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 Y Y1 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。
Roles of Notch signaling in the transcriptional contr ol 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 interactio n between two neighboring cells. After ligand binding Notch receptors are cleaved to release their intrace﹐ llular domains. Then the Notch intracellular domain (NIC) translocates from cytoplasm into nucleus and in teracts 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-in dependent pathway but that is poorly characterized at present.
In the previous study the Notch1 intracellular domain (N1IC) was demonstrated to associate with multifu﹐ nctional transcription factor YY1 to suppress the CBF1-dependent Notch signaling pathway. Based on thes e data it is possible that Notch signaling may be involved in the transcriptional control of YY1 via the ass﹐ ociation 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 exp ression 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-M﹐ yc 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.
