- 學位論文
探討在人類慢性骨髓性白血病細胞K562中由TRIM28介導的基因調控的分子機制
The molecular mechanism of TRIM28-mediated gene regulation in leukemia K562 cells
摘要
TRIM28，也稱為KAP1或TIF1-β，它會參與癌細胞的發展。Trim28通過 N 端的RBCC域與KRAB-ZNF鋅指蛋白的KRAB域相互作用，RING finger含有 E3 連接酶活性。 為了研究TRIM28在白血病細胞系K562中的功能作用，我們通過 CRISP/Cas9 基因組編輯生成了TRIM28剔除(KO)突變體。我們發現胚胎ε- (HBE) 和胎兒γ- (HBG) 珠蛋白在TRIM28 KO 細胞中增加。通過 RNA-seq 分析，我們知道 SOX6 作為 β 蛋白抑制物在TRIM28 KO突變體中被減少。此外，TRIM28 KO K562細胞中SOX6的過表達將恢復對HBE和HBG的抑制。我們證明剔除TRIM28通過增加microRNA而減少SOX6。我們還試圖解釋TRIM28在細胞增殖、細胞週期轉變和細胞凋亡中起著關鍵的調節作用。用藥物處理細胞會使細胞進入分化階段，這與TRIM28 KO細胞相似。 同時，我們還通過免疫沉澱和 LC-MS/MS分析鑒定了TRIM28相互作用蛋白。我們發現TRIM28與GID/CTLH E3連接酶複合物（包括 YPEL5、RanBP9、RMND5A、MAEA、WDR26、RBP10、ARMC8、TWA1 和 GID4）相關。我們已經證明TRIM28直接與 YPEL5相互作用，並發現了TRIM28的相關相互作用域。此外，我們已經檢查了 TRIM28可能調節RMND5A和MAEA的蛋白質穩定性。我們對GID/CTLH複合物和 TRIM28的E3連接酶活性的相互調節如何控制它們的底物穩定性感興趣。綜上所述，我們將從基因表達和蛋白質穩定性調控方面闡明TRIM28如何影響血癌細胞的生長和分化。
並列摘要
Tripartite motif-containing 28 (TRIM28), is also known as KAP1 (Krüppel-Associated Box (KRAB)-Associated Protein 1) or TIF1-β (Transcriptional Intermediary Factor 1 β), which is involved in cancer cell progression. Trim28 interacts with KRAB domain of KRAB-ZNF (zinc finger) proteins through N-terminal RING-B boxes-Coiled-Coil (RBCC) domain, and the RING finger contains E3 ligase activity. To investigate the functional role of TRIM28 in leukemia cell line K562, we have generated TRIM28 knockout (KO) mutant by CRISP/Cas9 genome editing. We found embryo ε- (HBE) and fetal γ- (HBG) globins were upregulated in TRIM28 KO cells. By RNA-seq analysis, we have known that SOX6 as a beta globin repressor was downregulated in TRIM28 KO mutant. Moreover, overexpression of SOX6 in TRIM28 KO K562 cells would restore the inhibition of HBE and HBG. We demonstrate knockout of TRIM28 downregulates SOX6 through increasing of microRNAs. We also try to explain that TRIM28 plays a key regulatory role in cell proliferation, cell cycle transition, and apoptosis. Treating cells with drugs will make the cells enter the stage of differentiation, which is similar to the TRIM28 KO cells. Meanwhile, we also identified TRIM28-interacting proteins by immunoprecipitation and LC-MS/MS analysis. We found TRIM28 associates with GID/ CTLH (glucose-induced degradation/ carboxy-terminal to LisH) E3 ligase complex (including YPEL5, RanBP9, RMND5A, MAEA, WDR26, RBP10, ARMC8, TWA1 and GID4). We have demonstrated that TRIM28 interacts directly with YPEL5 and found relative interacting domain of TRIM28. Moreover, we have examined that TRIM28 might modulates protein stability of RMND5A and MAEA. We are interested in how the mutual regulation of the E3 ligase activity of the GID/CTLH complex and TRIM28 control their substrate stabilities. Based on the above, we will clarify how TRIM28 affects the growth and differentiation of blood cancer cells in terms of gene expression and regulation of protein stability.