DNA 拓樸異構酶III 於染色質包裹組裝、轉錄重組、以及發育之角色

Type IA DNA topoisomerases in chromatin organization, transcription programming and development

賴婉容(Wan-Jung Lai)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03217

摘要

DNA 拓樸酶 III 屬於 type IA 拓樸異構酶，是所有生物都具有的酵素。脊椎動物有兩個同功酶：TOP3α 和 TOP3β。TOP3α 剔除的老鼠會在胚胎發育時期死亡，而 TOP3β 的剔除鼠則有提早老化的現象，說明了 TOP3 的重要性。然而，TOP3 的細胞及生理上扮演的角色與功能仍需要更進一步探討；在本論文中，我們分別利用細胞培養及斑馬魚模式系統來探討其功能。(一) 我們發現 TOP3α 缺乏的人類細胞株可能因為產生較鬆散的染色質，而呈現染色體分離時的缺陷、基因體的不穩定、以及對細胞週期藥物的高度敏感性。TOP3α 主要位於染色質上，TOP3α 缺乏細胞的染色質較容易被核酸酶所切割，更加支持其較鬆散的染色質結構，而此現象可以利用補回正常的 TOP3α來恢復，因此TOP3α 在染色質包裹組裝具有潛在的功能。這個論點亦被以下實驗所支持：(a) TOP3α 缺乏的人類細胞株在細胞分裂時會產生染色體分離缺陷；(b) TOP3α 缺乏的細胞在細胞週期間會累積雙股DNA 斷裂，並且造成片段狀的染色體。(二) 由於實驗室實驗發現 TOP3α 和 p53 具有蛋白功能及遺傳性的交互作用，因此我們假設 TOP3α 可能利用轉錄程式重組參與細胞分化及發育。於是我們利用斑馬魚模式生物系統來進一步探討TOP3 在發育中扮演的角色。首先我們發現斑馬魚 top3a 以及 top3b 的訊息RNA 於早期高度表現，發育後期則主要聚集於神經系統以及心臟，似乎有母源表現之現象。於胚胎早期外送 TOP3A 的表達質體 DNA 會產生胚胎腹型化的表徵，這些特徵包括：胚胎早期的腹型化調控基因表現增加，另一方面造成組織背型化延展缺陷，進而延遲胚胎的腸腔化，在胚胎發育後期產生嚴重的體節彎曲；而以寡聚核苷酸 (morpholino; MO) 抑制TOP3A 的表現則會產生胚胎背型化的缺陷，這些缺陷包括：胚胎早期的腹型化調控基因表現減少，尾節縮短，以及背側的心臟腫大的表徵。因此 TOP3A 在胚胎早期可能參與組織腹背決定於調控細胞的腹型化。有趣的是，TOP3A 和 p53 在斑馬魚胚胎以及纖維母細胞株中也具有蛋白及基因交互作用。重要的是，上述所有的缺失可以藉由同時注入 p53 MO 所回復。綜合以上結果，我們認為 TOP3 會與 p53 協同作用，調控斑馬魚發育及組織分化。

關鍵字

DNA拓樸異構酶III ； 染色質結構 ； p53 ； 斑馬魚 ； 背腹型化 ； 神經發育

並列摘要

Vertebrates possess two isoforms of type IA DNA topoisomerases (alpha and beta isozymes; TOP3α and TOP3β), the top3a and top3b knockout lead to embryonic lethality and reduced lifespan in mice, respectively. However, the cellular and biological functions of TOP3α remain unclear. In this dissertation, we use cell culture and zebrafish model system to explore the functions of TOP3. (I) Here, we showed TOP3α- knockdown (TOP3αKD) cells exhibit segregation defect, chromosome instability and hyper-sensitivity to cell cycle-disrupting agents which might result from the under-condensation of chromosome DNA. Moreover, TOP3α proteins mainly exist on chromatin and the role of TOP3α in chromatin organization is further supported by the enhanced chromatin digestion of TOP3αKD cells to nucleases. Furthermore, the enhanced accessibility to nuclease digestion observed in TOP3αKD cells can only be complemented by expression of functional TOP3α. Therefore, TOP3α have potential function in chromatin organization. This statement is further supported by the following experiments: (a) TOP3αKD cells have chromosome segregation defects during mitosis. (b) TOP3αKD cells accumulate DNA double strand breaks during cell cycle progression and cause chromosomal fragmentations. (II) Coupled with our previous findings about the physical, functional and genetic interactions of human TOP3α (hTOP3α) and p53, we speculate that TOP3 might participate in differentiation and development via facilitating transcriptional programming. In this regard, roles of TOP3 are studied in the well-developed zebrafish model. We showed that both zTOP3A and zTOP3B mRNAs are expressed during early embryogenesis and primarily localized to neuron systems and heart. Over-expression (zTOP3AOE) of zTOP3A showed ventralization phenotypes including expansion of BMP-regulated ventral marker genes during early embryogenesis while defected in convergence of dorsal-cellular tissues, which led to delayed gastrulation and server body curvature. In contrast, morpholino (MO)-mediated knockdown (zTOP3AMO) of zTOP3A display dose-dependent dorsalization phenotypes including reduced ventral marker genes and tail-segment shortages, while showed dorsal-cardiac edema. It suggests that zTOP3A is required for development of ventral tissues during dorsal-ventral patterning. In agreement with the functional interaction of p53 and hTOP3α, the association between TOP3A and p53 was further confirmed under physical conditions in zebrafish embryos and zebrafish fibroblast cell line, ZF4. In addition, we found that simultaneous zp53MO can rescue all the above developmental defects of zTOP3AMO and zTOP3AOE fish. Together, these results suggest that zTOP3A cooperatively functions with the p53-directed transcription program in zebrafish development and/or tissue differentiation.

並列關鍵字

DNA Topoisomerases III ； Chromatin structure ； p53 ； Zebrafish ； dorsal-ventral patterning ； Neuron development

參考文獻

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