COUP-TFI抑制人類TAO kinase1啟動子活性的結構與功能關係的研究

TAOK1 (TAO kinase 1) 屬於 Ste20p (Sterile 20 protein) 成員之一，其催化部位位於 N 端。該類基因最早是由大鼠 (Rat) 的腦部中發現，最初命名為 TAO1 (Thousand and One amino acids)，並且被證實在大鼠的腦部有很高的表現量，而人類的 TAO kinase 1 (hTAOK1) 是於人類胚胎的腦部分離出來。hTAOK1 已被證實會經由 JNK 路徑和 caspase- dependent way 來誘導細胞凋亡，這意味著其表達調控在作用機制中扮演著重要的角色。因此本篇論文主要研究方向著重於 hCOUP-TFI 抑制 hTAOK1 啟動子活性的結構與功能關係的研究。在 hTAOK1 基因啟動子區域的定位上，過去我們實驗室將其中三個 DNA 片段 -629/-1、-931/-1 和 -1,243/-1 選殖於SEAP基因前，分別轉殖在 HeLa 細胞中，此實驗顯示下游的 SEAP 基因有不同程度的表達，並且 hTAOK1 -1,243/-932 區域似乎與 hTAOK1 基因的轉錄作用的抑制有關。當 -1,243/-932 區域被安置在 SV40 early promoter 的上游時，下游的 SEAP 基因表達減少了 64％。此外，過去我們實驗室推測在 hTAOK1 基因 -38/-33 位置為可能的 TATA box 序列，其具有和 TBP (TATA-box-binding protein) 辨識結合的能力，且將此 -38/-33 區域突變後不僅是破壞轉錄的活性也破壞和 TBP 辨識結合的能力。過去我們實驗室利用 Yeast one-hybrid system 篩選到一個可能的調控蛋白- COUP-TFI (chicken ovalbumin upstream promoter transcription factor I)。COUP-TFI 屬於 steroid/thyroid hormone receptor superfamily 的孤兒接受器，在其蛋白序列上有兩個功能區域，分別是 DNA 結合區 (DNA binding domain, DBD) 和配體結合區 (ligand binding domain, LBD)。在我的研究利用 Yeast One-Hybrid 方法，實驗結果顯示 hTAOK1 基因的 -1,243/-932 DNA 片段和人類 COUP-TFI (hCOUP-TFI) 的 DBD 有很強的相互作用。因此利用刪除和突變的技術把 hTAOK1 啟動子區域 -1,243/-932 刪減為不同的小片段後，再利用 Yeast One-Hybrid 來決定可與 hCOUP-TFI 結合的最小片段。結果顯示與hCOUP-TFI 結合的最小片段是 hTAOK1 啟動子區域 -1,243/-1,188 和 -988/-932。根據文獻指出，COUP-TFI 可與不完美的 DNA 重複序列 AGGTCA 結合，藉此來調控基因轉錄的進行。而這樣的 AGGTCA 序列，分別位在 hTAOK1 啟動子區域 -1,225/-1,206 和 -953/-940。為了確認這些AGGTCA 序列是否可以和 hCOUP-TFI 結合，將此序列分別突變為 AAAAAA 。利用 Yeast One-Hybrid 進一步的篩選分析，結果顯示 hTAOK1 啟動子區域 -1,243/-1,188 的兩組 AGGTCA 序列皆同時突變時才會影響和 hCOUP-TFI 的作用；而在 hTAOK1 啟動子區域 -988/-932 只要突變其中一組 AGGTCA 序列時就會影響和 hCOUP-TFI 的作用。表示 hTAOK1 啟動子區域 -1,225/-1,206 和 -953/-940 的兩組不完美的 DNA 重複序列 AGGTCA 都可以和 hCOUP-TFI 結合。但hCOUP-TFI可能是以不同的方式和不同 hTAOK1 啟動子區域作用的。由於 hCOUP-TFI 可降低hTAOK1 轉錄的活性。故將 hTAOK1 啟動子區域 -1,225/-1,206 和 -953/-940 的兩組不完美 AGGTCA 重複序列突變，置於 SEAP 基因之前。利用 SEAP assay 來觀察 hCOUP-TFI 降低 hTAOK1 轉錄的活性的功能是否被抑制。根據 SEAP assay 的結果，當 hTAOK1 啟動子區域 -953/-940 的不完美AGGTCA 重複序列突變，hTAOK1 轉錄活性是明顯增加許多。此外， hCOUP-TFI 的過度表達也造成了 hTAOK1 啟動子 -1,243/-1 啟動子區域轉錄活性下降，且甚至低於背景值。本論文的實驗結果證實 hTAOK1 基因的 -1,243/-932 DNA 片段可和 hCOUP-TFI 的 DBD 結合，而與hCOUP-TFI 結合的最小片段，是 hTAOK1 啟動子區域 -1,243/-1,188 和 -988/-932。更進一步確認位在 hTAOK1 基因的 -988/-932 的 AGGTCA motif 可和 hCOUP-TFI 結合，並且hCOUP-TFI 可抑制 hTAOK1 啟動子相關活性。

