blmp-1抑制ced-9轉錄作用促進線蟲尾突細胞凋亡

Abstract

在動物體的發育過程中，計畫性細胞凋亡是一個具有高度保留性的過程。在線蟲當中，當egl-1/BH-3-only gene的轉錄作用啟動，會促使大部分的體細胞進行細胞凋亡，然而在尾突細胞中，會透過另一條途徑活化計畫性細胞凋亡。目前已知尾突細胞會透過PAL-1，這個轉錄因子結合至ced-3的啟動子，促使ced-3轉錄，使尾突細胞進行計畫性細胞凋亡，而這個過程證明細胞凋亡的啟動可以是和egl-1無關。然而，在沒有egl-1的時候如何調控細胞死亡的時間，其機制不太清楚。根據我們實驗室先前的研究發現，BLMP-1，一個鋅手指轉錄抑制因子，會參與在尾突細胞凋亡當中。同時發現在尾突細胞死亡前BLMP-1才開始表現，這表示blmp-1的表現時間點是被嚴格控制的，而且在尾突細胞凋亡中扮演重要的角色。為了瞭解blmp-1在計畫性細胞凋亡過程當中所扮演的角色，我們尋找其下游的因子。藉由遺傳分析發現，在blmp-1突變中，尾突細胞的異常存活需要ced-9/bcl-2。此外，利用dGFP在ced-3(n717)突變中的表現，ced-3(n717)是讓細胞死亡有缺陷的突變，讓尾突細胞得以存活下來讓我們觀察，我們發現ced-9在尾突細胞死亡之前開一直表現。而在ced-3(n717); blmp-1(RNAi)突變中，ced-9持續表現。同時我們也發現兩個(Pax)轉錄因子，會在生殖細胞促進ced-9的表現來保護細胞免於細胞凋亡，但是不參與在blmp-1所調控的尾突細胞凋亡當中，這意味著，blmp-1有可能是直接調控ced-9的轉錄，而ced-9的啟動子也發現具有blmp-1的潛在結合序列。我們利用electrophoretic mobility shift assay (EMSA)來驗證是否BLMP-1鋅手指蛋白能夠結合到ced-9啟動子上，而我們發現BLMP-1鋅手指蛋白能夠結合到ced-9啟動子來調控尾突細胞的凋亡。DRE-1是一個在SCF(Skp1-Cullin-F-box) ubiquitin連接酶複合物中的F-box蛋白，DRE-1已經被研究會透過ced-9促進尾突細胞凋亡，而我們利用遺傳分析證明dre-1和blmp-1透過平行的路徑促進尾突細胞的死亡。綜合上述的結果，我們知道blmp-1在核心細胞凋亡路徑中的位置，而我們的研究結果提供了一個時間上經由ced-9調控計畫性細胞凋亡的機制。

Programmed cell death (PCD) is a conserved cellular process during animal development. In Caenorhabditis elegans (C. elegans), transcriptional up-regulation of egl-1/BH-3-only gene promotes most somatic cell deaths, but tail-spike cell uses an alternative mechanism to initiate PCD where PAL-1, a transcription factor, binds to ced-3 promoter and re-initiates ced-3 transcription to promote tail-spike cell death, demonstrating that cell death onset could be egl-1-independent. Yet, little is understood about the temporal control mechanism of cell death in the absence of egl-1. Previously, our lab found that BLMP-1, a zinc finger transcriptional repressor, is required for the tail-spike death. The expression of BLMP-1 in tail-spike cell was observed just before its death, indicating that the timing of blmp-1 expression is tightly controlled and important for tail-spike cell death. To understand the role of blmp-1 in PCD, we looked for its downstream effectors. Our genetic analysis revealed that the abnormal survival of tail-spike cell in the blmp-1 mutant required ced-9/bcl-2. In addition, using dGFP transcriptional reporter in ced-3(n717) mutants as a death defective background which allow tail-spike cell to survive, we found that ced-9 is expressed in tail-spike cells just before its death. Moreover, ced-9 transcription persists in the tail spike cell in ced-3(n717); blmp-1(RNAi). These data indicated that ced-9 was repressed by blmp-1. While two paired box (Pax) transcription factors, pax-2 and egl-38, promote ced-9 transcription to protect cell from death in germline cells, we found that they were not involved in blmp-1-mediated tail-spike cell death, raising the possibility that blmp-1 may regulate ced-9 transcription directly. In support of this, the ced-9 promoter contains a potential BLMP-1 binding site. We employed electrophoretic mobility shift assay(EMSA) to test if BLMP-1 is able to bind to ced-9 promoter, we found that BLMP-1 Zn finger protein is able to bind to ced-9 promoter to mediate tail-spike cell death. DRE-1, the F-box protein of SCF (Skp1-Cullin-F-box protein) ubiquitin ligase complex, is previously identified to promote the tail-spike death through ced-9. Our epistasis analysis suggested that dre-1 acts in parallel of blmp-1 to promote the tail-spike cell. Taken together, we have characterized the position of blmp-1 in the core program cell death pathway. Our results provide a temporal regulatory mechanism of PCD through ced-9.