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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 呂勝春(Sheng-Chung Lee) | |
dc.contributor.author | Chiung-Wen Chang | en |
dc.contributor.author | 張瓊文 | zh_TW |
dc.date.accessioned | 2021-06-15T00:58:35Z | - |
dc.date.available | 2008-09-11 | |
dc.date.copyright | 2008-09-11 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-08-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42300 | - |
dc.description.abstract | 輔助轉錄調控因子TIF1beta(TIF1beta)/KAP1/TRIM28)是一個上位調控子,其功能與重要的角色為與heterochromtin protien 1 (HP1) 蛋白及其他染色質調控蛋白結合後,在某些特定的區域裡,調控染色質結構重組,使其鬆散或緻密,進而調控基因表現。其中TIF1beta 與HP1之間的交互作用,是經由位於TIF1beta序列上的HP1-box特性決定。這個具功能性的交互作用,對於生物在組織生長分化、個體發育、病毒抵抗,以及生理調控時的各項染色質的動態行為(動態特性)、穩定性的維持,乃至於基因表現的調控上,是很重要的。然而,闡述此等交互作用機制的文獻卻非常有限。TIF1beta目前被普遍認為是一個可經由多種'後轉譯修飾'的蛋白,修飾種類包括磷酸化、sumo化等。這些多種不同的修飾調控在不同的基因事件裡,皆扮演重要的角色。
本文的第一部分論述:經由PKC訊息調控途徑致使S473位置磷酸化後的TIF1beta,具有使G1-S細胞週期特異性基因活化的功能 。 TIF1beta/phorpho-Ser473 修飾在細胞周期的S-Phase與M-Phase中,被磷酸化的量顯著的提高,是一個受細胞周期調控的修飾位置。經由免疫化學反應及染色質免疫化學沉澱法分析發現,TIF1beta/Ser473 的磷酸化導致細胞週期調控的特異性基因cyclin A,cdc2,與cdc25A的活化。此誘導活化的發生,係由於TIF1beta與HP1的作用能力受到TIF1beta自身的S473被磷酸化所抑制。無法致磷酸話的TIF1beta/S473A與HP1beta共同存在於G1時期的細胞核間質裡,大量表現TIF1beta/S473A造成細胞停留在G2/M細胞週期的比例增加。TIF1beta/Ser473在細胞週期S phase 中的磷酸化,是經由PKCdelta訊息傳導途徑所媒介。TIF1beta/S473磷酸化的程度,在循環分裂的增殖細胞裡,明顯的比分化的細胞高。顯示TIF1beta/S473 磷酸化與去磷酸化,在細胞生長調控上,扮演重要的角色。 論文的第二部分論及:TIF1beta在細胞有絲分裂期間,透過自身的動態高度磷酸化調控其與染色質/染色體的相對分佈,具有維持細胞正常分裂增生的重要角色。 在TIF1beta的表現被基因功能性剔除後的HeLa(具有較低的p53功能活性)細胞株中觀察到細胞因此而產生不正常的外形改變化與細胞凋亡。這些現象係由這些細胞無法完成正常的有絲分裂所導致而成。經由流式細胞儀,免疫化學染色,和即時動態活細胞攝影術分析有絲分裂,這些細胞會產生錯誤的分裂溝決定位置,有絲分裂延遲或無法進行,與呈現染色質分佈不均等的現象。最後,細胞走向凋亡的途徑。 TIF1beta僅在M-phase的窗型期時,呈現高度磷酸化的狀態。利用LC/MS/MS分析技術,已經有至少三個磷酸化位置被分離鑑定出來。他们分別是位置S752,S757,和S473。TIF1beta透過自身的高度磷酸化狀態被排擠在具有高度緻密結構的染色體外圍的細胞質空間裡。在有絲分裂期間,TIF1beta會逐漸降低它的磷酸化狀態。有絲分裂後期,去高磷酸化的TIF1beta才具有與重新構建的染色質組成份子重新結合的能力。說明了藉由本身磷酸化的調節,TIF1beta在有絲分裂過程中具有無可替代的功能。 | zh_TW |
dc.description.abstract | The transcriptional intermediary factor TIF1beta(TIF1beta)/KAP1/TRIM28) is an epigenetic regulator, functions in gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein 1 (HP1) family and various other chromatin factors. The interaction between TIF1beta and HP1 depend on the HP1-box of TIF1beta. This functional interaction is crucial for chromatin dynamics, maintenance and gene regulation during differentiation, development, viral restriction, and cellular physiological regulation. However, the underlying mechanism of how the interaction is regulated remains poorly understood. TIF1beta is a multiply modificated proteins. The modifications include phosphorylation and sumoylation. These modifications play important role(s) in regulating its activity at different events.
