異位表達 Zelda 對 S2 細胞染色質景觀重塑的影響

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許芯恩(Hsin-En Hsu) 查詢紙本館藏

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異位表達 Zelda 對 S2 細胞染色質景觀重塑的影響
(The effect of ectopically expressing Zelda on remodeling chromatin landscape in S2 cells)

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至系統瀏覽論文 (2029-5-29以後開放)

摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 果蠅
★ 染色質可及性
★ 早期胚胎發育
★ 先鋒因子

關鍵字(英)

★ MZT
★ Drosophila
★ ATAC-seq
★ Zelda
★ pioneer factor
★ chromatin accessibility

論文目次

中文摘要 i
Abstract ii
誌謝 iv
目錄 v
圖目錄 ix
表目錄 xi
中英文對照表 xii
壹、前言 1
1.1 黑腹果蠅早期胚胎發育 1
1.2 早期胚胎發育的基因表現 2
1.2.1 母源-子源轉換期 (Maternal-to-zygotic transition, MZT) 2
1.2.2 合子基因組激活 3
1.3 黑腹果蠅轉錄因子Zelda與其功能 5
1.3.1 Zelda的發現 5
1.3.2 Zelda調控果蠅早期胚胎發育 6
1.3.3 Zelda影響母源軸向基因的作用 8
1.3.4 Zelda具有先鋒因子 (pioneer factor, PF) 的特性 9
1.4 Zelda對其他細胞的影響 10
1.4.1 Zelda對type II NB分化的影響 10
1.4.2 Zelda對胚胎晚期已分化細胞的影響 11
1.5 先鋒因子與重新編程 (reprogramming) 的關聯 12
1.6 ATAC-seq/MNase-seq介紹與區別 14
1.7 桿狀病毒表達系統Bac-to-Bac expression system 14
1.8 Self-transcribing active regulatory region sequencing (STARR-seq) 介紹 16
1.9 研究動機與目的 17
貳、實驗材料與方法 19
2.1 實驗材料 19
2.1.1 菌種與質體 19
2.1.2聚合酶連鎖反應引子 (Polymerase chain reaction primers) 20
2.1.3 細胞株 21
2.1.4 果蠅品系 21
2.2 實驗方法 21
2.2.1 質體抽取 (Alkaline lysis) 21
2.2.2 桿狀病毒質粒 (Bacmid) 抽取 21
2.2.3 果蠅genomic DNA (gDNA) 抽取 22
2.2.4 利用超音波細胞粉碎儀 (Ultrasonic processor) 將果蠅gDNA震斷並純化 23
2.2.5 DNA膠體電泳 (DNA gel electrophoresis) 23
2.2.6 電泳膠回收DNA 23
2.2.7 Sf9細胞培養 24
2.2.8 S2細胞培養 24
2.2.9 將bacmid DNA轉染 (transfect) 至Sf9細胞生產P0重組桿狀病毒 24
2.2.10 重組桿狀病毒擴增 (Baculovirus amplification) 25
2.2.11 半數細胞感染劑量 (50% tissue culture infective dose, TCID50) 26
2.2.12 在S2細胞中異位表達Zelda 26
2.2.13 將感染zld重組桿狀病毒的S2細胞進行sorting 27
2.2.14 ATAC-seq library製備 27
2.2.15 STARR-seq library製備 29
2.2.16 傳統法之大腸桿菌勝任細胞 (Competent cell) 製作 33
2.2.17 電穿孔法之大腸桿菌勝任細胞 (Competent cell) 製作 33
2.2.18 電菌及細菌轉形作用 (Electroporation and Transformation) 33
2.2.19 以限制酶切割 (Enzyme digestion) 確認Library DNA大小 34
2.2.20 收集STARR-seq library colony並製備stock 34
2.2.21 RNA-seq數據分析 34
2.2.22 ATAC-seq數據分析 35
2.2.23 CHIP-seq數據分析 35
參、實驗結果 36
3.1 以桿狀病毒表達系統異位表達Zelda 37
3.1.1 將bacmid轉染至Sf9細胞以製備P0-P2重組桿狀病毒 38
3.1.2 優化P3重組桿狀病毒的產率 38
3.1.3 計算多重感染複數 (multiplicity of infection, MOI) 39
3.2 測試不同MOI之重組桿狀病毒感染S2細胞之效率 39
3.3 以qRT-PCR確認PCR擴增的ATAC-seq library tagmented DNA片段 42
3.4 未感染的S2與感染Zelda重組病毒的S2細胞之RNA-seq分析 43
3.4.1 RNA-seq數據的前處理 44
3.4.2差異基因表現分析 (Differential expression analysis) 47
3.4.3 異位表達Zelda的S2細胞與早期胚胎的基因表現比較 47
3.5 Zelda對S2細胞染色質區域的影響 50
3.5.1 ATAC-seq數據的前處理 50
3.5.2以散點圖與箱型圖呈現Zelda對S2細胞中染色質區域的影響 51
3.5.3 Zelda對染色質開放程度的影響 51
3.5.4 染色質開放程度與基因表現之關係 52
3.6 染色質開放程度與Zelda結合位點的關聯 54
3.7製備STARR-seq library 58
3.7.1準備pSTARR-fly載體 58
3.7.2 製備果蠅gDNA片段 60
3.7.3 library的製備 60
3.7.4 測試不同條件下製備STARR-seq library 61
3.7.5 以EcoRI與HindIII切割單一菌落之質體 64
肆、實驗討論 67
4.1 於S2細胞中異位表達Zelda桿狀病毒之劑量 67
4.2 以TMM進行RNA-seq數據的標準化處理 67
4.3 異位表達Zelda的S2細胞中的基因表現 67
4.4 於異位表達Zelda的S2細胞中的早期基因與胚胎中的差異 68
4.5 染色質總體開放程度在異位表達Zelda的S2細胞中降低 69
4.6 未來研究與應用 70
參考資料 71
附錄 77
附錄一於S2細胞中異位表達Zelda的染色質開放程度與Zelda結合位點的關聯，以heatmap呈現 77

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指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2024-6-13

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