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研究生:駱又瑄
研究生(外文):You-Shen Luo
論文名稱:Pc2 於轉錄調控上的分子機制
論文名稱(外文):The molecular mechanism of Pc2 in transcriptional regulation
指導教授:陳全木陳全木引用關係
指導教授(外文):Chuan-Mu Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:56
中文關鍵詞:轉錄調控
外文關鍵詞:Pc2YY1ZFP42
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在真核生物中,細胞核是一個複雜的結構體,被組織成數種分離的次細胞核結構及功能性區域。這些次細胞核功能區,由染色質及蛋白質等數種不同的元素所組成,而參與在許多基因表現及細胞功能的調控上。核體(nuclear body)為其中一種次細胞核結構,是由蛋白質聚集形成之巨分子為主要組成、具有特定功能性的區域,能藉由免疫細胞化學(immuneocytochemistry)的方式,透過光學顯微鏡被觀察到。在過去的研究中發現,Pc2 能在細胞核中形成分離的點狀分佈,我們稱之為 Pc2 nuclear bodies。Pc2 (polycomb protein 2) 是櫛蛋白質家族(polycomb group,PcG)的成員蛋白之一,具有抑制轉錄活性之能力,然而,對於 Pc2 調控轉錄抑制的機制,以及此調控機制是否與 Pc2 nuclear bodies 的形成有關,目前仍不清楚。因此,本論文將針對 Pc2 nuclear bodies 的形成及 Pc2 於轉錄作用的調控機制兩部份進行探討。

在第一部份,為探討 Pc2 nuclear bodies 的形成機制,我們藉由純化 Pc2 蛋白質複合體以尋找可能的方向。結果發現,許多與細胞核基質(nuclear matrix)相關的蛋白質分子存在於 Pc2 蛋白複合體中,而推測 Pc2 nuclear bodies 的形成可能與細胞核基質的連結有關。因此,進一步以細胞分層實驗(cell fractionation)證明,Pc2 蛋白主要存在於細胞核基質的層次中。另一方面,為尋找出特定的 Pc2 nuclear bodies 形成功能域(Pc2 nuclear bodies-formation domain),我們藉由 Pc2 的系列剔除突變法(serial deletion mutant),利用蛋白質分佈狀態的分析而找出 Pc2 nuclear bodies 形成功能域(Pc2 nuclear bodies-formation domain),並再次利用細胞分層實驗證明,Pc2 nuclear bodies 的形成是基於與細胞核基質的連結。

第二部份,在 Pc2 調控轉錄作用方面,我們發現,Pc2 能吸引 YY1 及 ZFP42 此二具有 DNA 結合及轉錄調控能力的鋅指蛋白(zinc finger protein)進入 Pc2 nuclear bodies。再以共同免疫沈澱法(co-immunoprecipitation)進行檢測,證明 Pc2 與 YY1 及 ZFP42 皆具有交互作用。而藉由轉錄活性檢測(transcriptional assay)的結果顯示,Pc2 能增加 YY1 於其結合啟動子(binding-promoter)上之轉錄抑制活性,這暗示 Pc2 nuclear bodies 可能透過與轉錄調控因子的交互作用,作為一個抑制轉錄活性的中心。此外,雖然由 Pc2 nuclear bodies-formation domain 所形成的 nuclear bodies,其大小和型態與完整的 Pc2 蛋白所形成之 nuclear bodies 不盡相同,但利用 Pc2 nuclear bodies 能夠吸引 YY1 及 ZFP42 進入其中的特性,藉由免疫螢光染色法檢測,發現僅由 Pc2 nuclear bodies-formation domain 所形成的 nuclear bodies 亦能吸引 YY1 及 ZFP42 進入其中,證明這些 nuclear bodies 與一般型的 Pc2 nuclear bodies 具有相似的性質。

本論文結果顯示,Pc2 抑制轉錄活性的機制,可能是由特定的 Pc2 body-formation domain 與細胞核基質產生連結,而形成 Pc2 nuclear bodies,作為一個轉錄抑制的中心,透過吸引 YY1 及 ZFP42 進入 Pc2 nuclear bodies 而抑制它們所結合的啟動子。


