臺灣博碩士論文加值系統

真核生物細胞在細胞複製S期必須將衛星DNA及組蛋白所組成的高度緊密結構--臂間異染色質（pericentromeric heterochromatin）--鬆開並進行複製，待複製完成後再立即恢復緊密的狀態，以確保染色體在細胞分裂過程能夠正常分離。若是臂間異染色質無法被恢復成緊密的結構，則會導致染色體異常分離，造成微核（micronuclei）的產生，進而引發癌症。過去研究證明，heterochromatin protein 1α（HP1α）會結合上H3K9me3，幫助臂間異染色質的形成，此過程透過類泛素化修飾（SUMOylation）促進新生成的HP1α形成臂間異染色質。類泛素化修飾過程需要專一性的SUMO E3 ligase，協助類泛素化蛋白對HP1α進行修飾。然而，HP1α SUMO E3 ligase尚未被發現。實驗室前人發現KAP1可能作為支架蛋白，幫助兩個可能的HP1α SUMO E3 ligase--Pc2及PIASy增強HP1α類泛素化修飾，但對於Pc2及PIASy是如何調控HP1α類泛素化修飾仍不清楚。因此本篇論文將研究在細胞複製S期Pc2及PIASy調控HP1α類泛素化修飾之角色探討。我們認為異染色質在S期進行DNA複製，需要透過Pc2及PIASy的幫忙增強HP1α類泛素化修飾，進而結合到異染色質。為了證明此假說，我們利用conjugation assay證明在S期Pc2及PIASy的SUMO E3 ligase活性會增強HP1α類泛素化修飾。接著利用nascent chromatin capture assay證明HP1α和類泛素化蛋白會結合在DNA複製後新生成的染色質，且削弱Pc2的表現量會導致較少的HP1α結合在新生成的染色質。另外，利用螢光顯微鏡觀察結果顯示，在S期削弱Pc2的表現量，會降低HP1α結合上異染色質區域的程度；以及削弱Pc2後，微核的比例明顯上升，暗示著染色體的不穩定性提高了。進一步探討Pc2和PIASy是否透過KAP1的協助幫助HP1α類泛素化修飾，我們利用co-immunoprecipitation發現KAP1和PIASy有交互作用，但和Pc2沒有交互作用。接著利用conjugation assay證明在S期KAP1增強HP1α類泛素化修飾，但同時表現PIASy失去SUMO E3 ligase活性，則降低KAP1幫助HP1α類泛素化修飾，暗示著KAP1作為支架蛋白協助PIASy增強HP1α類泛素化修飾。另一方面，最新研究顯示，HP1α結合上H3K9me3受到H3K27me3的正向調控，根據此研究，我們推測H3K27me3表現增強也將增強HP1α類泛素化修飾，進而結合上H3K9me3，而此過程可能是透過Pc2結合上H3K27me3來幫助HP1α類泛素化修飾；藉由conjugation assay，我們證明了當EZH2 (H3K27 methyltransferase)大量表現時能夠增強HP1α類泛素化修飾;但同時表現H3K27 demethylase-UTX、JMJD3以及Pc2，發現失去H3K27me3，Pc2仍可以增強HP1α類泛素化修飾。綜合以上結果顯示，(1) Pc2及PIASy在S期會藉由SUMO E3 ligase活性增強HP1α的類泛素化修飾。(2) 削弱Pc2的表現量會導致HP1α結合上異染色質及新生成染色質的能力下降。(3) 削弱Pc2提高微核產生的比例，暗示著降低染色體的穩定性。(4)KAP1和PIASy有交互作用，但和Pc2沒有，及KAP1作為PIASy的支架蛋白幫助HP1α類泛素化修飾。(5) EZH2會增強HP1α類泛素化修飾，暗示著增加H3K27me3可以增強HP1α類泛素化修飾。(6)失去H3K27me3，Pc2仍可以增強HP1α類泛素化修飾。

DNA replication happens during S phase in eukaryotic cells. In this period, pericentric heterochromatin(PCH), which is a compacted structure constituted of DNA and histone, have to loosen for DNA replication, and immediately returns to its compacted status after the replication. This process ensures that chromosome is normally separated. Disability in reforming compacted structure of PCH results in abnormal chromosome segregation proceeded by generation of micronuclei, and leads to cancer. HP1α (Heterochromatin Protein 1α) can target to H3K9me3 and is critical for formation of PCH. Previous studies demonstrate that HP1α can be regulated by SUMOylation. However, the HP1α SUMOylation SUMO E3 ligase, which can specifically identify HP1α, remains unknown. Our lab found that KAP1 might function as a scaffold protein for supporting two HP1α SUMO E3 ligases, Pc2 and PIASy, to promote HP1α SUMOylation. Whereas, the regulation mechanism of Pc2 and PIASy on HP1α SUMOylation remains unclear. In this thesis, we studied the regulation of HP1α SUMOyaltion by Pc2 and PIASy in S phase. Here we propose that Pc2 and PIASy might promote HP1α SUMOylation thus target it to heterochromatin region. To prove this hypothesis, we identified that SUMO E3 ligase activity of Pc2 and PIASy could promote HP1α SUMOylation in S phase by conjugation assay. Furthermore, using nascent chromatin capture assay we found that HP1α associated with nascent chromatin in S phase and knockdown Pc2 decreased this association in S phase. In immunofluorescence assay we observe that knockdown Pc2 decreased HP1α locating on heterochromatin region in S phase. Moreover, knockdown Pc2 increased micronuclei, suggesting that the instability of chromosome is enhanced. To further determine if Pc2 and PISAy promote HP1α SUMOylation through KAP1, we use co-immunoprecipitation and found that KAP1 could interact with PIASy, while not with Pc2. Then using conjugation assay we found that KAP1 could promote HP1α SUMOylation, but when cotransfected with E3 ligase-defected PIASy mutant, KAP1 loses this ability. This result suggests that KAP1 function as a scaffold protein for helping PIASy to promote HP1α SUMOylation. On the other hand, recent study shows that targeting of HP1α on H3K9me3 is positively regulated by H3K27me3. We suppose that increase in H3K27me3 could elevate HP1α SUMOylation, which is mediated by Pc2, thus further promote its binding with H3K9me3. Result from conjugation assay shows that EZH2, which is H3K27 methyltransferase, could promote HP1α SUMOylation. While cotransfecting H3K27 demethylase-UTX and JMJD3 with Pc2 could not block off HP1α SUMOylation by Pc2. In summary, our results show that：(1) SUMO E3 ligase activity of Pc2 and PIASy promote HP1α SUMOylation in S phase. (2) Knockdown of Pc2 decreases HP1α targeting on heterochromatin region and associating with nascent chromatin in S phase. (3) Knockdown of Pc2 results in increase of micronuclei, which indicate that the stability of chromosomes is reduced. (4) KAP1 can interact with PIASy, while not with Pc2. (5) H3K27 methyltransferase EZH2 can also promote HP1α SUMOylation. (6) Despite of depleting H3K27me3, Pc2 still can promote HP1α SUMOylation.

