利用CRISPR/Cas9 基因編輯技術研究TRIM28 在人類慢性骨髓性白血病細胞中之功能

Abstract

第一部份： TRIM28量化調控之轉錄抑制，影響慢性骨髓性白血病細胞—K562的細胞生長。 TRIM28被認為是胚胎幹細胞中的必要基因，在小鼠的生殖細胞中剔除Trim28基因會造成老鼠胚胎在受孕後8.5天時死亡。相較於正常的組織之中，TRIM28已被證明會在許多腫瘤中呈現過量表現，此過量表現會促進癌細胞的增生和轉移。K562是一種急性轉化期的慢性骨髓性白血病的癌細胞，其染色體組型呈現不正常的類三倍體並且伴隨著不尋常的急性生理狀態，是常用來模擬紅血球細胞和血小板的前驅細胞。在此次的研究中，我們利用CRISPR/Cas9系統對K562細胞中TRIM28基因的套數加以編輯，並發現藉由改變基因體中等位基因的套數來降低TRIM28的表現量可以抑制K562細胞的生長，並促進期其對抗癌藥物的敏感性。 第二部分： 在TRIM28蛋白RBCC結構區域上的後轉錄性修飾是一關鍵因子，用以調控KRAB-鋅指蛋白與TRIM28的結合。 TRIM28（也稱作KAP1或TIF1-β）是一個表觀遺傳學上重要的調控者，已知可以藉由與異染色質蛋白（HP1）和鋅指蛋白（ZFPs or ZNFs）的交互作用來調控基因表現。Krüppel-associated box (KRAB)結構區域由約75個胺基酸所組成，存在於三分之一的人類C2H2類型的鋅指蛋白中。TRIM28藉由其RBCC （RING finger, B-box, and coiled coil）結構區域與KRAB結構區域相接合。作為轉錄抑制子，TRIM28和KRAB-鋅指蛋白的交互作用，可調控KRAB-鋅指蛋白所結合之特定區域中的基因表現。與許多的TRIM蛋白相同，TRIM28被認為對必須形成聚合體化後才具有共同抑制子之功能。然而，調控其聚合體化的詳細機制目前尚未可知。 在人類與小鼠的TRIM28蛋白的RBCC結構區域上發現三個可以被乙醯化的賴氨酸位置，分別是人類的266、304、340殘基和小鼠相對應的267、305、341殘基。在此次研究中，我們證實了Trim28 K305Q突變蛋白（模仿賴氨酸305被乙醯化之老鼠Trim28蛋白）無法與KRAB-鋅指蛋白結合，但並不影響其聚合體結構之形成。

PART I: A Dosage-Dependent Regulation of Transcriptional Repression by TRIM28 Affects the Cell Proliferation in Chronic Myeloid Leukemia Cells– K562. TRIM28 is considered as an essential gene in embryonic stem cells, and the knockout of Trim28 in mouse germ-line cells cause embryonic lethal at day E8.5. Compared with normal tissue, overexpression of TRIM28 has been demonstrated in many tumors, which promotes proliferation and metastasis of the cancer cells. K562, a model of common progenitor of erythroblast and megakaryocyte, is a human chronic myeloid leukemia (CML) cell line in terminal blast crisis in which near-triploidy with complex karyotypic abnormalities occurred together with unusual blast morphology. In this study, we used CRISPR/Cas9 system to edit the copy number of TRIM28 gene in K562 cells, and revealed that down-regulating the expression of TRIM28 through genomic alternation of copy number allele would suppress the proliferation of K562 Cells, and induce the sensitivity of anti-cancer drugs. Part II: The Post-translational Modification of TRIM28 at RBCC Domain is Critical to switch the interaction of KRAB-ZFPs and TRIM28. TRIM28 (also known as KAP1 or TIF1-β) is a key epigenetic modifier known to regulate gene expression via interaction with HP1 and zinger finger proteins (ZFPs or ZNFs). The Krüppel-associated box (KRAB) domain is about 75 amino acids found in approximately one-third of human C2H2-type ZNFs. The RBCC (RING finger, B-box, and coiled coil) domain of TRIM28 is responsible for its interaction with KRAB domain. Interactions between TRIM28 and KRAB-ZNFs have been shown to function as transcriptional repressor to regulate gene expression at the KRAB-ZFPs specific binding loci. Similar to many TRIM proteins, oligomerization of TRIM28 was considered as a requirement for its co-repressor activity. However, the underlying mechanism(s) governing its oligomerization is poorly understood. Three acetylated lysine residues of TRIM28 from both human (K266, K304, and K340) and mouse (K267, K305, and K341) have been identified. In present study, we demonstrated that Trim28 K305Q mutant protein (the mimic of mouse Trim28/Ac-K305) resulted in failure of interacting with KRAB-ZFPs, but did not affect the forming of its oligomer structure.