In eukaryotic cell, the nucleus is a complex organelle organized into several specialized subnuclear structures and functional domains. These subnuclear domains are composed by chromatin, proteins, and various components, which intimately linked to gene expression and cellular functions. One of these subnuclear structures is the nuclear body, which is composed of proteins forming macromolecular aggregates. Previous study has found that in human nucleus Pc2 (polycomb protein 2) distributes as separate speckles, called "Pc2 nuclear bodies". Pc2 is a polycomb group (PcG) protein able to repress transcription. However, the mechanism how Pc2 regulates transcriptional repression remaines elusive. In this thesis, we studied the molecular mechanism of Pc2 in transcriptional regulation from the perspective of Pc2 nuclear bodies.
Through characterization of Pc2 protein complex in HEK 293 cells, we found that Pc2 associates with various nuclear matrix proteins, and it is enriched Pc2 major in nuclear matrix by cell fractionation. In addition, we identified the domains responsible for the formation of the Pc2 nuclear bodies by analyzing the distribution of Pc2 serial deletion mutants, and further proved that Pc2 nuclear bodies-formation intimately associates with nuclear matrix.
Immunofluorescence experiment shows that YY1 and ZFP42, two zinc finger proteins that bind DNA and regulate transcription are recruited into Pc2 nuclear bodies. Interaction happen between Pc2 and YY1, and Pc2 and ZFP42 as demonstrated by co-immunopreciptitation assay. Moreover, results of transcriptional assay showed that Pc2 increases the repressional activity of YY1 on it’s target promoter. These results implied that Pc2 nuclear bodies might serve as a repressional center for transcription. In addition, although size and distribution patterns differ from each other, bodies formed by deletion-form Pc2 containing body-formation domain could also recruit YY1 and ZFP42. These deletion form bodies therefore might have similar capacity to full-length Pc2 bodies.
In summary, these results suggest that Pc2 nuclear bodies function as a transcriptional repression center through recruiting YY1 and ZFP42 and it is tetheried with the nuclear matrix.

壹、緒論……………………………………………………………………1
一、前言……………………………………………………………………1
二、真核細胞的轉錄調控機制……………………………………………1
(一) 真核生物的基因表現 …………………………………………...1
(二) 轉錄作用的調控 …………..…………………………………….2
(三) 次細胞核分區(subnuclear compartment) ……………………3
1. 基因體組織(genome organization)……………………………4
2. 核體(nuclear body) ……………………………………………5
3. 細胞核基質(nuclear matrix)……………………………………7
三、Pc2 背景介紹…………………………………………………………8
(一) 櫛蛋白質家族(Polycomb Group,PcG)………………………8
1. 櫛蛋白家族複合體(PcG complex)………………..……………8
2. 櫛蛋白家族的次細胞核分佈………………..………………….10
(二) Pc2 的發現及結構 ………………..……………………………11
(三) Pc2 的功能 ………………..……………………………………11
1. Pc2 的轉錄調控活性………………..…………………………..11
2. Pc2 的 SUMO E3 活性………………..……………………….12
四、研究目的……………………………………………………………12
五、研究策略……………………………………………………………13
(一) 探討 Pc2 nuclear bodies 的形成機制…………………………13
(二) 探討 Pc2 的轉錄抑制調控…………………………………….13
貳、材料與方法…………………………………………………………..15
一、質體構築……………………………………………………………15
(一) 本論文構築之質體………………..…………………………….15
(二) 其它質體………………..……………………………………….16
二、細胞培養與基因轉移感染…………………………………………16
三、共同免疫沈澱法……………………………………………………17
四、膠體電泳法及西方墨點法………………...…………………….…18
五、免疫螢光染色法…………………………...…………………….…19
六、轉錄活性檢測………………………………………………………20
七、蛋白質複合體純化…………………………………………………21
八、細胞分層法(cell fractionation)………………………………….21
參、結果…………………………………………………………………..23
一、Pc2 nuclear bodies 的形成…………………………………………23
(一) Pc2 蛋白複合體…………………………………………………23
(二) Pc2 蛋白主要存在於細胞核基質層次……………………….….23
(三) Pc2 nuclear bodies-formation 功能域……………………………24
(四) Pc2 nuclear bodies 的形成與細胞核基質有緊密的關聯 …….25
二、Pc2 nuclear bodies 與轉錄作用之調控……………………………26
(一) Pc2 吸引 YY1 及 ZFP42 進入 Pc2 nuclear bodies ………..26
(二) Pc2 與 YY1 及 ZFP42 具有交互作用………………………26
(三) Pc2 能增加 YY1 於其結合啟動子上之轉錄抑制活性………27
(四) YY1 及 ZFP42 能被 Pc2 nuclear bodies-formation domain
所形成之 bodies 吸引而存在共同分佈(colocalization)…..28
肆、討論……………………………………………………………………..30
一、 Pc2 蛋白質複合體 ……………………………………………….30
(一) YY1 未能在 Pc2 蛋白複合體中被偵測到……………………30
(二) 纖維層蛋白(lamin)……………………………………………30
二、 Pc2 nuclear bodies …………………………………………………31
(一) 胺基端及羧酸端的 Pc2 nuclear bodies 形成功能域與細胞核基質的連結……………………………………………………………………31
(二) Pc2 可能以多聚體(polymer)的形式形成 nuclear bodies……31
(三) Pc2 nuclear bodies-formation domain……………………………31
三、 Pc2 與轉錄因子的交互作用……………………………………..32
(一) Pc2 與轉錄因子的交互作用……………………………………32
(二) Pc2 可能與 H3K27me3 結合而調控轉錄作用…………………..33
(三) Pc2 nuclear bodies 與轉錄作用調控
四、 Pc2 的 E3 ligase 活性……………………………………………33
伍、圖表……………………………………………………………………34
陸、參考文獻 ………………………………………………………………47
柒、附圖……………………………………………………………………51


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