測試以異位表達 Zelda 之 S2 細胞為平台進行 STARR-seq 分析 Zelda 依賴增強子活性

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張鈺(Yu Chang) 查詢紙本館藏

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測試以異位表達 Zelda 之 S2 細胞為平台進行 STARR-seq 分析 Zelda 依賴增強子活性
(Testing S2 cells ectopically expressing Zelda as a platform for mapping Zelda dependent-enhancers by STARR-seq)

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至系統瀏覽論文 (2027-9-16以後開放)

摘要(中)

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（ChIPseq）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（STARRseq）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-STARRseq 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。

摘要(英)

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 immunoprecipitationsequencing（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 Bacto-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）-STARRseq]. 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

關鍵字(中)

★ 異位表達
★ 基因表現
★ 增強子

關鍵字(英)

★ Zelda
★ S2 cells
★ STARR-seq
★ enhancer
★ maternal-to-zygotic transition
★ zygotic genome activation

論文目次

中文摘要 i
Abstract iii
致謝 v
中英文對照表 x
一、緒論 1
1. 果蠅早期胚胎發育 1
2. Maternal to zygotic transition（MZT） 3
2.1 母源物質的降解（maternal material degradation） 3
2.2合子基因組的啟動（Zygotic genome activation, ZGA） 4
3. Zelda在MZT之功能 5
3.1 Zelda調控軸向發育 5
3.2 Zelda作為先驅因子克服核小體屏障 7
3.3 Zelda目標基因的類型 9
4. 增強子的鑒定與預測 9
4.1增強子敲擊法 11
4.2 STARR-seq分析 11
4.3 STARR-seq步驟 12
5. zen之增強子與調控 13
6. 以S2細胞作為STARR-seq轉染分析平台 15
7. 研究動機與目的 17
二、實驗材料與方法 18
實驗材料 18
1. 菌株 18
2. Schneider 2（S2）細胞株 18
3. 帶有zelda基因之病毒 18
4. 藥物 19
5. 引子 19
6. STARR-seq 專用載體 20
實驗方法 22
1、勝任細製做（E. coli） 22
2、使用已知的增強子片段製做STARR-seq控制組建構 22
3、抽取質體DNA（Preparation before Plasmid DNA isolation） 23
4、萃取特定的DNA片段（Gel extraction of specific DNA fragments） 24
5、S2細胞培養、馴化以及保存（Schneider 2 cells handeling） 26
6、Transfection of S2 Cell 27
7、抽取S2細胞之RNA與RNA電泳 27
8、使用iScript™ cDNA Synthesis Kit合成cDNA 28
9、RT-qPCR測試基因表現量（RT-qPCR test the gene expression） 29
10、Baculovirus病毒制備（P3 Virus preparation for the infection of S2 cells） 30
12、ectopic expression of Zelda in S2 cells 30
三、實驗結果 31
3.1建構測試STARR-seq系統之增強子轉殖質體 32
3.2 S2細胞之馴化 35
3.3以桿狀病毒轉染S2細胞異位表達Zelda 36
3.4 以qRT-PCR測試增強子與啟動子組合在有無Zld的S2細胞中之活性 38
3.4.1 在表現Zelda的S2細胞中，已知zen增強子的活性 39
3.4.2 在表現Zelda的S2細胞中，Act5c增強子的活性 39
四、實驗結論與討論 41
4.1 使用ESF培養基序列稀釋馴化S2細胞以及使用病毒感染S2細胞 41
4.2.1 凝膠電泳分析RT-qPCR之產物 43
4.3在S2細胞異位表現Zld，足以進行STARR-seq分析定位Zld-dependent增強子 44
4.4 所測試的增強子對核心啟動子有偏好 44
五、參考資料 47

