PARP1與ZSCAN4交互作用對端粒長度調節之探討

Abstract

Zinc finger and SCAN domain containing 4 (Zscan4)為大量表現於二細胞時期鼠胚之基因。在體外培養中僅約1-5%的胚幹細胞可表達此基因。Zscan4之主要功能包含維持基因組穩定與調控端粒長度之動態平衡等，並可藉由非端粒酶之同源重組之方式延長端粒長度。然而，Zscan4延長端粒之分子機制至今仍未明瞭。透過免疫沈澱串聯質譜儀的方式，poly [ADP-ribose] polymerase 1 (PARP1)被預測為ZSCAN4潛在的結合蛋白之一，具有酵素活性，可將NAD+上之ADP-ribose接至目標蛋白上。過去研究顯示PARP1為偵測DNA斷裂並參與在DNA修復之重要蛋白；更重要的是，剔除Parp1基因後，會導致小鼠端粒變短且提早老化。因此，本研究之目的為藉由探討ZSCAN4與其潛在結合蛋白PARP1之間的交互作用，以期進一步了解ZSCAN4延長端粒之機制。在本研究中，首先產製Flag-Zscan4與Ha-Parp1之重組質體，透過FLAG與HA抗體分別進行共同免疫沈澱，確認PARP1為一ZSCAN4之結合蛋白。接著，根據不同蛋白結構域將Zscan4與Parp1分別截斷為三個部分，包括Zscan4的SCAN、Linker sequence及Zinc finger （ZF) domain；與Parp1之DNA binding (DB)、Automodification (AM)及Catalytic domain，更進一步發現ZSCAN4以SCAN domain與PARP1之DB domain與AM domain結合，且ZSCAN4也會以ZF domain與PARP1之AM domain結合。為了解PARP1在ZSCAN4延長端粒中扮演之角色，進一步產製了ZSCAN4過表達之胚幹細胞株，並發現相較於控制組細胞，此細胞株之增殖速率較慢且端粒長度有較長的特性。接著，在施予PARP的抑制劑處理後，控制組胚幹細胞中發現端粒有顯著延長的現象；然在ZSCAN4過表達的胚幹細胞株中則觀察到細胞喪失其原本端粒長度較長的特性，顯示PARP1可能在ZSCAN4延長端粒的機制中扮演著雙重的角色。後續以短髮夾RNA降低Parp1的表現量後，觀察到端粒調控相關之基因被激活，Zscan4的表現量有顯著的提升，且端粒有延長的現象，進一步證實了PARP1具有抑制Zscan4表現的能力。最後透過端粒酶半缺失（Terc+/-）之核移殖胚幹細胞（ntESCs）模擬端粒綜合症的患者，施予PARP的抑制劑，期能補救其端粒較短之缺陷。結果顯示，試驗之三株Terc+/- ntESCs中，其中一株ntESCs有顯著延長端粒的效果。綜上所述，本研究驗證PARP1為一與ZSCAN4結合之蛋白，並進一步揭露其交互作用之蛋白功能域；另也發現PARP1在調控ZSCAN4與端粒長度上可能扮演著雙重的角色，然而Zscan4如何參與延長端粒更詳盡之分子機制仍待更進一步的實驗釐清。由於Zscan4為藉由非端粒酶途徑調控端粒長度之重要蛋白，若能深入探討Zscan4延長端粒之分子機制，盼能為端粒綜合症患者提供有效治療之策略或方向。

Zinc finger and SCAN domain containing 4 (Zscan4) is a gene that expressed in 2-cell stage embryos and sporadically in a subpopulation of embryonic stem cells (ESCs). Zscan4 participates in the regulation of telomere homeostasis and genomic stability. However, the detailed mechanism of Zscan4 involved in telomere elongation is still unclear. Poly [ADP-ribose] polymerase 1 (PARP1) is known to sense DNA damage and activate several DNA repair pathways. The objective of this study is to investigate the mechanisms of PARP1 interacts with ZSCAN4 and participates in telomere regulation. By serial immunoprecipitations, our data showed that PARP1 not only interacted with SCAN domain of ZSCAN4 through DNA binding domain and auto-modification domain but also interacted with zinc finger domain of ZSCAN4 by auto-modification domain. To investigate whether PARP1 is associated with ZSCAN4 in telomere homeostasis, Zscan4 over-expressed ESCs (OEZ4 ESCs) were generated. We found that OEZ4 ESCs show slower proliferation rate and significantly longer telomere length compared to the vector control. Moreover, inhibition of PARP1 activity by treating 3-aminobenzamide (3-AB), the PARP inhibitor, led to shorten telomeres in OEZ4 ESCs, indicating PARP1 may play dual roles to interact with Zscan4 and participates in telomere regulation. Next, knockdown Parp1 by shRNA in ESCs showed elongation of telomere length and activation of genes involved in telomere regulation, suggesting PARP1 regulates telomere homeostasis. Lastly, treating 3-AB in Terc+/- ntESCs, which mimic the telomere syndrome patient, we further reveal that shorten telomeres has been elongated. In conclusion, this study uncovers PARP1 as a ZSCAN4-associated protein and participates in telomere regulation, and provides novel insights on the molecular interaction between DNA damage and telomere regulation.