透過免疫共沉澱和質譜分析鑑定 Zelda 輔助因子

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姓名

黃鈺婷(Yu-Ting Huang) 查詢紙本館藏

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生命科學系

論文名稱

透過免疫共沉澱和質譜分析鑑定 Zelda 輔助因子
(Identification of Zelda co-factors by co-immunoprecipitation and mass-spectrometry analysis)

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至系統瀏覽論文 (2029-4-24以後開放)

摘要(中)

動物早期胚胎發育的控制首先由母體產物（mRNA和蛋白質）所主導，直到母源子源轉換期（maternal to zygotic transition, MZT），胚胎發育轉為由合子基因調控。因此，在MZT期間有兩個主要事件：（1）母體成分的降解；（2）合子基因組的活化（zygotic genome activation, ZGA）。在果蠅的早期發育中，第一波的ZGA大約有500個合子基因被活化，第二波大約有2000個合子基因被活化。在這個過程中，母源轉錄因子 Zelda（Zld）扮演著關鍵角色。經過多數研究顯示Zelda不僅是一種轉錄活化因子（transcriptional activator），還是一種表觀遺傳調節因子（epigenetic regulator），能夠在MZT期間打開特定區域的染色質，促進基因組的活化。儘管我們瞭解其功能，但對於Zelda的功能性結構與分子機制依然尚未完全釐清，特別是Zelda是否能直接與轉錄機制或表觀遺傳調節因子相互作用，或是通過輔助因子發揮作用仍然未知。為了瞭解Zelda的分子機制，實驗室先前利用桿狀病毒表現系統，在昆蟲細胞中異位表現帶有6xHis-tag的Zelda蛋白，隨後進行了Ni-NTA蛋白純化。以純化的Zelda與果蠅早期胚胎的總蛋白萃取物進行免疫共沉澱（Co-IP），並通過質譜分析（mass spectrometry），鑑定出了134種蛋白質。
　　在我的研究中，我改善了純化的方式，使蛋白的產量及純度增加，可供後續Zelda結構的解析，例如Cryo-EM分析。此外為了縮小Zelda候選輔助因子的範圍，由於Zelda作用於染色質，我進一步純化果蠅早期胚胎核蛋白，再重複免疫共沉澱實驗，與質譜分析，將輔助因子的候選範圍縮小至98種。根據 Gene Ontology term（GO-term）analysis和基因表現數據，大多數蛋白質為核蛋白，並在胚胎發育的前兩個小時內表達。未來可針對功能與表觀遺傳和轉錄調控有關的蛋白進行進一步驗證，以便更佳了解Zelda功能和早期胚胎中合子基因組重新編程的機制。

摘要(英)

Early embryonic development in all animals is first controlled by maternal products until the maternal-to-zygotic transition (MZT). During MZT, there are two major events: degradation of maternal components and zygotic genome activation (ZGA). In the early development of Drosophila, ZGA occurs in two waves. The first wave (minor ZGA) activates approximately 500 zygotic genes, while the second wave (major ZGA) activates around 2000 zygotic genes. In this process, the maternal transcription factor Zelda (Zld) plays a pivotal role. With multiple researches, it has been suggested that Zelda is not only a transcriptional activator but also an epigenetic regulator, capable of opening specific chromatin regions and facilitating genome activation during MZT. However, the structural and molecular mechanisms of Zelda remain incompletely elucidated. Particularly whether Zelda interacts directly with transcriptional machinery or epigenetic regulatory factors, or acts through co-factors, is still unknown. To tackle these questions, our laboratory previously utilized a Bac-to-Bac baculovirus expression system to ectopically express Zelda protein tagged with 6xHis-tag in insect cells, followed by Ni-NTA protein purification. Purified Zelda was used for co-immunoprecipitation (Co-IP) with total protein extracts from early Drosophila embryos, followed by mass spectrometry (MS) analysis. 134 proteins were identified.
In my study, I first optimized Zelda production for future Zelda structural study, such as Cryo-EM analysis. Higher yield and more purified proteins were obtained. To narrow down the candidates of Zelda co-factors, I repeated the Co-IP experiments and MS analysis with nuclear protein extracts from early embryos, since Zelda mainly works on chromosome. 98 proteins were recovered. Based on Gene Ontology (GO) term analysis and expression data, most protein were nuclear (cellular component) and expressed in the first two hours of embryonic development. A handful of proteins were functional related to epigenetic and transcriptional regulation. Further verification of these proteins is required and will provide better understanding of Zelda function and the mechanism of reprogramming zygotic genome in early embryos.