目錄
中文摘要……………………………......……..……….…...……………1
Abstract……………………………......………………..…...……………3
壹、緒論……………………………......……………….…...……………5
一、前言…………………………………......…………...………………5
二、細胞複製S期…………………………......…...……………………5
三、染色質（chromatin）的介紹………………………......…………...…6
（ㄧ）染色質的結構…………………..…………………….……..6
（二）異染色質的複製及形成………………………………...…..6
四、Heterochromaitn Protein 1（HP1）的背景介紹……..……………..9
（ㄧ）HP1的發現與結構…………………...…….………………9
（二）HP1的家族………………………………….……..….…..10
（三）HP1α的功能…………………………..…….……..….…..10
五、類泛素化 (SUMO) 背景介紹………………....…………………..11
（ㄧ）類泛素化蛋白的發現與結構………..…………....…..……11
（二）類泛素化家族…………………………………..…………..11
（三）類泛素化修飾功能……………………..…………………..12
（四）類泛素化修飾途徑………………..…..………………...….13
六、SUMO E3 ligase…………………………………..………………..14
（ㄧ）Polycomb group protein 2（Pc2）背景介紹…………..…..14
（二）Protein inhibitor of activated STAT y（PIASy）背景介紹..14
（三）KRAB-associated protein 1（KAP1）背景介紹…………..15
七、研究目的……………….……………………………….………….16
八、研究策略…………….……………………………….…………….16
貳、材料與方法…………….…………………………………………...19
ㄧ、質體（plasmid）DNA…………..……………………………19
二、細胞培養（Cell culture）………..……………………………19
三、基因轉移感染（Transfection）……….……….……………19
四、細胞同步化（Cell synchronization）………..……………….20
五、免疫沈澱法（co-immunoprecipitaion）……………………..20
六、西方默點法（Western Blot）………..………………………21
七、免疫螢光染色法（Immunofluorescense）……………………22
八、類泛素化結合實驗（Conjugation assay/SUMOylation assay）……..………………………………………..….…………..22
九、新生成染色質捕捉技術（Nascent Chromatin Capture assay）………..…………………………….…………..…………..23
參、結果…………………………..………...…………...………………24
ㄧ、SUMO E3 ligase -- Pc2和PIASy會增強HP1α類泛素化修飾…..……………………………………...…………...………..…24
二、SUMO E3 ligase -- Pc2會藉由SUMO E3 ligase活性增強HP1α類泛素化修飾………………………………………………...……...24
三、SUMO E3 ligase -- PIASy會藉由SUMO E3 ligase活性增強HP1α類泛素化修飾…………………………………………..........……25
四、細胞複製早、中S時期Pc2藉由SUMO E3 ligase活性增強HP1α類泛素化修飾……………………...…………………………...…25
五、細胞複製中、晚 S 時期PIASy藉由SUMO E3 ligase活性增強HP1α類泛素化修飾………...……………………………...……..26
六、在細胞複製 S 時期 HP1α和SUMO1類泛素化蛋白皆會結合在新生成的染色質上………………………………………......……27
七、在細胞複製 S 時期削弱Pc2的表現導致較少的 HP1α結合在新生成的染色質上……………………………………………..……28
八、削弱Pc2的表現降低HP1α坐落於異染色質區域的程度………………………………………………………...……...…28
九、在細胞複製 S 時期削弱Pc2的表現降低 HP1α坐落於異染色質區域的程度……………………….……………………...…..……29
十、削弱 Pc2 的表現導致微核的產生…………………………….…30
十一、KAP1和Pc2沒有交互作用……………………..………….….30
十二、KAP1和PIASy有交互作用………………………..……….…30
十三、在細胞複製S期KAP1透過PIASy的SUMO E3 ligase活性增強HP1α類泛素化修飾……………………………….…….….…31
十四、H3K27甲基轉移酶-- EZH2增強HP1α類泛素化修飾.……...32
十五、H3K27去甲基酶-- UTX和JMJD3不影響Pc2增強HP1α類泛素化修飾.………………………………………..………………...32
肆、討論……………………………………………..…………………33
伍、參考文獻…………………………………….……..………………40
陸、圖表……………..………………………………..…………………46
柒、附圖……………..………………………………..…………………64

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