參考文獻

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Cai, H. N., Arnosti, D. N., & Levine, M.（1996）. Long-range repression in the Drosophila embryo. Proc Natl Acad Sci U S A, 93（18）, 9309-9314. https://doi.org/10.1073/pnas.93.18.9309
Chen, H., Xu, Z., Mei, C., Yu, D., & Small, S.（2012）. A system of repressor gradients spatially organizes the boundaries of Bicoid-dependent target genes. Cell, 149（3）, 618-629. https://doi.org/10.1016/j.cell.2012.03.018
Chung, Y. T., & Keller, E. B.（1990）. Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter. Mol Cell Biol, 10（12）, 6172-6180. https://doi.org/10.1128/mcb.10.12.6172-6180.1990
Darbo, E., Herrmann, C., Lecuit, T., Thieffry, D., & van Helden, J.（2013）. Transcriptional and epigenetic signatures of zygotic genome activation during early Drosophila embryogenesis. BMC Genomics, 14, 226. https://doi.org/10.1186/1471-2164-14-226
Doyle, H. J., Kraut, R., & Levine, M.（1989）. Spatial regulation of zerknullt: a dorsal-ventral patterning gene in Drosophila. Genes Dev, 3（10）, 1518-1533. https://doi.org/10.1101/gad.3.10.1518
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Foo, S. M., Sun, Y., Lim, B., Ziukaite, R., O′Brien, K., Nien, C. Y., Kirov, N., Shvartsman, S. Y., & Rushlow, C. A.（2014）. Zelda potentiates morphogen activity by increasing chromatin accessibility. Curr Biol, 24（12）, 1341-1346. https://doi.org/10.1016/j.cub.2014.04.032
Freese, N. H., Norris, D. C., & Loraine, A. E.（2016）. Integrated genome browser: visual analytics platform for genomics. Bioinformatics, 32（14）, 2089-2095. https://doi.org/10.1093/bioinformatics/btw069
Fu, S., Nien, C. Y., Liang, H. L., & Rushlow, C.（2014）. Co-activation of microRNAs by Zelda is essential for early Drosophila development. Development, 141（10）, 2108-2118. https://doi.org/10.1242/dev.108118
Gotze, M., & Wahle, E.（2014）. Smaug destroys a huge treasure. Genome Biol, 15（1）, 101. https://doi.org/10.1186/gb4156
Graveley, B. R., Brooks, A. N., Carlson, J. W., Duff, M. O., Landolin, J. M., Yang, L., Artieri, C. G., van Baren, M. J., Boley, N., Booth, B. W., Brown, J. B., Cherbas, L., Davis, C. A., Dobin, A., Li, R., Lin, W., Malone, J. H., Mattiuzzo, N. R., Miller, D., . . . Celniker, S. E.（2011）. The developmental transcriptome of Drosophila melanogaster. Nature, 471（7339）, 473-479. https://doi.org/10.1038/nature09715
Grubb, B. J.（2006）. Developmental Biology, Eighth Edition. Scott F. Gilbert, editor. Integrative and Comparative Biology, 46（5）, 652-653. https://doi.org/10.1093/icb/icl011
Harrison, M. M., Li, X. Y., Kaplan, T., Botchan, M. R., & Eisen, M. B.（2011）. Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet, 7（10）, e1002266. https://doi.org/10.1371/journal.pgen.1002266
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Tadros, W., Goldman, A. L., Babak, T., Menzies, F., Vardy, L., Orr-Weaver, T., Hughes, T. R., Westwood, J. T., Smibert, C. A., & Lipshitz, H. D.（2007）. SMAUG is a major regulator of maternal mRNA destabilization in Drosophila and its translation is activated by the PAN GU kinase. Dev Cell, 12（1）, 143-155. https://doi.org/10.1016/j.devcel.2006.10.005
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Xu, Z., Chen, H., Ling, J., Yu, D., Struffi, P., & Small, S.（2014）. Impacts of the ubiquitous factor Zelda on Bicoid-dependent DNA binding and transcription in Drosophila. Genes Dev, 28（6）, 608-621. https://doi.org/10.1101/gad.234534.113
Zabidi, M. A., Arnold, C. D., Schernhuber, K., Pagani, M., Rath, M., Frank, O., & Stark, A.（2015）. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature, 518（7540）, 556-559. https://doi.org/10.1038/nature13994

指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2022-9-26

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