與分裂的結果。以人體為例，從一個受精卵發育成人，大約需要經歷30萬億次細胞分裂【1】。據粗略估計，一個成年個體每天大約發生500億次有絲分裂。細胞在有絲分裂期的核心任務是準確地分配它在之前的S期複製的染色體，確保每個子細胞得到一份完整的基因組拷貝。這個過程必須高度精確，否則會造成染色體和基因組的不穩定性，進而可能導致細胞的癌變。如圖1所示，在S期DNA複製完成後，每條染色體形成兩條相同的姐妹染色單體，它們靠黏連蛋白複合體（Cohesin）和DNA連環（DNA catenation）順著染色體長軸緊密結合在一起。有絲分裂期染色體的分離需要去除姐妹染色單體間的粘連（cohesion）和DNA連環（catenation），這一過程分兩步完成。首先，在有絲分裂的前期和前中期，伴隨著Wapl從染色體臂上移除Cohesin的過程，DNA拓撲異構酶IIα（TOP2A）得以去除雙鏈DNA之間的連環和纏繞，從而解除姐妹染色體臂部的物理聯結【2】。在此期間，著絲粒區的Cohesin在Sgo1、Sororin和Haspin等蛋白的保護下逃脫Wapl對其的移除【3】，並拮抗TOP2A對DNA連環的去除作用【4,5】。隨後，在有絲分裂中期向後期轉換的過程中，隨著蛋白水解酶Separase對Cohesin的剪切，TOP2A須快速高效地去除著絲粒區染色體間的DNA連環，以使得姐妹染色單體及時分離並在紡錘體的牽引下向細胞的兩極移動。圖1：有絲分裂期姐妹染色體的分離（左），以及TOP2A（中）和H2ApT120（右）在染色體上的動態分布和定位。因此，為了確保有絲分裂期染色體的精確分離，TOP2A必須在染色體分離前就聚集於著絲粒區。事實上，早在二三十年前科學家們就已經發現，TOP2A分布於有絲分裂期染色體的臂部並高度富集於著絲粒區【6-8】。後續的研究進一步發現，類泛素化修飾促進TOP2A在著絲粒區的定位及姐妹染色體的精確分離【9】。那麼，TOP2A在著絲粒區定位的受體分子是什麼呢？這個問題的答案一直不為人們所知。2019年11月26日，浙江大學生命科學學院汪方煒課題組在The EMBO Journal雜誌上發表論文「Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability」，揭示了有絲分裂期組蛋白磷酸化通過招募TOP2A保護基因組穩定性的重要機制和功能。為了解決上述重要科學問題，汪方煒實驗室的博士生張妙同學開展了基於RNA幹擾技術的篩選工作，並發現了紡錘體檢查點蛋白Bub1對於TOP2A在著絲粒區定位的重要性（圖2A）。進而，利用小分子化合物抑制Bub1的激酶活性後，TOP2A在著絲粒的定位幾乎消失（圖2B），說明Bub1的激酶活性對於TOP2A的著絲粒定位是必需的。圖2：敲減Bub1的表達（A）或利用小分子化合物抑制Bub1的激酶活性（B）去除TOP2A在有絲分裂前中期染色體著絲粒區的富集。

已知的Bub1激酶底物有三個，它們分別是紡錘體檢查點蛋白Cdc20【10,11】、組蛋白H2A【12】以及端粒蛋白TRF1【13】。那麼，Bub1是通過磷酸化哪個已知或未知的蛋白底物發揮作用的呢？張妙同學發現，敲減Cdc20或TRF1的表達並不影響TOP2A的著絲粒定位，而TOP2A和Bub1介導的組蛋白H2A第120位蘇氨酸的磷酸化（簡稱H2ApT120）在有絲分裂前期、前中期及中期細胞的著絲粒區有較好的共定位，提示了H2ApT120參與調控TOP2A定位的可能性。接著，張妙同學進行了一系列精巧的實驗操作，將H2ApT120人為地產生於細胞周期不同時期的染色體的不同區域，發現它都能招募TOP2A至染色體的相應區域，說明H2ApT120對TOP2A在著絲粒定位是必要且充分的。繼而，通過在細胞內表達多種不同的TOP2A突變體，張妙同學發現，位於TOP2A的C-gate區的coiled-coil結構域和羧基端的ChT結構域對於TOP2A定位於著絲粒區是重要的（圖3）。

