Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97558
標題: SUMOylation 對 HP1β 座落到異染色質區域之功能的探討
The study of SUMOylation of HP1β in heterochromatin loading
作者: 劉思龍
Sih-Long Liu
關鍵字: 異染色質;類泛素化修飾;heterochromatin;Heterochromatin protein 1;HP1β;SUMOylation
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摘要: 
異染色質( heterochromatin )是細胞核中結構緊緻的染色質( chromatin )區域,能抑制基因的表現和維持基因組的穩定( Genome stability )。在過去的研究中,我們已經知道 Heterochromatin protein 1 (HP1) family 的蛋白在維持異染色質結構上扮演了重要的角色。HP1通過其胺基端的 chromodomain 辨認 H3K9me3 並與之結合,而後會吸引 SUV39h1 或 G9a 等組蛋白甲基轉移酶( histone methylatransferase )對周圍的組蛋白進行甲基化,生成 H3K9me3 進而使更多的 HP1 能結合到組蛋白上,通過其羧基端的 chromoshadow domain 與鄰近的 HP1 交互作用,維持異染色質的結構。而在這兩個功能域之間還存在一個可被進行類泛素化修飾( SUMOylation )的 hinge domain,目前對其功能我們只知道 HP1 家族中的 HP1α 需要被 SUMO1 修飾其 hinge domain 後才能在 S phase 穩定的結合到異染色質上,顯示類泛素化修飾參與在 HP1α 座落異染色質的過程。近年的研究發現剔除 HP1亞型 HP1β 會導致染色體的斷裂以及嚴重的 mitotic defects,顯示 HP1β 在維持基因組的穩定上扮演了重要的角色。然而, HP1β 座落到異染色質區域的機制仍有許多未知。由於 HP1β 與 HP1α 有著類似的序列,因此在本篇論文中我們假設 HP1β 可能也會受到類泛素化修飾的調控,增進其與異染色質穩定結合的能力,進而幫助 HP1β 座落到異染色質區域。為了證明這個假說。首先要偵測能對 HP1β 進行類泛素化修飾的 SUMO 家族成員有哪些,以及這些成員是否有參與 HP1β 座落到異染色質區域。我們利用 conjugation assay 顯示 SUMO1、SUMO2 與 SUMO3 皆能對 HP1β 進行類泛素化修飾。並在 NIH-3T3 細胞中削弱 SUMO1 與 SUMO3 的表現後利用免疫螢光顯示 HP1β 座落到異染色質區域的比例明顯降低。顯示 SUMO1 與 SUMO3 確實有參與在 HP1β 座落到異染色質區域的過程中,而後,為了構築不會被類泛素化修飾的 HP1β 突變蛋白,我們利用 conjugation assay 偵測 HP1β 會被類泛素化修飾的功能域為何,結果顯示 SUMO1 的修飾發生在 hinge domain,而 SUMO2 與 SUMO3 的修飾則在 hinge-chromoshadow domain。通過線上工具預測 SUMO1、SUMO2 與 SUMO3 的修飾位,並構築不會被類泛素化修飾的 HP1β 突變蛋白,我們發現 SUMO2 與 SUMO3 的修飾位在 HP1β 的第 155 個離胺酸,失去 SUMO2 與 SUMO3 的類泛素化修飾會明顯的降低 HP1β 座落到異染色質區域的比例並導致微核的產生。這個結果顯示 SUMO2 與 SUMO3 的類泛素化修飾有參與 HP1β 座落到異染色質的過程。而我們也同樣利用 conjugation assay 證明參與 HP1α 類泛素化修飾的 E3 連接酵素 PIASy 以及 SUV39H1 同樣能幫助 HP1β 的類泛素化修飾,顯示 HP1β 可能是以和 HP1α 同樣的機制進行類泛素化修飾。綜合以上結果顯示,(1) SUMO2 與 SUMO3 的類泛素化修飾參與 HP1β 座落到異染色質的過程。(2) SUMO2 與 SUMO3 的修飾位在 HP1β 的第 155 個離胺酸。(3) HP1β 可能通過和 HP1α 同樣的機制進行類泛素化修飾。

HP1 plays an important role in maintaining heterochromatin structure. In mammalian cells, there are three HP1 subtypes, termed HP1α, HP1β, and HP1γ. Although previous research mostly focused on HP1α, recent in vitro studies found that HP1β has the ability to condense chromatin. Moreover, HP1β knockout cells show serious mitotic defects in that chromosomes break at the pericentric regions. These results suggest HP1β has important functions on pericentric heterochromatin, so the mechanism of HP1β loading on pericentric regions becomes an important issue. However, how HP1β loading on pericentric regions remains poorly understood. Base on the studies that SUMOylation promote HP1α loading on pericentric heterochromatin and the similar structure between HP1β and HP1α, it is possible that SUMOylation of HP1β is involved in the loading on heterochromatin. Here, we show that SUMO1, SUMO2, and SUMO3 can SUMOylate HP1β in vivo. Knockdown either SUMO1 or SUMO3 decreased HP1β on DAPI foci in NIH3T3 cell. These results suggest SUMO1 and SUMO3 are involved in HP1β loading on pericentric heterochromatin. Immunofluorescence result showed that SUMO2/3 colocalized with HP1β and DAPI foci, but SUMO1 can not. Mapping the SUMOylation domains shows that SUMO1 SUMOylation was on the hinge domain and SUMO2/3 SUMOylation was on the hinge domain and chromoshadow domain. We use a web tool call 'GPS-SUMO' to predict the possible SUMOylation site. In our result, we found that K155 is a HP1β SUMO2 and SUMO3 SUMOylation site. Mutation on this site caused the percentage of HP1β locating on DAPI foci decreasing . And this mutation also increased the number of micronuclei containing cells. To demonstrate the mechanism of HP1β SUMOylation, our result show that PIASy and SUV39H1 increased HP1β SUMOylation. These factors found in HP1α SUMOylation mechanism. In summary, our results show that: (1) SUMO2 and SUMO3 SUMOylation are involved in heterochromatin loading of HP1β. (2) HPβ SUMO2 and SUMO3 SUMOylation site is K155. (3) HP1β shares the same SUMOylation pathway as HP1α.
URI: http://hdl.handle.net/11455/97558
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