以桿狀病毒載體系統 建構與異源表達果蠅Zelda基因及其功能分析;Construction and ectopic expression of Drosophila Zelda using baculovirus system for functional analysis

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Title:	以桿狀病毒載體系統建構與異源表達果蠅Zelda基因及其功能分析;Construction and ectopic expression of Drosophila Zelda using baculovirus system for functional analysis
Authors:	康致甄;Kang, Chih-Chen
Contributors:	生命科學系
Keywords:	果蠅早期發育;MZT;Drosophila;ZGA;Zelda;pioneer factor
Date:	2022-06-27
Issue Date:	2022-07-13 18:18:07 (UTC+8)
Publisher:	國立中央大學
Abstract:	大多數動物早期胚胎發育是由母源基因產物來支持與調控，這些產物在卵子生成的過程中生成並寄存於卵子內。而合子基因組則維持不轉錄狀態，直到母源-子源轉換期 (maternal-to-zygotic transition, MZT) 才開始被激活表現，稱為zygotic genome activation (ZGA)，此時胚胎發育轉由合子基因組接管調控。在果蠅的MZT期間，初期約500-900個基因被激活，隨後約有2000-3000個基因激活[1, 2]。先前研究發現一母源轉錄因子Zelda在果蠅MZT時期扮演關鍵的角色，經由結合特定序列 (CAGGTAG及相關位點)，激活多數的早期基因。目前已知Zelda的作用方式包括: 1. 以轉錄激活因子活化目標基因，2. 增加染色質開放程度，以利其他轉錄因子與加強子 (enhancer) 結合並促進轉錄[2, 3]，3. 降低核小體的屏障[2, 4]，因此Zelda被視為先鋒因子(pioneer factor)。然而，其結構與分子功能機制仍未明朗，Zelda蛋白的表現與純化的困難更增加研究上的挑戰。本實驗主要目的是利用桿狀病毒表現系統為工具，於果蠅胚胎晚期的細胞株Schneider 2 (S2) cells異位表達6xHis-tagged Zelda 蛋白，以期未來得以Cryo-EM作結構分析、分子功能區 (functional domains) 分析、及交互因子的研究。除了重組病毒的建構、病毒增殖及轉染、Zelda蛋白表現等各項條件的探討，本實驗並以RNA-seq及qRT-PCR檢視轉染異位表達Zelda對S2 cells基因組的影響。結果顯示大量基因 (5588) 表現量上升，僅有少部分下降，表現量上升的基因與文獻1到3小時受Zelda影響的基因相互比較[3]，有463個基因重疊 (50%)，特別是MZT初期表現基因。將表現量增加的基因按表現差異倍率排序，前300個基因進行基因功能分類分析，軸向發展相關基因佔多數，細胞化、細胞週期運轉、性別決定相關的基因亦包含在內，顯示異位表達Zelda可以重新激活這些早期胚胎發育基因，此結果呼應先前推測Zelda具有重塑染色質 (chromatin remodeling) 與細胞重新編程 (reprogramming) 的潛力。利用果蠅成蟲幹細胞標記初步探討異位表達Zelda是否足以使已分化細胞重新編程，結果發現其相關基因表現量皆有上升。綜合本研究及目前已知的斑馬魚MZT關鍵因子與誘導多能幹細胞 (induced pluripotent stem cells, iPSCs) 之核心誘導因子[5]，顯示以先驅因子重塑染色質，促進相關基因組表現，可能為細胞重新編程的策略之一。 ;In all animals, the initial embryonic development is controlled by the maternal gene products, while zygotic gene expression remains silent until later time point at maternal to zygotic transition (MZT). During MZT, a major proportion of the maternal components is removed and the zygotic genome is activated (ZGA). During Drosophila MZT, about 500-900 genes are first activated (minor wave), followed by a major wave of gene expression (around 2000-3000 genes)[1, 2]. A maternal transcription factor, Zelda, was reported as the key activator of early ZGA[2, 4]. Zelda, is not only a strong activator but also acts as an epigenetic regulator which opens chromatin during MZT through a specific motif (CAGGTAG and its related sites), reduces nucleosome barriers and in turn facilitates chromatin accessibility[2, 3]. In that, Zelda is considered as a pioneer factor. However, the underlined molecular and structural functions of Zelda remain unclear. The difficulties to robustly express and purify Zelda protein make it even more challenging. In this study，we successfully induced 6xHis-tagged full length Zelda proteins in Schneider 2 (S2) cells using the Bac-to-Bac expression system. S2 cells are derived from the late-stage Drosophila embryos and do not express Zelda. The conditions and timeline for virus production and transfection were optimized. Resulted Zelda protein will be subjected to future Cryo-EM analysis, functional domain dissection, interactor screening and global enhancer mapping by STARR-seq. We also applied RNA sequencing (RNA-seq) and qRT-PCR to characterize the effects of ectopically expressed Zelda on the gene expression of S2 cells. A large number of genes (5588) have been up-regulated. 463 up-regulated genes were overlapped with Zelda-dependent targets (50%) expressed in embryo during MZT[3]. Most of them are related to embryonic morphogenesis. Moreover, axis patterning genes were enriched in GO term analysis of top 300 up-regulated genes. Cellularization, cycle regulators, and sex determination related genes were also included. This result suggested that ectopically expressing Zelda can reactivate early embryonic development genes, and have the potential to reprogram differentiated cells. In fact, Drosophila adult stem cell markers were up-regulated by Zelda in S2 cells[5]. In all, Zelda, the key MZT factors of Zebrafish and iPSC (induced pluripotent stem cells) core reprogramming factors in mamalians share a similar feature as the pioneer factors which remodel the chromatin landscape and collectively and specifically activate subsets of genes, hinting a paradigm strategy to induce the pluripotency of cells.
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