| 摘要: | 在早期胚胎發育中，母源-子源轉換期 (MZT) 是一個關鍵過程，其中涉及兩個主要 事件:合子基因組的活化 (ZGA) 和母體成分的降解。在果蠅胚胎中，轉錄因子 Zelda (Zld) 被認為是啟動 ZGA 的關鍵活化因子，在 MZT 期間 Zelda 扮演著多重角色，主要 為 (1) 作為強大的活化因子直接活化 ZGA，(2) 作為先鋒因子，增強染色質的可及性， 助益其他轉錄因子與基因體的結合，進而促進轉錄作用。先前針對早期果蠅胚胎，藉 由染色質免疫沉澱定序 (ChIP-seq) 和微球菌核酸酶定序 (MNase-seq) 的分析已經確認 Zelda 作為先鋒因子調節染色質景觀的能力。目前，我們探討了 Zelda 是否有潛力在已 分化的果蠅細胞中重新編程轉錄組，我們利用 Bac-to-Bac 表達系統在果蠅晚期胚胎細 胞 S2 細胞中異位表達 Zelda，由 RNA-seq 數據顯示 Zelda 能夠重新激活大約 75%的早期 基因，以及成體幹細胞的標記，證明了 Zelda 重新編程的潛力，在本研究中，我進一步 探討 Zelda 是否能夠在 S2 細胞中重塑其染色質景觀。
首先我優化了重組桿狀病毒的產量以及 Zelda 在 S2 細胞中的表達效率，使重組桿 狀病毒的感染能力增加約 100 倍。其次，我進行了轉座酶可及染色質測序 (ATAC-seq) 以檢查 Zelda 對 S2 細胞染色質的影響，並與先前實驗室結果和文獻比較，研究染色質 開放性與基因表達、Zelda 結合等等的相關性。整體而言，於對照組及實驗組中分別發 現了 62,414 和 28,275 個開放區域，將 Zelda 對染色質的影響可大致分為三類: (1) 原本 較封閉的區域在表達 Zelda 後變得更加開放與集中，開放程度與 Zelda 結合強度呈現正 相關，並與 ATAC 峰值附近的上調基因相關，這表示 Zelda 有能力在 S2 細胞中增加染 色質的可及性。 (2) 原本開放、且鄰近基因激活的區域，因 Zelda 的存在而變得封閉， 推測這些區域具有較高的可及性，致使 Zelda 與這些區域結合。有趣的是 Zelda 的結合 導致染色質變得封閉且基因下調，是否此為 Zelda 的直接影響還需進一步的研究。 (3) 無論 Zelda 存在與否都沒有變化的區域。未來進一步分析三個類別之間的差異可能會提 供更多有關 Zelda 功能的解析，並能定義出染色質受 Zelda 影響的特徵。;In early embryonic development, the maternal-to-zygotic transition (MZT) is a crucial process, which involves two main events, the zygotic genome activation (ZGA) and the degradation of maternal components. In Drosophila embryos, the transcription factor Zelda (Zld) is known as the key activator to initiate ZGA. During MZT, Zelda serves multiple roles, mainly (1) as a strong activator that directly triggers ZGA, (2) as a pioneer factor, which enhances chromatin accessibility, thus facilitating the binding of the other transcription factors and promoting transcription. Previous analyses using Chromatin Immunoprecipitation- Sequencing (ChIP-seq) and Micrococcal Nuclease-Sequencing (MNase-seq) in early Drosophila embryos, have confirmed its role as a pioneer factor in regulating the chromatin landscape. Currently, we asked whether Zelda has the potential to reprogram the transcriptome in differentiated Drosophila cells. We were able to use the Bac-to-Bac expression system to ectopically express Zelda in the Drosophila late-stage embryonic cells, S2 cells. RNA-seq data revealed that Zelda could re-activate approximately 75% of early genes, along with markers of adult stem cells, indicating a potential for reprogramming. In my study, we asked whether Zelda is able to remodel chromatin landscape in S2 cells.
I first optimized the yield of recombinant baculovirus and the efficiency of Zelda expression in S2 cells, by attaining approximately 100-fold increase in infectivity. Secondly, I conducted ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to examine the effects of Zelda on chromatin in S2 cells, and investigated its correlation to the expression and Zelda binding profiles from previous lab results and literatures. Overall, 62,414 and 28,275 open regions were recovered in the control group and experimental group, respectively. The effects of Zelda on chromatin could be categorized into three classes. (1) Regions that were originally more closed became more open and concentrated in the presence of Zelda. The openness was positively correlated with the strength of Zelda binding and associated with up-regulated genes nearby the ATAC peaks. This suggested that Zelda has the ability to increase chromatin accessibility in S2 cells. (2) Regions that were originally open and associated with gene expression became closed within Zelda expression. Zelda may bind to these regions due to their high-accessibility. Intriguingly, Zelda binding led to more closed chromatin and down-regulated genes. Whether it is the direct effect of Zelda requires further investigation. (3) Regions that were unchanged with or without the presence of Zelda. Further analysis of the differences among the three classes may provide more insights of Zelda function and define Zelda-responsive features of chromatin. |
