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研究生:連黃偉
研究生(外文):Huang-Wei Lien
論文名稱:斑馬魚的類鋅指蛋白219(ZNF219L)參與調控脊索相關基因表現
論文名稱(外文):A novel zinc finger protein 219-like (ZNF219L) is involved in the regulation of zebrafish notochord related genes expression
指導教授:韓玉山韓玉山引用關係
口試委員:李明亭黃鵬鵬張茂山鄭嘉雄
口試日期:2013-12-11
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:漁業科學研究所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2013
畢業學年度:102
語文別:英文
論文頁數:60
中文關鍵詞:鋅指蛋白219脊索斑馬魚第二型膠元蛋白α1a
外文關鍵詞:zinc finger protein 219notochordzebrafishcollagen type 2 alpha 1atranscriptional regulationsynuclein-γ2 (sncgb)Sox9a
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脊索 (notochord) 為脊椎動物軀幹發育時重要的結構,在發育早期具有支撐軀幹發育以及誘導神經管 (neural tube) 形成之功能,故研究脊索發育的相關基因調控機制相當的重要。在此篇研究,我們根據哺乳類具有九個C2H2 類型鋅指區域的鋅指蛋白219 (ZNF219) 的序列比對並且選殖出斑馬魚的類鋅指蛋白219 (znf219L)。透過胚胎整體包埋原位雜合反應技術(whole-mount in situ hybridization),我們發現斑馬魚的類鋅指蛋白219的mRNA主要表現於發育早期的中後腦交界 (midbrain-hindbrain boundary, MHB)、後腦 (hindbrain)、以及脊索。使用Morpholino抑制斑馬魚胚胎發育初期的類鋅指蛋白219基因表現會導致脊索部份的發育異常現象以及造成內生性第二型膠元蛋白α1a(col2a1a)基因在脊索的表現量下降。 此外,利用冷光&;#37238;分析實驗 (luciferase assay) 以及體外結合力測試實驗 (in vitro binding experiments),我們發現類鋅指蛋白219會透過其第六以及第九個C2H2鋅指區域專一辨認後結合至第二型膠元蛋白α1a &;#21843;動子(promoter) 上的GGGGG區域且提升其&;#21843;動子活性。統整以上實驗結果,斑馬魚的類鋅指蛋白219具有調控第二型膠元蛋白α1a基因表現於斑馬魚脊索之功能。
另一部份,斑馬魚的synuclein-γ2 (sncgb) 基因於先前研究指出會專一表現於脊索,然而其&;#21843;動子的調控機制並未有完整的研究。於此部份,我們發現sncgb基因為另一個受類鋅指蛋白219調控之目標基因。此外,實驗結果也指出除了斑馬魚類鋅指蛋白219外,sox9a也共同參與了sncgb基因調控。增加類鋅指蛋白219 以及sox9a的基因表現量皆可以促進 sncgb基因的表現量。加上類鋅指蛋白219 會與sox9a有物理性的交互作用且透過morpholino同時抑制二者基因表現量會造成協同性的降低sncgb基因表現量。故以上的實驗結果指出類鋅指蛋白219 與sox9a會協同性的共同調控sncgb基因表現於斑馬魚早期胚胎時期的脊索。
總結以上研究成果,我們確定了斑馬魚的類鋅指蛋白219參與調控脊索相關基因表現將有助於探討脊索早期發育的機制。


The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.
On the other hand, zebrafish synuclein-γ2 (sncgb) has been reported to be expressed specifically in the notochord. However, the mechanism by which the sncgb gene promoter is regulated has not been described. In this part, we demonstrate that sncgb gene was another target gene regulated by ZNF219L. ZNF219L and sox9a are involved in the regulation of sncgb gene expression. Furthermore, we observed that over-expression of both ZNF219L and Sox9a resulted in increased sncgb expression. In addition, ZNF219L is physically associated with Sox9a, and simultaneous morpholino knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression in the notochord. Thus, these results reveal that coordination of ZNF219L with Sox9a is involved in the regulation of notochord-specific expression of sncgb.
Taken together, ZNF219L is involved in the regulation of zebrafish notochord related genes expression and our studies provided better understanding of the mechanism of notochord related genes regulation in early developmental stage.


Contents
中文摘要---------------------------------i
Abstract---------------------------------ii
Abbreviation-- ---------------------------iv
Introduction-- ---------------------------1
The notochord structure- ------------------1
The collagen type 2 alpha 1 (col2a1) gene-----2
The zinc finger protein 219 (ZNF219) gene--- -3
The synuclein genes family------------------4
The zebrafish synuclein-γ2 (sncgb) gene------4
Zebrafish as an experimental model ---------5
Specific aims-- ---------------------------7
Materials and metods----------------------9
Materials--------------------------------9
Fish------------------------------------9
Cell cultures-----------------------------9
Rapid Amplification of cDNA Ends (RACE) -----9
Cloning of full-length and deletion mutations
of zebrafish znf219L----------------------10
Luciferase reporter gene assay--------------11
Total RNA isolation and reverse-transcription
polymerase chain reaction (RT-PCR) analysis
of zebrafish znf219L mRNA-----------------12
Morpholino oligonucleotide (MO) injection-----12
Whole-mount in situ hybridization-----------13
Microinjection of zebrafish embryos ---------13
Oligonucleotide precipitation assay ---------14
In vitro binding assay using recombinant
His-tagged Sox9a protein and ZNF219L-------15
Western blot------------------------------15
Results----------------------------------15
Cloning of znf219L cDNA from zebrafish -----16
Genomic organization of the zebrafish
znf219L gene-----------------------------16
Expression of zebrafish znf219L in various
adult tissues and at different
developmental stages----------------------18
Expression patterns of znf219L mRNA in
zebrafish embryos -----------------------18
Knockdown of znf219L caused partial
notochord abnormalities -------------------18
Zebrafish col2a1a is down-regulated in the
notochord of znf219L morphants-------------20
ZNF219L regulates col2a1a promoter activity
via binding to GGGGG motifs-----------------21
Zinc finger protein 219-like (ZNF219L) is
required for regulation of sncgb promoter
activity in vitro and in vivo-------------------23
Sox9a is required for regulation of sncgb
promoter activity in vitro and in vivo -----------24
Zebrafish ZNF219L associates with Sox9a-------24
Simultaneous knockdown of znf219L and sox9a
results in a synergistic decrease of sncgb
expression in the notochord------------------25
Discussion---------------------------------26
Conclusion and perspective-------------------30
Figures------------------------------------32
References---------------------------------56


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