臺灣博碩士論文加值系統

基因的轉錄抑制是由不同的轉錄因子專一性地針對其標的基因進行抑制調控，表觀因子則是會進一步透過形成異質染色體的方式維持住基因的轉錄抑制，然而表觀因子要如何接收訊息知道哪些基因需要被維持住轉錄抑制呢?在這邊我利用果蠅tll的轉錄抑制當作模型研究這個過程的分子機制。已知tll的轉錄抑制是由Ttk69、GAF和Hsf所組成的蛋白質抑制複合體結合在tor-RE上所進行的調控，進一步從BioGRID資料庫中注意到PRC1可能可以經由Ttk69、Lwr、Scm和Z被招募到tor-RE，所以我們藉由免疫沉澱法的實驗去證實這些蛋白質的確結合在一起，再藉由遺傳實驗得知tll的轉錄抑制是需要來自果蠅媽媽的這些基因活性，也就是說PRC1有參與tll的轉錄抑制初期階段。此外，根據實驗室的初步結果推論當PRC1被招募到tor-RE上後，會因為Ttk69、GAF和Lolal的交互作用而被運送到在tor-RE上游的multiple Pho-binding sites。已知這三個蛋白質皆有一個BTB domain且同屬於“ttk Group”這個家族，藉由電腦軟體模擬以及分析的結果支持這些蛋白質可以利用他們的BTB domain形成多聚物來運送PRC1。綜上所述，這些結果提供了一個基礎讓我們可以更了解tll的轉錄抑制是如何轉交給PcG表觀因子以維持其轉錄抑制。

Transcription repression is mediated originally by gene-specific repressors. Epigenetic repression is responsible for the maintenance of the inactivated status of target genes via heterochromatin formation. The gap between gene-specific repression and epigenetic repression has been unknown at the molecular level. The tll repression is as a model to study the transition. tll is activated due to the relief of the repression, mediated by Ttk69, GAF and Hsf proteins binding at tor-RE. Data from BioGRID imply that PRC1 is recruited to tor-RE due to the interactions among Ttk69, Lwr, Scm and Z. Here, cross-linked co-IP experiments showed that these proteins associate together. Further, genetic interaction experiments showed that the maternal activities of lwr, Scm, z and PRC1 are required for the tll repression. It indicates that PRC1 is recruited to regulate the initiation of the tll repression. In addition, according to our preliminary results, it is speculated that after PRC1 is recruited, it will be transported to the multiple Pho-binding sites due to the interactions among Ttk69, GAF and Lolal proteins. Ttk69, GAF and Lolal are in the same BTB subgroup, “ttk Group”. In silico analysis was adopted to examine the possibility that Ttk69, GAF and Lolal associate together. The result of structural alignment supported that the BTB domains of Ttk69, GAF and Lolal form a hetero-oligomer. Taken together, the results provide a cornerstone to understand how the tll repression is transferred to PcG proteins-mediated repression.

摘要 i
Abstract ii
Table of content iii
List of Figures vi
Abbreviation vii
I.Introduction 1
I-1. Transcription initiation in eukaryotes 1
I-2. Early elongation and RNA pol II pausing 3
I-3. Transcription repression 4
I-4. Chromatin structure and epigenetic repression 5
I-5. Polycomb group proteins-mediated repression 7
I-5-1. Polycomb group proteins 7
I-5-2. Polycomb response element 8
I-5-3. “Transfer Mechanism”, proposed by Bienz and Müller 9
I-6. Early embryogenesis in Drosophila melanogaster 9
I-7. Studies on the transcription regulation of Drosophila tll gene 10
I-7-1. tll expression is mainly initiated by the tor pathway 11
I-7-2. Ttk69, GAF and Hsf proteins form a repressive complex binding at tor-RE to respond the activation of the tor pathway 12
I-7-3. PcG proteins maintain the tll repression at stages 6 and after 12
I-7-4. Ttk69 associate with GAF and Lolal to transfer PRC1 to the multiple Pho-binding sites 14
I-8. Specific aim 14