關鍵字(中)

★ 果蠅
★ 母源轉錄因子 Zelda
★ 早期胚胎發育
★ 蛋白質間交互作用
★ Zelda輔助因子

關鍵字(英)

★ Drosophila
★ maternal transcription factor Zelda
★ early development
★ Protein-protein interaction
★ Zelda cofactor

論文目次

目錄
中文摘要 i
Abstract ii
致謝 iv
目錄 v
圖目錄 viii
表目錄 ix
中英文對照表 x
一、前言 1
1.1 黑腹果蠅（Drosophila melanogaster）早期胚胎發育 1
1.2 母源子源轉換期（Maternal to zygotic transition, MZT） 1
1.3 果蠅轉錄因子Zelda 2
1.3.1　Zelda調控果蠅早期胚胎發育 2
1.3.2　Zelda具有pioneer factor的功能 3
1.4 Zelda的蛋白結構與功能 4
1.4.1　 Zelda蛋白的結構解析 4
1.4.2　預測Zelda的結構與功能 6
1.5 與Zelda共同作用的蛋白質 8
1.6 Bac-to-bac expression system重組桿狀病毒表達系統 12
1.7 研究動機與目的 13
二、實驗方法與材料 15
2.1 實驗材料 15
2.1.1 菌種與質體 15
2.1.2 聚合酶連鎖反應引子（Polymerase chain reaction primers） 15
2.1.3 細胞株 15
2.1.4 果蠅品系 15
2.2 實驗方法 15
2.2.1抽取質體DNA（alkaline lysis） 15
2.2.2大腸桿菌勝任細胞製作 16
2.2.3 以膠體電泳回收DNA 16
2.2.4 限制酶切割 17
2.2.5 DNA接合反應（DNA ligation） 17
2.2.6 萃取桿狀病毒質粒（bacmid） 17
2.2.7 細菌轉形作用（Transformation） 17
2.2.8 細胞培養 18
2.2.9 細胞轉染作用（Transfection） 18
2.2.10 病毒擴增（Virus amplification） 18
2.2.11 半數細胞感染劑量（50% tissue culture infective dose，TCID50） 19
2.2.12 利用桿狀病毒表現系統產出目標蛋白（Bac to bac expression system） 20
2.2.13 以Ni-NTA Agarose純化蛋白質 20
2.2.14免疫共沉澱（Co-Immunoprecipitation） 20
2.2.15 SDS-PAGE分析 22
三、實驗結果 23
3.1 建構pFB_actpRed、pFB_ActpZldczfRed重組病毒質體 23
3.2 建構 pFB_actpRed、pFB_actpZld-Red bacmid 25
3.3 轉染bacmid至昆蟲細胞生產重組病毒 26
3.4 利用pPH_HZld_DsRed 病毒生產Zelda 蛋白 28
3.5 利用Ni-NTA agarose beads 純化 Zelda 蛋白 29
3.6 萃取果蠅早期胚胎核蛋白 32
3.7 Zelda蛋白與早期胚胎核蛋白免疫共沉澱（Co-IP） 33
3.8 蛋白質質譜分析（MS-analysis）及Gene Ontology（GO）Term分析 34
3.9 Protein-protein interaction （PPI） network分析 37
四、實驗討論 40
4.1 利用Bac-to-Bac expression system生產Zelda蛋白 40
4.2 利用Ni-NTA純化6x His-tag Zelda蛋白質 40
4.3 萃取果蠅早期胚胎核蛋白與Zelda免疫共沉澱 41
4.4 與Zelda有交互作用的蛋白質 41
4.5 未來應用 45
參考資料 46
附錄 50
附錄表一：實驗所使用之菌種與質體 50
附錄表二：Primer sequences 50
附錄表三：PCR篩選Bacmid之時間與溫度設定 51
附錄表四：DNA膠體電泳相關試劑 51
附錄表五：以Ni-NTA純化蛋白相關試劑 52
附錄表六： SDS-PAGE相關試劑 52

