線蟲遠頂細胞遷移中dpy-24的功能探討及其異時性調控

Abstract

細胞遷移在動物發育過程中扮演很重要的角色。在線蟲(Caenorhabditis elegans)雌雄同體中，兩顆遠頂細胞的遷移是很好的研究模型，它們在幼蟲期會進行三個時期不同方向的遷移，牽引成蟲中兩個對稱U型的性腺的形成。先前研究發現，第二時期的背向遷移發生於L3晚期，是由Netrin受器UNC-5的轉錄啟始所啟動。此外，異時性基因也會參與決定遠頂細胞的時間身分：DAF-12/賀爾蒙受器、DRE-1/F-box蛋白質、LIN-29/鋅指轉錄調控子共同促進第二和第三時期的執行。LIN-42/Period(日光週期蛋白質)則防止第二和第三時期過早於L2執行。我們實驗室先前分離並分析了dpy-24突變，發現其會提早DTC第二時期的背向遷移；DPY-24蛋白質是一鋅指轉錄調控子，含有一個PR domain和五個鋅指，只會在遷移的第一時期表現在DTC中，並且會抑制unc-5的轉錄。在進入第二時期時，daf-12、dre-1、lin-29共同作用造成dpy-24表現量的下降。然而，DPY-24如何抑制unc-5的轉錄以及DPY-24的表現量如何被降低仍然未被探討清楚。在我的研究中，透過結構與功能分析，發現DPY-24的PR domain、鋅指、以及其以外的區域對於DPY-24在遠頂細胞中的功能都是重要的。我更進一步發現，DPY-24的鋅指在EMSA (Electrophoretic Mobility Shift Assay)實驗中可直接透過兩個接合位置與unc-5啟動子接合，表示DPY-24可能直接抑制unc-5的轉錄。此外，DAF-12和LIN-29的鋅指也都具有接合到unc-5啟動子的能力，而當daf-12和lin-29都突變後，unc-5的轉錄即消失了，表示DAF-12和LIN-29可能透過接合到unc-5啟動子來直接啟動unc-5的轉錄。至於dpy-24本身的調控，透過表現綠色螢光蛋白的基因轉殖線蟲，偵測dpy-24在各個基因表現階層的變化量，發現DAF-12和LIN-29共同抑制dpy-24的轉錄，而DRE-1則降低DPY-24蛋白質的穩定性；此外，dpy-24表現也會被自己的3’UTR所抑制。最後，我發現dpy-24在第一時期的轉錄需要LIN-42，並且dpy-24會透過正回饋機制維持自己的轉錄活化。這些實驗結果有助於我們了解細胞遷移中的時間調控機制。亦即在遠頂細胞遷移的第一時期，LIN-42啟動dpy-24的轉錄，進而抑制lin-29和unc-5的轉錄來避免遠頂細胞過早的背向遷移。在第二時期，DAF-12和LIN-29在轉錄層次降低DPY-24的表現量，而DRE-1則在後轉譯層次降解DPY-24；這些機制疏解了DPY-24對於unc-5的抑制，並且容許LIN-29和DAF-12啟動unc-5的轉錄，最終導致遠頂細胞的背向遷移。

Cell migration plays an essential role during animal development. In the hermaphrodite of Caenorhabditis elegans, two distal tip cells (DTCs) undergo three sequential phases of linear migration during larval stages and lead the formation of bi-lobed U-shaped gonad arm. Previous studies have shown that the initiation of the ventral-to-dorsal phase Ⅱ migration occurs in the late L3 stage and is controlled by the transcriptional up-regulation of the dorsal guidance receptor UNC-5. On the other hand, the heterochronic genes control the temporal identity of DTCs: DAF-12/nuclear hormone receptor, DRE-1/F-box protein, and LIN-29/zinc finger transcription factor function redundantly to promote phase Ⅱ and phase Ⅲ migration in the L3 stage. In contrast, LIN-42/Period prevents phase Ⅱ and phase Ⅲ to occur precociously in L2. Our laboratory has previously isolated and characterized a dpy-24 mutation, which results in precocious DTC dorsal turn in early L3 stage. Previous studies have shown that dpy-24 represses the transcription of unc-5 to prevent precocious DTC dorsal turn. DPY-24 contains one PR domain and five zinc fingers and is detected in DTCs prior to, but not during or after, dorsal phase Ⅱ migration. In late L3, lin-29, dre-1, and daf-12 function redundantly to down-regulate the dpy-24 level to promote DTC dorsal turn. However, how DPY-24 represses unc-5 transcription and how the DPY-24 level is maintained during phase Ⅰ and is down-regulated during the phase Ⅰ to Ⅱ transition have not been explored. My structural and functional analysis of DPY-24 reveals that the PR and zinc finger domains and the region outside of these domains are required for its complete function in regulating DTC migration. Furthermore, I demonstrated that DPY-24 zinc fingers are able to bind to unc-5 promoter in EMSA (electrophoretic mobility shift assay), suggesting that DPY-24 may repress unc-5 transcription directly. In addition, DAF-12 and LIN-29 each can bind to the unc-5 promoter and when both are mutated, no unc-5 transcription is observed. Therefore, DAF-12 and LIN-29 may activate unc-5 transcription by directly binding to its promoter. As for temporal regulation of dpy-24, I generated transgenic worms carrying dpy-24::GFP reporters and found that DAF-12 and LIN-29 repress dpy-24 transcription, and DRE-1 decreases DPY-24 protein stability. Furthermore, dpy-24 expression is also repressed through its 3’UTR. Finally, dpy-24 transcription during phase Ⅰ is activated by LIN-42 and maintained by a positive feedback loop. These results elucidate the molecular mechanism of temporal regulation during cell migration. In phase Ⅰ, LIN-42 activates dpy-24 transcription, which in turn blocks lin-29 and unc-5 transcription and hence prevents DTC from dorsalward turning. In phase Ⅱ, DPY-24 is down-regulated by LIN-29 and DAF-12 at the transcriptional level and by DRE-1 at the post-translational level, which relieves the repression of unc-5 by DPY-24 and allows LIN-29 and DAF-12 to activate unc-5 transcription, leading to DTC dorsal turn.