II.Materials & Methods 16
II-1. Fly genetics 16
II-2. Whole-mount in situ hybridization of embryos 16
II-2-1. Embryo fixation 16
II-2-2. RNA hybridization 17
II-2-3. Probe detection 18
II-2-4. Color development 19
II-2-5. Sample mounting 19
II-3. Wild-type embryo extract preparation for western blotting 20
II-4. Western blotting to detecting the results of co-immunoprecipitation 21
II-5. In silico analysis 22
II-5-1. The BTB domains of Ttk69, GAF, Psq and Lolal proteins 22
II-5-2. The BTB domains of the GAF-Ttk69 and Lolal-GAF heterodimers 23

III.Results 24
III-1. Ttk69 associates with PcG proteins through Lwr and Pits 24
III-1-1. Experimental design to show that PcG proteins are recruited via Lwr and Pits 24
III-1-2. Antibodies against Scm, Z, Pc, Ph, E(z), Esc, Pho and Sfmbt proteins were acquired to detect the proteins in the immunoprecipitated protein complexes 24
III-1-3. PRC1 and PRC2 associate with Ttk69 via Lwr and Pits respectively 25
III-2. Lwr, Scm and Z participate in the initiation of the tll repression 25
III-2-1. Experimental design to examine the maternal roles of lwr, Scm and z in the tll regulation 25
III-2-2. The maternal activities of lwr, Scm and z are required for the initiation of the tll repression 26
III-2-3. The zygotic activities of lwr, Scm and z are also involved in the tll regulation 27
III-3. PRC1 participates in the initiation of the tll repression 28
III-4. The interactions among Ttk69, GAF and Lolal may help PRC1 to be transported to the multiple Pho-binding sites 29
III-4-1. Experimental design to examine the possibility that Ttk69, GAF and Lolal associate together 29
III-4-2. In silico analysis on the BTB domains of Ttk69, GAF, Psq and Lolal proteins 30
III-4-3. In silico analysis on the BTB domains of the GAF-Ttk69 and Lolal-GAF heterodimers 31

IV.Discussion 33
IV-1. Z protein plays a dual role in the tll regulation 34
IV-2. The recruitment of PRC1 is through a transcriptional factor, Ttk69 34
IV-3. PRC1 mediates the initiation of the tll repression by means of the maintenance of RNA pol II at the promoter 35
IV-4. PRC2 may not participate in the initiation of the tll repression 36
IV-5. PhoRC may participate in the tll regulation 36

V.References 38

Figures 44

Supplementary Figures 66


 
List of Figures
Fig. 1 The expression patterns of tll at stages 4-6
Fig. 2 Ttk69 interacts with PRC1, PRC2 and PhoRC through Lwr and Pits
Fig. 3 modENCODE data support that PRC1 maintains the tll repression at stages 6 and after
Fig. 4 The Pho and GAF –binding sites, located upstream from tor-RE, are conserved among Drosophila species
Fig. 5 Examples to illustrate how to collect embryos from mutant females in the dosage-dependent genetic interaction experiments
Fig. 6 The gel used for separating the immunoprecipitated protein complexes in the cross-linked co-IP experiments
Fig. 7 An example how to use PyMOL software for structural alignments
Fig. 8 Antibodies against Pc, Ph, Scm, Z, E(z), Esc, Pho and Sfmbt proteins are available to detect the immunoprecipitated protein complexes in the cross-linked co-IP experiments
Fig. 9 PcG proteins were detected in the immunoprecipitated protein complexes using anti- Lwr or Pits antibody
Fig. 10 Strategies used to study the maternal and maternal/zygotic defects on the tll posterior patterns in the dosage-dependent genetic interaction experiments
Fig. 11 The activities of lwr, Scm and z were involved in the regulation of the posterior tll repression
Fig. 12 The maternal activity of PRC1 was required for the tll repression
Fig. 13 In silico analysis on the Ttk69, GAF, Psq and Lolal BTB monomers
Fig. 14 The interaction between the Miz1-BCL6 BTB heterodimers is through their beta 1 and beta 5 stands
Fig. 15 In silico analysis on the GAF-Ttk69 and Lolal-GAF BTB heterodimers

Fig. S1 Lwr interacts with Ttk69, Scm and Z
Fig. S2 The multiple Pho-binding sites, located upstream from tor-RE, are crucial to maintain the posterior tll repression at stages 6 and after

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