參考文獻

1. Yamaguchi, M. and H. Yoshida, Drosophila as a Model Organism. Adv Exp Med Biol, 2018. 1076: p. 1-10.
2. Kennison, J.A. and J.W. Tamkun, Early Drosophila development. Curr Opin Cell Biol, 1990. 2(6): p. 991-5.
3. Tadros, W. and H.D. Lipshitz, The maternal-to-zygotic transition: a play in two acts. Development, 2009. 136(18): p. 3033-42.
4. Vastenhouw, N.L., W.X. Cao, and H.D. Lipshitz, The maternal-to-zygotic transition revisited. Development, 2019. 146(11).
5. Lee, M.T., A.R. Bonneau, and A.J. Giraldez, Zygotic genome activation during the maternal-to-zygotic transition. Annu Rev Cell Dev Biol, 2014. 30: p. 581-613.
6. Hamm, D.C. and M.M. Harrison, Regulatory principles governing the maternal-to-zygotic transition: insights from Drosophila melanogaster. Open Biol, 2018. 8(12): p. 180183.
7. Pilot, F., et al., Developmental control of nuclear morphogenesis and anchoring by charleston, identified in a functional genomic screen of Drosophila cellularisation. Development, 2006. 133(4): p. 711-23.
8. De Renzis, S., et al., Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo. PLoS Biol, 2007. 5(5): p. e117.
9. Lott, S.E., et al., Noncanonical compensation of zygotic X transcription in early Drosophila melanogaster development revealed through single-embryo RNA-seq. PLoS Biol, 2011. 9(2): p. e1000590.
10. Chen, K., et al., A global change in RNA polymerase II pausing during the Drosophila midblastula transition. Elife, 2013. 2: p. e00861.
11. Saunders, A., et al., Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription. Genes Dev, 2013. 27(10): p. 1146-58.
12. Kwasnieski, J.C., T.L. Orr-Weaver, and D.P. Bartel, Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns. Genome Res, 2019. 29(7): p. 1188-1197.
13. ten Bosch, J.R., J.A. Benavides, and T.W. Cline, The TAGteam DNA motif controls the timing of Drosophila pre-blastoderm transcription. Development, 2006. 133(10): p. 1967-77.
14. Liang, H.L., et al., The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila. Nature, 2008. 456(7220): p. 400-3.
15. Nien, C.Y., et al., Temporal coordination of gene networks by Zelda in the early Drosophila embryo. PLoS Genet, 2011. 7(10): p. e1002339.
16. Harrison, M.M., et al., Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet, 2011. 7(10): p. e1002266.
17. Lee, M.T., et al., Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature, 2013. 503(7476): p. 360-4.
18. Leichsenring, M., et al., Pou5f1 transcription factor controls zygotic gene activation in vertebrates. Science, 2013. 341(6149): p. 1005-9.
19. Loh, Y.H., et al., The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet, 2006. 38(4): p. 431-40.
20. mod, E.C., et al., Identification of functional elements and regulatory circuits by Drosophila modENCODE. Science, 2010. 330(6012): p. 1787-97.
21. Foo, S.M., et al., Zelda potentiates morphogen activity by increasing chromatin accessibility. Curr Biol, 2014. 24(12): p. 1341-1346.
22. Sun, Y., et al., Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation. Genome Res, 2015. 25(11): p. 1703-14.
23. Hamm, D.C., E.R. Bondra, and M.M. Harrison, Transcriptional activation is a conserved feature of the early embryonic factor Zelda that requires a cluster of four zinc fingers for DNA binding and a low-complexity activation domain. J Biol Chem, 2015. 290(6): p. 3508-18.
24. Dubochet, J., J. Frank, and R. Henderson, The nobel prize in Chemistry 2017. Chimica Oggi-Chemistry Today, 2018. 36(3): p. 28-29.
25. Cheng, Y., et al., A primer to single-particle cryo-electron microscopy. Cell, 2015. 161(3): p. 438-449.
26. Earl, L.A., et al., Cryo-EM: beyond the microscope. Curr Opin Struct Biol, 2017. 46: p. 71-78.
27. Jumper, J., et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021. 596(7873): p. 583-589.
28. Varadi, M., et al., AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res, 2022. 50(D1): p. D439-D444.
29. Laver, J.D., et al., Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity. Nucleic Acids Res, 2013. 41(20): p. 9438-60.
30. Carnesecchi, J., et al., Multi-level and lineage-specific interactomes of the Hox transcription factor Ubx contribute to its functional specificity. Nat Commun, 2020. 11(1): p. 1388.
