透過免疫共沉澱和質譜分析鑑定 Zelda 輔助因子;Identification of Zelda co-factors by co-immunoprecipitation and mass-spectrometry analysis

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题名:	透過免疫共沉澱和質譜分析鑑定 Zelda 輔助因子;Identification of Zelda co-factors by co-immunoprecipitation and mass-spectrometry analysis
作者:	黃鈺婷;Huang, Yu-Ting
贡献者:	生命科學系
关键词:	果蠅;母源轉錄因子 Zelda;早期胚胎發育;蛋白質間交互作用;Zelda輔助因子;Drosophila;maternal transcription factor Zelda;early development;Protein-protein interaction;Zelda cofactor
日期:	2024-05-02
上传时间:	2024-10-09 15:25:58 (UTC+8)
出版者:	國立中央大學
摘要:	動物早期胚胎發育的控制首先由母體產物（mRNA和蛋白質）所主導，直到母源子源轉換期（maternal to zygotic transition, MZT），胚胎發育轉為由合子基因調控。因此，在MZT期間有兩個主要事件：（1）母體成分的降解；（2）合子基因組的活化（zygotic genome activation, ZGA）。在果蠅的早期發育中，第一波的ZGA大約有500個合子基因被活化，第二波大約有2000個合子基因被活化。在這個過程中，母源轉錄因子 Zelda（Zld）扮演著關鍵角色。經過多數研究顯示Zelda不僅是一種轉錄活化因子（transcriptional activator），還是一種表觀遺傳調節因子（epigenetic regulator），能夠在MZT期間打開特定區域的染色質，促進基因組的活化。儘管我們瞭解其功能，但對於Zelda的功能性結構與分子機制依然尚未完全釐清，特別是Zelda是否能直接與轉錄機制或表觀遺傳調節因子相互作用，或是通過輔助因子發揮作用仍然未知。為了瞭解Zelda的分子機制，實驗室先前利用桿狀病毒表現系統，在昆蟲細胞中異位表現帶有6xHis-tag的Zelda蛋白，隨後進行了Ni-NTA蛋白純化。以純化的Zelda與果蠅早期胚胎的總蛋白萃取物進行免疫共沉澱（Co-IP），並通過質譜分析（mass spectrometry），鑑定出了134種蛋白質。　　在我的研究中，我改善了純化的方式，使蛋白的產量及純度增加，可供後續Zelda結構的解析，例如Cryo-EM分析。此外為了縮小Zelda候選輔助因子的範圍，由於Zelda作用於染色質，我進一步純化果蠅早期胚胎核蛋白，再重複免疫共沉澱實驗，與質譜分析，將輔助因子的候選範圍縮小至98種。根據 Gene Ontology term（GO-term）analysis和基因表現數據，大多數蛋白質為核蛋白，並在胚胎發育的前兩個小時內表達。未來可針對功能與表觀遺傳和轉錄調控有關的蛋白進行進一步驗證，以便更佳了解Zelda功能和早期胚胎中合子基因組重新編程的機制。 ;Early embryonic development in all animals is first controlled by maternal products until the maternal-to-zygotic transition (MZT). During MZT, there are two major events: degradation of maternal components and zygotic genome activation (ZGA). In the early development of Drosophila, ZGA occurs in two waves. The first wave (minor ZGA) activates approximately 500 zygotic genes, while the second wave (major ZGA) activates around 2000 zygotic genes. In this process, the maternal transcription factor Zelda (Zld) plays a pivotal role. With multiple researches, it has been suggested that Zelda is not only a transcriptional activator but also an epigenetic regulator, capable of opening specific chromatin regions and facilitating genome activation during MZT. However, the structural and molecular mechanisms of Zelda remain incompletely elucidated. Particularly whether Zelda interacts directly with transcriptional machinery or epigenetic regulatory factors, or acts through co-factors, is still unknown. To tackle these questions, our laboratory previously utilized a Bac-to-Bac baculovirus expression system to ectopically express Zelda protein tagged with 6xHis-tag in insect cells, followed by Ni-NTA protein purification. Purified Zelda was used for co-immunoprecipitation (Co-IP) with total protein extracts from early Drosophila embryos, followed by mass spectrometry (MS) analysis. 134 proteins were identified. In my study, I first optimized Zelda production for future Zelda structural study, such as Cryo-EM analysis. Higher yield and more purified proteins were obtained. To narrow down the candidates of Zelda co-factors, I repeated the Co-IP experiments and MS analysis with nuclear protein extracts from early embryos, since Zelda mainly works on chromosome. 98 proteins were recovered. Based on Gene Ontology (GO) term analysis and expression data, most protein were nuclear (cellular component) and expressed in the first two hours of embryonic development. A handful of proteins were functional related to epigenetic and transcriptional regulation. Further verification of these proteins is required and will provide better understanding of Zelda function and the mechanism of reprogramming zygotic genome in early embryos.
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