In the thesis part one, these studies conclude that the PKCdelta pathway mediated phosphorylation on TIF1beta/S473 is important for activation of G1-S cell cycle gene. The Ser473 of TIF1beta locates near the HP1b-binding motif, PXVXL. Phosphorylation of TIF1beta at Ser473 is cell cycle regulated which is elevated during S phase and M-phase. The dynamical alteration of which is functionally associated with cell cycle genes expression. Phosphorylation of TIF1beta/Ser473 coincides with the induction of cyclin A, cdc2, and cdc25A, due to the interaction with HP1beta is compromised by TIF1beta/phospho-S473 itself. Non-phsophorylated form TIF1beta/S473A preferentially associated with HP1beta in G1-phase nuclear matrix and affect cell cycle progression with more cell population stall in G2/M-phase while it is over-expressed. The phosphorylation of TIF1beta/Ser473 in early S phase is mediated by the PKCdelta pathway and is closely related to cell proliferation. In the thesis part two, these studies conclude that TIF1beta is functionally important for mitotic progression through its dynamically regulated hyperphosphorylation during mitosis. The abnormal cell morphology and apoptosis phenotype in TIF1beta knockdown HeLa cells is due to the failure to complete the mitotic phase. The failure in cleavage furrow determination, mitotic delay, gradually having unevenly distributed chromatin, and finally apoptosis, are revealed by FACs, immunostaining, and Time Lapse Cinematography. TIF1beta exhibits a hyperphosphorylation state only in M-phase. At least three phosphorylation sites, S757, S752, and S473 are identified by LC/MS/MS. The exclusion of TIF1beta from M-phase chromosome is due to its hyperphosphorylation. The re-orchestration of TIF1beta with reorganized chromatin is tightly correlated with its gradually decreased phosphorylation level during mitosis. The functional TIF1beta in M-phase may coincide with the mitotic failure observed in TIF1beta knockdown HeLa cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:58:35Z (GMT). No. of bitstreams: 1 ntu-97-D89448001-1.pdf: 6599109 bytes, checksum: f3b0d833e7fe6cf6aea77cdf6a2dfe33 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | Summary P7
中文摘要 P9 Abstract (Part I) Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1beta/KAP1 P11 Abstract (Part II) TIF1beta is functional important through its dynamically regulated hyperphosphorylation in mitotic progression P13 1. Introduction Part I Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1beta/KAP1 1.1 General functions of TIF1beta and HP1beta P15 1.2 Functions of TIF1beta with HP1beta in differentiation, developement and viral restriction P17 1.3 Post-translational modifications on TIF1beta P20 1.3-1 Phosphorylation at S824 P20 1.3-2 Sumoylations P21 1.3-3 Phosphorylation at S473 P22 1.4 Cell cycle regulation P22 1.5 PKC family kinases P24 Part II TIF1beta is functional important through its dynamically regulated hyperphosphorylation in mitotic progression 1.6 Apoptosis phenotype reported in TIF1beta knockdown cells P26 1.7 Similar morphology due to mitotic failure P28 1.8 Chromatin structure and histone modifications in M- phase P29 1.9 Mitosis onset and exit P31 1.10 Cytokinesis & Roscovitine P33 1.11 Molecular transport during Mitosis P35 1.12 Transcription-related factors during Mitosis P37 2. Materials and Methods 2.1 Antibodies P39 2.2 Plasmids P39 2.3 Chemicals P41 2.4 Cell cultures P41 2.5 Biochemical Analysis: cell lysate extraction, Immunoprecipitation, recombinant proteins, in vitro pull-down, and western blotting P42 2.6 CIP P43 2.7 In vitro transcription and translation P43 2.8 Chromatin immunoprecipitation assays P44 2.9 Immunostaining P46 2.10 Flow cytometry P47 2.11 RNA extraction and Real-time PCR P47 2.12 Nuclear matrix extraction P48 2.13 Time Lapse Cinematography P49 2.14 In gel digestion P49 Part I Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1beta/KAP1 3. Results 3.1 Characterization of TIF1beta and phosphorylated TIF1beta/Ser473 Antibodies (Fig. 1A-1C) P51 3.1-1 Characterzation of TIF1beta/S473 in M-phase (Fig. 1D) P52 3.2 Phosphorylation of TIF1beta/Ser473 is dynamically regulated during cell cycle progression (Fig. 2A-2D) P52 3.2-1 TIF1beta/S473A is preferentially colocalized with HP1 beta in nuclear matrix (Fig. 2E) P54 3.2-2 Supporting resultes from references - The fluctuation and expression timing of cyclins during cell cycle (Table. 