31. Rives-Quinto, N., et al., Sequential activation of transcriptional repressors promotes progenitor commitment by silencing stem cell identity genes. Elife, 2020. 9.
32. Larson, E.D., et al., Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila. Nat Commun, 2021. 12(1): p. 7153.
33. Rieder, L.E., et al., Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP. Genes Dev, 2017. 31(14): p. 1494-1508.
34. Duan, J., et al., CLAMP and Zelda function together to promote Drosophila zygotic genome activation. Elife, 2021. 10.
35. Reichardt, I., et al., The tumor suppressor Brat controls neuronal stem cell lineages by inhibiting Deadpan and Zelda. EMBO Rep, 2018. 19(1): p. 102-117.
36. Larson, E.D., et al., Premature translation of the Drosophila zygotic genome activator Zelda is not sufficient to precociously activate gene expression. G3 (Bethesda), 2022. 12(9).
37. Jenkins, V.K., et al., Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol, 2022. 2540: p. 1-34.
38. Schafer, M., et al., Expression of a gene duplication encoding conserved sperm tail proteins is translationally regulated in Drosophila melanogaster. Mol Cell Biol, 1993. 13(3): p. 1708-18.
39. Ni, J.Q., et al., Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods, 2008. 5(1): p. 49-51.
40. Morikawa, R.K., et al., Different levels of the Tripartite motif protein, Anomalies in sensory axon patterning (Asap), regulate distinct axonal projections of Drosophila sensory neurons. Proc Natl Acad Sci U S A, 2011. 108(48): p. 19389-94.
41. Comeron, J.M., R. Ratnappan, and S. Bailin, The many landscapes of recombination in Drosophila melanogaster. PLoS Genet, 2012. 8(10): p. e1002905.
42. Emelyanov, A.V., et al., Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization. Genes Dev, 2014. 28(18): p. 2027-40.
43. Ando, T., et al., Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum. Curr Biol, 2019. 29(9): p. 1512-1520 e6.
44. Shokri, L., et al., A Comprehensive Drosophila melanogaster Transcription Factor Interactome. Cell Rep, 2019. 27(3): p. 955-970 e7.
45. Kanca, O., et al., An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination. Elife, 2022. 11.
46. David, J.R., et al., Evolution of assortative mating following selective introgression of pigmentation genes between two Drosophila species. Ecol Evol, 2022. 12(4): p. e8821.
47. Tjia, S.T., G.M. zu Altenschildesche, and W. Doerfler, Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells. Virology, 1983. 125(1): p. 107-17.
48. Pennock, G.D., C. Shoemaker, and L.K. Miller, Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol Cell Biol, 1984. 4(3): p. 399-406.
49. Lee, D.F., et al., A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells. J Virol, 2000. 74(24): p. 11873-80.
50. Kang, C.C., Construction and ectopic expression of Drosophila Zelda using baculovirus system for functional analysis, in The Department of Life Sciences. 2022, National Central University.
51. Huang, Y.T., Construction of Drosophila Zelda using baculovirus system and screening for proteins interacting with Zelda, in The Department of Life Sciences. 2022, National Central University.
52. Ramakrishnan, M.A., Determination of 50% endpoint titer using a simple formula. World J Virol, 2016. 5(2): p. 85-6.
53. ATCC Virology Culture Guide. Available from: https://www.atcc.org/resources/culture-guides/virology-culture-guide.
54. Racaniello, V. Multiplicity of infection. 2011; Available from: https://virology.ws/2011/01/13/multiplicity-of-infection/.
55. Huang, P.H., Anti-Zelda antibody production and Zelda expression analysis, in The Department of Life Sciences. 2023, National Central University.
56. Bonnet, J., et al., Quantification of Proteins and Histone Marks in Drosophila Embryos Reveals Stoichiometric Relationships Impacting Chromatin Regulation. Dev Cell, 2019. 51(5): p. 632-644 e6.
57. Shannon, P., et al., Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 2003. 13(11): p. 2498-504.
58. Rhee, D.Y., et al., Transcription factor networks in Drosophila melanogaster. Cell Rep, 2014. 8(6): p. 2031-2043.

指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2024-5-2

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