探討 Notch 訊息傳導路徑對於轉錄因子 YY1 的調控作用所扮演之角色

1. 探討Notch訊息傳導路徑對於轉錄因子YY1的調控作用所扮演之角色探討Notch訊息傳導路徑對於轉錄因子YY1的調控作用所扮演之角色 • Notch訊息傳導路徑可調控許多的細胞活動﹐包括生長、分化、細胞週期、細胞凋亡…等。Notch為細胞膜上之受體蛋白﹐結構為異二聚物的型式﹐Notch受體蛋白與ligand結合後﹐會引發Notch訊息傳導路徑向下傳遞﹐Notch受體經酵素切割﹐而釋放出Notch受體的活化型式(NIC)。NIC由細胞質進入到細胞核中﹐藉由與轉錄因子CBF1結合﹐而結合於特定之DNA序列上﹐藉此調控基因的表現﹐此為CBF1-dependent的Notch訊息傳導路徑﹐但Notch訊息傳導路徑也可能經由CBF1 independent的路徑﹐但其詳細機制目前並不清楚。之前的研究﹐已觀察到Notch1受體的活化型式(N1IC)﹐與轉錄因子YY1具有直接結合作用﹐且YY1會抑制藉由CBF1的Notch訊息傳導路徑。基於以上發現﹐YY1可調控Notch訊息傳導路徑﹐是否Notch訊息傳導路徑同樣可能會影響YY1之生物功能﹐故本論文擬探討N1IC對於YY1調控轉錄作用之過程中﹐是否具有調控的功能。藉由報導基因分析結果發現﹐YY1抑制帶有野生株 YY1結合序列之報導基因的表現﹐此抑制現象會因為N1IC的表現而被逆轉﹐此結果顯示N1IC或許可對YY1下游基因有正向調控的作用。此外﹐藉由oligoprecipitation 及 Chromatin immunoprecipitation (ChIP) 實驗發現﹐N1IC藉由與YY1結合﹐而一起結合於YY1 結合序列上。此外在Notch訊息傳導路徑中﹐很重要的轉錄因子CBF1並沒有參與在此過程中﹐故N1IC對於YY1調控轉錄作用之調控﹐可能是經由CBF1-independent的型式。本論文同時探討N1IC是否會影響YY1下游基因之表現﹐藉由西方墨點法﹐發現內生性之c-Myc蛋白表現﹐會因為N1IC的存在而增加﹐而c-Myc之表現已被報導受YY1所調控﹐藉由報導基因分析結果發現﹐N1IC可提升具有c-Myc啟動子序列的報導基因活性﹐同時藉由ChIP實驗﹐發現N1IC與c-Myc啟動子序列﹐在細胞內有直接的結合關係。綜合以上結果指出﹐Notch藉由與基因啟動子序列之交互作用﹐而影響YY1所調控之基因—cMyc的表現﹐且此作用可能藉由CBF1-independent的型式而達成。

2. Roles of Notch signaling in the transcriptional control of transcription factor YY1 • Notch signaling participates in several cellular processes such as proliferation﹐differentiation﹐cell cycle and apoptosis. Notch is a heterodimeric receptor. Notch signaling is initiated by a receptor-ligand interaction between two neighboring cells. After ligand binding﹐Notch receptors are cleaved to release their intracellular domains. Then the Notch intracellular domain (NIC) translocates from cytoplasm into nucleus and interacts with transcription factor CBF1 on the specific DNA element to regulate target gene expression. This pathway is known as the CBF1-dependent pathway. Genetic evidence points to the existence of a CBF1-independent pathway but that is poorly characterized at present.In the previous study﹐the Notch1 intracellular domain (N1IC) was demonstrated to associate with multifunctional transcription factor YY1 to suppress the CBF1-dependent Notch signaling pathway. Based on these data﹐it is possible that Notch signaling may be involved in the transcriptional control of YY1 via the association between these two proteins. In this study﹐N1IC was shown to activate the activity of reporter gene containing two copies of wild type YY1 response elements. Furthermore, the complex containing N1IC and YY1﹐but not CBF1﹐bound on YY1 response element by oligoprecipitation and Chromatin immunoprecipitation (ChIP) assay. These data indicated that the reporter gene containing YY1-response elements was activated by Notch signaling via a CBF1-independent pathway. And these data also showed the possibility that N1IC could modulate YY1 target gene expression. The expression of endogenous c-Myc was also found to be increased in the presence of N1IC. N1IC promoted the activity of reporter gene containing c-Myc promoter. Furthermore﹐the N1IC-YY1 complex bound on c-Myc promoter by ChIP assay. Taken together﹐these results indicated that the gene expression modulated by YY1 is regulated by Notch signaling via a CBF1-independent manner.