關鍵字

Yeast one-hybrid assay ；分泌性鹼性磷酸酶；啟動子； hTAOK1 ； Ste20p ； hCOUP-TFI

並列摘要

TAOK1 (TAO kinase 1) is a member of the Ste20p (Sterile 20 protein) family with its kinase domain located at N-terminus. TAOK1, originally named TAO1 (thousand and one amino acids), was first found to be highly expressed in the rat brain. hTAOK1 (human TAO kinase 1) was initially cloned from human fetal brain, and has been shown to induce apoptosis through JNK- and caspase -dependent ways in our laboratory. This implies that the regulation of its expression should play an important role in the mechanism of its action. Therefore, my study was focusing on the structure-function relationship of hCOUP-TFI for repressing the promoter activity of hTAOK1. Over the past years, our laboratory has determined the promoter region hTAOK1 gene and its possible associated regulator, human COUP-TFI. It has been demonstrated that three genomic DNA fragments -629/-1, -931/-1 and -1,243/-1 of hTAOK1 promoter were shown to induce the expression of the downstream SEAP gene to different extents in HeLa cells, and the region -1,243/-932 seems to repress the transcription of hTAOK1 gene. When -1,243/-932 was placed upstream of SV40e promoter, the expression of the downstream SEAP was decreased by 64％. In addition, our laboratory also demonstrated that the putative TATA box of hTAOK1 gene at positions -38/-33 interacted with TATA-box-binding protein (TBP), and the mutations at this region abolished not only the transcriptional activity but also the interaction with TBP. Moreover, our laboratory used the Yeast one-hybrid system to screen the human brain cDNA library, and a putative regulatory gene, COUP-TFI (chicken ovalbumin upstream promoter transcription factor I), was isolated for interaction with the promoter region -1,243/-932. hCOUP-TFI belongs to the steroid/thyroid hormone receptor superfamily of orphan receptors. It contains two functional domains, DNA binding domain (DBD) and ligand binding domain (LBD). My studies used the Yeast One-Hybrid method to show that human COUP –TFI (hCOUP –TFI) was selected for its strong interaction with DNA fragment -1,243/-932 through its DNA-binding domain. In this study, the promoter region -1,243/-932 was divided into smaller ones by the deletion and mutation analysis. When Using the Yeast One-Hybrid method to determine the minimal sequences for interaction with hCOUP-TFI, the results showed that nucleotides at positions -1,243/-1188 and -988/-932 contain the sequences for binding hCOUP-TFI. hCOUP-TFI has been reported to recognize an imperfect repeat of AGGTCA motif to repress or activate the transcription of downstream genes; similarly, the promoter regions -1,243/-1,188 and -988/-932 of the hTAOK1 gene do contain such sequences at the positions -953/-940 and -1,225/-1,206 of the template strand. To determine whether the AGGTCA motif located at the positions -953/-940 and -1,225/-1,206 of the template strand are associated with the binding of hCOUP-TFI, the AGGTCA sequences at these positions were mutated to AAAAAA, and their interaction with hCOUP-TFI in the yeast cells was tested. To determine whether the AGGTCA motif located at the positions -953/-940 and -1,225/-1,206 of the template strand are associated with the binding of hCOUP-TFI, the AGGTCA sequences at these positions were mutated to AAAAAA, and their interaction with hCOUP-TFI in the yeast cells was tested. The yeast one-hybrid assay showed that the mutations on both half-sites within the region -1,243/-1,188 were necessary to abolish the interaction with hCOUP-TFI, whereas the mutations on either one of the half-sites within the region -988/-932 were enough to abolish the interaction, suggesting that both imperfect direct repeats of AGGTCA sequence located at positions -1,225/-1,206 and -953/-940 were able to bind hCOUP-TFI, but may have interacted in different ways. If the binding of COUP-TFI to the AGGTCA sequences of the promoter region -1,243/-932 decreases the transcriptional activities of hTAOK1, the mutations on these DNA sequences should interfere with the repressive effect of hCOUP-TFI in the promoters. Therefore, mutations were introduced into these imperfect direct repeats of AGGTCA motif located at regions -1,225/-1,206 and -953/-940 and equal molecular amounts of the resultant constructs were used to transfect HeLa cells. The SEAP activity of the transfected cells with mutations on nucleotides -953/-940, indicating that the mutations counteracted the repressive effect of the promoter region -1,243/-932. Moreover, the overexpression of hCOUP-TFI caused a decrease in the transcriptional activity of the promoter region -1,243/-1 down to the background level. Both yeast one-hybrid and SEAP assays indicated that the binding of hCOUP-TFI to the imperfect direct AGGTCA sequences of the promoter region -1,243/-932 may be associated with the repression in hTAOK1 promoter activity.

並列關鍵字

SEAP ； hTAOK1 ； Ste20p ； Promoter ； hCOUP-TFI ； Yeast one-hybrid assay

參考文獻

Alberghina, L. and A. M. Colangelo (2006). "The modular systems biology approach to investigate the control of apoptosis in Alzheimer''s disease neurodegeneration." BMC Neurosci 7 Suppl 1: S2.

Bagrodia, S. and R. A. Cerione (1999). "Pak to the future." Trends Cell Biol 9(9): 350-5.

Benn, S. C. and C. J. Woolf (2004). "Adult neuron survival strategies--slamming on the brakes." Nat Rev Neurosci 5(9): 686-700.

Blinder, D., S. Bouvier, et al. (1989). "Constitutive mutants in the yeast pheromone response: ordered function of the gene products." Cell 56(3): 479-86.

Bourne, H. R., D. A. Sanders, et al. (1990). "The GTPase superfamily: a conserved switch for diverse cell functions." Nature 348(6297): 125-32.

國際替代計量

COUP-TFI抑制人類TAO kinase1啟動子活性的結構與功能關係的研究

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