| 摘要: | 自然界幾乎所有生物的胚胎發育初期是由母體提供的物質進行調控，合子基 因則不表現，直到 maternal-to-zygotic transition（MZT）時期，母源成分開始降 解，數千個早期合子基因被激活，稱之為 zygotic genome activation（ZGA）。ZGA 的啟動一直是一個重要的議題，黑腹果蠅（Drosophila melanogaster）的轉錄因子 Zelda（Zld）是最先被發現的 ZGA master regulator。Zld 透過辨認、結合 CAGGTAG 或相關序列（TAG sites）激活早期合子基因組（Liang et al., 2008; ten Bosch et al., 2006），然而 Zelda 的分子機制仍未完全瞭解。先前染色質免疫沉澱測序（ChIP seq）在果蠅胚胎初期偵測到超過上萬個 Zld 與染色質結合的區域（Harrison et al., 2011），然而僅有上千個合子基因被 Zld 激活，顯示僅有部分 Zld 結合區域是具 功能性的增強子，如何區分並定位 Zld-dependent 增強子及定量其活性仍有待研 究。 近年一項名為 Self-transcribing active regulatory region sequencing（STARR seq）的新技術，能同時將全基因組中的增強子定量、定序且分析其活性。Arnold 等人以果蠅之 Schneider 2（S2）細胞進行 STARR-seq 分析（S2-STARR-seq）， 並以 DHS-seq、FAIRE-seq 以及 ChIP-seq 證實此系統的有效性既正確性（Arnold et al., 2013）。為了定位果蠅全基因組 Zld-dependnet 增強子及其活性，本研究（1） 先以 Bac-to-Bac 系統使 S2 細胞異位表達 Zld，再評估以此為平台進行 STARR- ii seq 的可行性[S2（+Zld）-STARR-seq]。（2）我們選取 2 個 Zld 調控之早期軸向 發育基因 zen 的增強子（4zld、VRE），及 2 個位於 Act5c 基因，在先前 S2-STARR seq 分析中具增強子活性的片段（Act5c2、Act5c3），克隆到包含核心起動子及 GFP reporter 的 STARR-seq 載體中。Arnold 等人的研究發展了 3 種不同核心啟動 子之 STARR-seq 載體，分別是早期發育相關之啟動子 Fly 和 pnr，以及一管家基 因啟動子 RpS12，本實驗測試了這 3 種載體與 4 個增強子的組合。（3）先後以 Zld 重組桿狀病毒感染及 STARR-seq 轉染的 S2 細胞，最後藉由 RT-qPCR 比較 在 S2 細胞中 Zld 的存在與否對增強子活性之影響。 我們的研究結果顯示原本在 S2-STARR-seq 分析中不具活性的 4zld 與 VRE， 在表現 Zld 的 S2 細胞中足以驅動報導基因的表現，亦即為有活性的增強子。此 外，不同的增強子片段對核心啟動子有偏好，4zld 及 VRE 與早期發育相關的 Fly 或 pnr 啟動子的組合展現較大的活性。因此建議未來 S2（+Zld）-STARR-seq 的 研究以 Fly 為啟動子進行，期能鑑識果蠅全基因組增強子以及對應活性，進一步 找出功能性增強子的序列特徵。;In most organisms, early embryonic development is regulated by mRNA and protein provided by the mother. No transcription occurs until the maternal-to-zygotic transition（MZT）. During MZT, there are two events: the degradation of maternal components, and the activation of zygotic genome（ZGA）. The first discovered master regulator which initiates ZGA is Zelda（Zld）in Drosophila melanogaster （Liang et al., 2008）. Zld activates early zygotic genome through the TAG sites （CAGGTAG and related motifs）enriched in the upstream of early genes（ten Bosch et al., 2006）. In the previous studies, chromatin immunoprecipitation sequencing（ChIP-seq）with anti-Zld antibody detected more than ten thousand of Zld-bound regions in early embryos（Harrison et al. al., 2011）. However, just a thousand of zygotic genes or more were Zld-dependent, suggesting only partial bound regions are functional. How to map these enhancers and determine their activities requires more studies. A new technology, self-trancribing active regulatory region sequencing （STARR-seq）, could simultaneously map and quantify enhancer activities in the whole genome scale. Arnold et al. performed STARR-seq analysis in Drosophila S2 cells（s2-STARR-seq）and the feasibility of this system was validated by DHS-seq, FAIRE-seq, and ChIP-seq（Arnold et al., 2013）. Utilizing this approach, we aimed to identify genome-wide functional Zld-dependent enhancers in S2 cells. (1) Since iv Zld is not expressed in S2 cells, we first ectopically expressed Zld in S2 cells by Bac to-Bac system. (2) We tested two Zld-responsive enhancers of zen（4zld, VRE）and two Act5C enhancers（Act5c2, Act5c3）as positive controls to test the possibility to perform STARR-seq in S2 cells ectopically expressing Zld [S2（+Zld）-STARR seq]. We also tested three STARR-seq-vectors containing different core promoters, including development-related promoters, Fly and pnr, and a housekeeping gene promoter, RpS12. (3) The strength of enhancer-driven GFP in the presence or absence of Zld in S2 cells was tested by RT-qPCR. Our results showed that with the help of Zld, the activity of 4Zld and VRE had significant increased, indicating that Zld-dependent enhancers are able to drive GFP expression with the presence of Zld. Also, developmental enhancers showed preference for developmental core promoters. Experiment timeline and conditions were suggested. This platform will be further used to perform whole-genome S2 （Zld）-STARR-seq analysis, for identifying functional Zld-dependent enhancers, in the hope to investigate their features corresponding to activities |