圖3：人源TOP2A蛋白的結構域示意圖。

那麼，H2ApT120對TOP2A的招募作用是直接還是間接的呢？為了解答這個疑問，張妙同學利用林世賢實驗室提供的獨特技術，從一株改造過的大腸桿菌中表達純化了第120位蘇氨酸被替換為磷酸化的絲氨酸的組蛋白H2A，並在梁材同學的幫助下證實，與野生型的H2A蛋白相比，磷酸化的H2A蛋白與TOP2A蛋白在體外的結合能力明顯增強。這些實驗結果不僅展示了H2ApT120對TOP2A的直接招募作用，而且也為探究TOP2A在著絲粒區的功能提供了重要的實驗手段。通過破壞H2ApT120與TOP2A的結合，選擇性地去除TOP2A在著絲粒區的定位而不影響其在染色體臂上的分布，張妙同學發現，著絲粒區姐妹染色體DNA的去連環（decatenation）作用明顯受阻，主要表現為有絲分裂後期細胞中由超細DNA構成的染色體橋（Ultra-fine anaphase DNA bridges）的顯著增加(註：此類染色體橋容易發生DNA損傷）。因此，H2ApT120與TOP2A的結合對於解除有絲分裂期姐妹染色單體間的DNA連環是重要的。這一系列研究結果表明，當細胞進入有絲分裂時，Bub1對組蛋白H2A的磷酸化修飾招募TOP2A至著絲粒，促進著絲粒區DNA連環的去除，進而保證了染色體的精確分離和基因組的穩定性（圖4）。基於TOP2抑制劑在癌症化療中的廣泛應用【14】，以及近期報導的Bub1小分子抑制劑對於癌細胞增殖的抑制作用【15】，這項研究也為靶向TOP2A和Bub1的癌症治療提供了新的視角。

圖4：TOP2A在有絲分裂期著絲粒區定位的分子機制及功能。 

汪方煒教授是論文的通訊作者，博士生張妙為第一作者，汪方煒實驗室的博士生梁材和陳親富以及林世賢實驗室的趙紅霞同學等也有貢獻。劉洪（杜蘭大學，Tulane University）Proper chromosome segregation is essential for maintaining chromosome stability as well as preventing many human diseases, such as cancer. During mitotic cell cycle, chromatin is duplicated at S phase (sister chromatids). Duplicated sister chromatids are linked together by a protein complex cohesin, which is called sister-chromatid cohesion, and by intertwined DNA fibers, which is referred to as DNA catenation. Maintenance of these sister-chromatid linkages till late mitosis, especially sister-chromated cohesion, is critical for chromosome stability. At late mitosis (anaphase onset), these sister-chromatid linkages must be resolved to allow sister chromatids to segregate from each other. Failure in doing so results in lagging chromosomes and/or ultra-fine anaphase bridges, which also significantly jeopardize chromosome stability. Therefore, proper orchestration of sister-chromatid cohesion and DNA catenation is critical for protecting chromosome stability. However, how this is achieved is poorly understood.An interesting piece of work from Dr. Wang’s lab has identified a key orchestrator, Bub1, for these two processes. Extensive previous work has shown that Bub1, a mitotic kinase, enriches an essential cohesion protector, Shugoshin, to centromeres to protect sister-chromatid cohesion there by phosphorylating histone H2A (pH2A). Shugoshin is targeted to centromeres by directly binding Bub1-phosphorylated histone H2A. These previous studies have established Bub1/pH2A as an important regulatory pathway for centromeric cohesion protection. Topoisomerase II, an important enzyme that resolves DNA catenation, is one of scaffold chromosome proteins. Interestingly, this enzyme has long been noticed to be enriched at centromeres during mitosis, but the biological functions and regulation of this pool of Topoisomerase II largely remain unknown. Using elegant cell-biological and biochemical tools, Dr. Wang and his colleagues convincingly demonstrated that this very Bub1/pH2A also enriches Topoisomerase II to centromeres. Interestingly, like Shugoshin, the centromeric enrichment of Topoisomerase II is also through its direct binding with pH2A. They further showed that the Bub1/pH2A-enriched pool of Topoisomerase II at centromeres is important for timely and proper resolution of catenated DNA fibers at anaphase, thus reducing ultra-fine anaphase bridges that endanger chromosome stability. These novel findings extend our understanding horizontal on the molecular mechanisms underlying chromosome segregation. Especially, they center Bub1 as a master conductor to orchestrate two important biological processes. In addition, this study also provokes several important follow-up questions. What is the relative binding affinity of these two readers, Shugoshin and Topoisomerase II, toward phosphorylated histone H2A? Knowing the answer to this question could help determine the ratio between two proteins at centromeres, which might be a critical factor to regulate chromosome segregation during mitosis. 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