2F) P55 - FACs analysis of cell cycle distribution for 0 – 44 hours of thymidine released HeLa cells (ref, Figure 2G) P55 3.3 Phosphorylation of TIF1beta/Ser473 compromises interaction between HP1beta and TIF1beta P55 3.4 TIF1beta regulates key genes expression during G1 to S- phase cell cycle progression (Fig. 4A-4D, Table 4E) P57 3.5 Phosphorylation of TIF1beta/Ser473 in S phase is mediated by PKC pathway (Fig. 5A-5H) P60 3.6 Level of phosphorylated TIF1beta/Ser473 is reduced in megakaryocytic differentiated K562 cells (Fig. 6A) P61 3.6-1 The steady state level of TIF1beta/phospho-S473 in different mouse tissue (Fig. 6B) P62 4. Discussion P63 5. List of Figures and Tables (legends included) 5.1 Figure 1A 1B 1C (3.1) P67 5.1-1 Figure 1D (3.1-1) P68 5.2 Figure 2A 2B 2C 2D (3.2) P69 5.2-1 Figure 2E (3.2-1) P71 5.2-2 Figure 2F 2G (3.2-2) P72 5.3 Figure 3A 3B 3C 3D (3.3) P73 5.4 Figure 4A 4B 4C 4D Table 4E (3.4) P75 5.5 Figure 5A 5B 5C 5D 5E 5F 5G 5H (3.5) P77 5.6 Figure 6A (3.6) P79 5.6-1 Figure 6B (3.6-1) P80 Part II TIF1beta is functional important through its dynamically regulated hyperphosphorylation in mitotic progression 3. Results 3.1 HeLa cell is morphologically abnormal when TIF1beta is knockdown by siRNA (Fig. 1A-1D) P82 3.2 Abnormal cell morphology observed in interphase is due to mitotic failure in TIF1beta knockdown cells (Fig. 2A- 2D) P82 3.2-1 Live cell images of mitotic HeLa progression by Time Lapse Cinematography (Supplemental Videos 2E, 2F) P84 3.3 TIF1beta is dynamically regulated by phosphorylation during mitosis (Fig. 3A-3C) P85 3.3-1 The predicted phosphorylation sites on TIF1beta (Table 3D) P85 3.3-2 HP1 binding domain is relatively important for the nuclear localization of TIF1beta (Fig. 3E) P86 3.3-3 S757 and S752 are responsible for the hyperphosphorylation state of TIF1beta in prometaphase (Fig. 3F, 3G) P86 3.4 The hypo-phosphorylated TIF1beta is important for its reassociation with reorganized/decondensed chromatin in the end of mitosis (Fig. 4A-4D) P87 3.4-1 TIF1beta is dispersed into mitotic cytoplasm with cdk1 (Fig. 4E) P90 3.5 TIF1beta may shuttle with Giantin or GRASp65 in mitotic cytoplasm (Fig. 5A, 5B) P90 3.6 TIF1beta indirectly interacts with actinin 4 in M-phase (Fig 6A, 6B) P90 4. Discussion P92 5. List of Figures and Tables (legends included) 5.1 Figure 1A 1B 1C 1D (3.1) P97 5.2 Figure 2A 2B 2C 2D (3.2) P98 5.2-1 Supplemental Videos 2E 2F (3.2-1) P100 5.3 Figure 3A 3B 3C (3.3) P101 5.3-1 Table 3D (3.3-1) P102 5.3-2 Figure 3E (3.3-2) P103 5.3-3 Figure 3F 3G (3.3-3) P104 5.4 Figure 4A (3.4) P105 5.4 Figure 4B 4C (3.4) P106 5.4 Figure 4D (3.4) P107 5.4-1 Figure 4E (3.4-1) P108 5.5 Figure 5A 5B (3.5) P109 5.6 Figure 6A 6B (3.6) P110 6. Reference P111 Appendix P122 | |
dc.language.iso | en | |
dc.title | 輔助抑制子TIF1β/KAP1於Ser473位置磷酸化後對其結合HP1蛋白能力和本身抑制功能之調控 | zh_TW |
dc.title | Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1β/KAP1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 李芳仁(Fang-Jen Lee),張?仁(Ching-Jin Chang),阮麗蓉(Li-Jung Juan),陳宏文(Hung-Wen Chen) | |
dc.subject.keyword | 磷酸化,細胞週期,染色質/染色體,細胞分化,有絲分裂,組蛋白, | zh_TW |
dc.subject.keyword | TIF1beta/KAP1,heterochromatin protein 1(HP1),phosphorylation,cell cycle,E2F,PKC,differentiation,mitosis,histone code,chromatin,cdk1, | en |
dc.relation.page | 122 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-08-02 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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