Klf10為Krüppel-like factors家族的成員之一，能夠以其鋅指的結構與基因啟動子結合並扮演一轉錄調控的角色。先前的研究指出，Klf10可以模擬TGF-造成上皮細胞抑制增生與細胞凋亡的現象，顯示其對TGF-訊號傳導有其重要性；利用ChIP-chip探尋可能為Klf10下游相關基因，其中Sei-1為目標基因之一，Klf10可能透過結合在其啟動子而調控其轉錄。已知Sei-1可藉由與CDK4/Cyclin D 和E2f1/DP1的交互作用來調控細胞週期，而異位性表現Sei-1可以活化p21基因係經由p53機制而抑制細胞的生長；除此之外，Sei-1基因剔除鼠其胰島數目顯著有減少的情形。
本論文首先利用ChIP-chip(Chromatin immunoprecipitation-chip )與ChIP-PCR確認Klf10確實與Sei-1 啟動子上游的Sp1/klf 結合位有交互作用，並陸續以RT-PCR、西方墨點法(Western blot)和啟動子活性分析(promoter activity assay)，觀察兩者之關係，結果顯示當在細胞中大量表現Klf10其Sei-1表現量亦隨之增加，其中在啟動子活性分析實驗中經點突變Klf10結合在Sei-1啟動子的位置，能夠使Sei-1啟動子的活性恢復至正常水準，再次顯示Klf10對於促進Sei-1的重要性；另有研究指出，Sei-1對於p53並非影響其轉錄層面，而是藉由穩定其蛋白質而進一步促進p21表現。另一方面，Klf10促進p21表現已知亦非經由p53或結合在其啟動子上。我們進一步在細胞以Klf10與Sei-1 shRNA，RT-PCR實驗進行分析結果顯示Klf10係藉由調控Sei-1表現而影響p21的轉錄。有鑑於Klf10與Sei-1此二基因皆對於胰臟有影響，在實驗中最後利用免疫組織化學染色觀察小鼠胰臟，結果顯示Klf10基因剔除鼠Sei-1表現量較少，且其胰島面積與數目較正常小鼠相對減少，結果有如Sei-1基因剔除鼠。綜觀上述實驗結果，Klf10可能透過調控Sei-1進而影響相關組織的生理功能。

Klf10 is a group of Krüppel-like transcription factors which generally bind to the promoter of target genes through the zinc-finger structure, and regulate their transcription. Previous study has indicated that increased intracellular levels of Klf10 mimic the anti-proliferative and apoptotic effects of TGF-β1 on epithelial cell growth, suggesting that Klf10 is an important factor for mediating TGF-β1 signaling. From ChIP-chip, which have been adopted to elucidate the novel target genes and signaling cascades of KLF10, Sei-1 is one of the target genes that may be regulated by Klf10 through promoter binding. The Sei-1 is a nuclear factor that implicated in cell cycle regulation through interaction with CDK4/CyclinD and E2F-1/DP-1 complexes. Ectopic expression of Sei-1 protein results in activation of the p21 gene and inhibition of cell growth via p53 mechanism; moreover, its knockout mice show a significant decrease in the amount of pancreatic β-islet.
In this study, the ChIP-chip and ChIP-PCR were adopted to confirm whether Klf-10 directly binds to the Sp1/Klf binding sites on the upstream of Sei-1 promoter. Moreover, we also conducted RT-PCR, Western blotting, and promoter activity assay to investigate the regulation of Sei-1 by Klf10. Results demonstrated that Klf10 acts as a transcriptional activator on Sei-1 promoter where overexpression of Klf10 induces Sei-1 mRNA and protein expression in cells. After co-transfected with Klf10, Sei-1 promoter activity was significantly increased in which nearest binding site mutation showed loss of promoter activity. Results again suggested an important activating role of Klf10 on Sei-1 promoter. It has been known that Sei-1 is the key factor in cell cycle, it is unlikely to regulate p53 expression at the transcriptional level, and that it up-regulates the p53 activity probably by protein stabilization of p53, which in turn activates the p21 gene. Interestingly, Klf10 has been known as a dose-dependently activated p21 transcription that was independent of p53 and Sp1/Klf binding sites in p21 promoter. We attempted to confirm the presence of p21 using RT-PCR assay and Sei-1 shRNA in synchronized cells, which suggests that Klf10 activates p21 transcription indirectly through regulating the activity of Sei-1 promoter. In light of the influential effect of these two genes on pancreas, we compare the phenotype of pancreas between wild type and Klf10 knockout mice in the end of our study. Immunohistochemistry staining results indicated lower Sei-1 expression level and decreases in the number and amount of β-islet, as in the Sei-1 knockout mice, in our Klf10 knockout mice. Taken together, my experiments conclude that Klf10 up-regulates Sei-1 and thus affects related physiological functions.

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