探討Ikbkap/ Elp1 在基因組不穩定性中扮演的角色

基因組不穩定性通常在細胞複製或分裂時發生，起因於染色體損壞未被修復且不斷的累積，而基因組的不穩定性已知與許多癌症有關。生物體為將細胞內的遺傳物質準確傳遞到子代，染色體的損壞修復機制實為重要。Ikbkap 基因編碼 IKAP/Elp1 蛋白質，為延伸因子 (Elongator complex, Elp1-6) 蛋白的其中一個子單元，依據蛋白質結構分析 Elp1 之功能顯示，其結構為維持延伸因子蛋白之構型所必要。延伸因子最初被發現參與第二型RNA聚合酶轉譯作用，而後更被發現參與許多細胞內生理活動。延伸因子調控細胞之方式目前認為主要是透過組蛋白乙醯化 (Histone Acetylation) 或轉運核糖核酸修飾 (tRNA modification)。先前的研究顯示 Ikbkap 參與小鼠精細胞減數分裂時染色體的聯會、雙股 DNA 斷裂修復與重組。在 Ikbkap 缺失的小鼠睪丸組織中，與DNA修復相關的基因表現量下降，這些結果說明了Ikbkap 在精細胞中可能參與DNA損壞修復。為探討 Ikbkap 在體細胞中是否也參與基因組穩定性的維持，我們以基因剔除之方式觀察小鼠纖維母細胞(Mouse Embryonic Fibroblast, MEFs) 。我們發現在 MEF 細胞中缺乏 Ikbkap ，且經過輻射線照射過後細胞生長曲線下降、在彗星試驗中發現 Ikbkap 缺乏會影響 DNA雙股斷裂後的修復，在染色體分析實驗發現 Ikbkap 缺失相較於 Ikbpap 正常表現的組別，異常的染色體比例顯著增加、但 Ikbkap 缺失且給予照射輻射線後 24 小時並不會導致細胞死亡。故本篇研究認為 Ikbkap 基因影響 DNA 斷裂後的細胞存活，且與染色體修復有關。此外，我們利用帶有綠螢光報導基因的 U-2 OS 細胞來探討 Ikbkap 是否參與 DNA 雙股斷裂修復，發現 Ikbkap 參與在染色體同源雙股斷裂的修復而不是在非同源性末端接合的修復。後續之研究將會著重於 Ikbkap 如何參與 DNA 修復與相關機制的探討。

關鍵字

基因組不穩定性；輻射；同源重組DNA修復；染色體型態；細胞凋亡

並列摘要

Genomic instability refers to an increased tendency of alterations in the genome during cell cycle progression, and likely contributes to the development of many cancers. To faithfully transmit genetic information to the progeny, efficient DNA repair pathways are required for the maintenance of genomic stability. Ikbkap gene encodes IKAP/Elp1, which functions as a scaffold subunit of the Elongator complex (Elp1-6). The Elongator complex was initially described as an RNA polymerase II-associated transcription elongation factor, but it has been shown later to be involved in a variety of cellular functions through regulation of histone acetylation and tRNA modification. A previous study has shown that Ikbkap regulates meiotic synapsis, double strand break (DSB) repair and recombination during male meiotic progression. Downregulation of DNA repair-related genes was observed in Ikbkap deficient testes, suggesting that Ikbkap plays a crucial role in DNA damage response (DDR) in germ cells. To investigate whether Ikbkap maintains genomic stability in somatic cells, we deleted Ikbkap in mouse embryonic fibroblasts (MEFs). Comet assays show that Ikbkap-/- MEFs are hypersensitive to DNA damage, like -irradiation. Ikbkap-/- MEFs exhibited a proliferative impediment in response to -irradiation. In addition, a significant accumulation of chromosomal abnormalities, including breaks was observed in Ikbkap-/- MEFs compared to controls. Although apoptotic rate was unaffected in Ikbkap-/- MEFs one day post -irradiation, these data shows that IKAP function is essential for the ability to survive DNA damage, and implicating IKAP in repair in U-2 OS cells. Furthermore, we revealed that Ikbkap is required for DNA repair by homologous recombination (HR), but not non-homologous end joining (NHEJ) using U-2 OS cell lines harbouring a green fluorescent protein reporter for DSB repair. These finding define a function of Ikbkap in DNA repair. Further investigation will be focused on the underlying DNA repair mechanisms.

並列關鍵字

genomic instability ； irradiation ； homologous recombination DNA repair ； chromosome morphology ； apoptosis

參考文獻

Abdel-Fattah, W., Jablonowski, D., Di Santo, R., Thuring, K. L., Scheidt, V., Hammermeister, A., Ten Have, S., Helm, M., Schaffrath, R., & Stark, M. J. (2015). Phosphorylation of Elp1 by Hrr25 is required for elongator-dependent tRNA modification in yeast. PLoS Genet, 11(1), e1004931.

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Altieri, F., Grillo, C., Maceroni, M., & Chichiarelli, S. (2008). DNA damage and repair: from molecular mechanisms to health implications. Antioxid Redox Signal, 10(5), 891-937.

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Attardi, L. D., de Vries, A., & Jacks, T. (2004). Activation of the p53-dependent G1 checkpoint response in mouse embryo fibroblasts depends on the specific DNA damage inducer. Oncogene, 23(4), 973-980.

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探討Ikbkap/ Elp1 在基因組不穩定性中扮演的角色

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