Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97554
標題: PIASy 與 SUV39H1 促進 HP1α 類泛素化修飾在異染色質上功能之探討
The study of PIASy and SUV39H1 for HP1α SUMOylation during heterochromatin loading
作者: 鄭珮汝
Pei-Ru Cheng
關鍵字: 異染色質;類泛化修飾;Heterochromatin;HP1α;HP1α SUMOylation;PIASy;SUV39H1
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HP1α 類泛素化修飾在 pericentric heterochromatin 形成之探討。國立大學分子生物學研究所碩士學位論文。 葉,(2015). Pc2 和 PIASy 在細胞複製 S 期對 HP1α 類泛素化修飾之角色探討。國立大學分子生物學研究所碩士學位論文。
摘要: 
染色質分為真染色質以及異染色質,其中異染色質結構纏繞緊緻,可維持基因組的穩定性並調控基因表現。已知在異染色質形成的過程中,HP1α 會辨認並結合 H3K9me3 以幫助異染色質形成。此過程中 HP1α 需被類泛素化修飾 (SUMOylation)。SUMOylation 的過程中 SUMO E3 ligase 能夠專一性促進目標蛋白類泛素化,過去研究發現 SUV39H1 以及 PIASy 能夠促進 HP1α SUMO1 SUMOylation,但是其中詳細的機制仍不清楚。另外,實驗室發現 SUMO2、 SUMO3 能夠座落於 DAPI foci ,顯示 SUMOylation 在異染色質形成的過程中可能為重要的機制。因此本篇研究探討 PIASy 及 SUV39H1 在 HP1α 被不同 SUMO 蛋白 (SUMO1、 SUMO2 及 SUMO3) 類泛素化修飾的機制,並進一步探討 SUMO 蛋白對 HP1α 座落在異染色質區域的影響。首先,我利用 conjugation assay 觀察到 HP1α 被 SUMO2/3 的修飾較弱於 SUMO1 的修飾,並發現 SUV39H1 與 PIASy 可以增強 HP1α SUMO2/3 SUMOylation。為探討 PIASy 是否做為 HP1α 的 SUMO E3 ligase,我利用 co-immunoprecipitation (Co-IP),發現 HP1α 會透過 chromo shadowdomain (CSD) 與 PIASy 進行交互作用。當我使用失去 SUMO E3 ligase 活性的 PIASy RINGmt,發現 PIASy RINGmt 不僅無法促進 HP1α 的 SUMOylation,還會阻礙野生型 PIASy 促進 HP1α SUMOylation,顯示 PIASy 是做為 HP1α SUMO E3 ligase。另一方面,我發現沒有 E3 ligase 活性的 SUV39H1 也能夠與 PIASy 進行交互作用,暗示 SUV39H1 應藉由 PIASy 促進 HP1α SUMOylation。因此,我進一步探討並發現 PIASy RINGmt 能夠阻礙 SUV39H1 促進的 HP1α SUMOylation,表示 SUV39H1 是藉由 PIASy 促進 HP1α SUMOylation。另外,為了瞭解 HP1α SUMOylation 的功能,我構築 HP1α 的結構域,發現 HP1α chromodomain (CD)、hinge domain (HR) 以及 chromo shadow domain (CSD) 皆可以被 SUMOylation,暗示 HP1α 上不只一個 SUMOylation site。接著我將 HP1α 上可能為 SUMOylation site 的 lysine (K) 突變成 arginine (R),分析 HP1α 被 SUMOylation 的位點,結果顯示 HP1α K159R 的 SUMOylation 大幅減弱,表示 HP1α K159 是 SUMOylation site 之一。另外,我利用 HP1α- SUMO fusion protein 觀察是否能夠增加 HP1α 座落在異染色質區域,我發現當 SUMO protein fusion 在 HP1α 的羧基端時,會提高與 H3K9me3 重疊的比率,顯示 SUMOylation 對於 HP1α 上到異染色質區域十分重要。最後,為證明 HP1α SUMOylation 會影響到異染色質的穩定性,我發現 HP1α K159R 會提高微核的比例,顯示 HP1α 失去 SUMOylation 會降低染色體的穩定性。本篇論文發現: 1. PIASy 是 HP1α 的 SUMO E3 ligase,並且在促進 HP1α SUMOylation 的過程中 SUV39H1 可能參與在其中。2. HP1α 上不只有一個 SUMOylation site,而其中之一是 HP1α CSD 上的 K159。3. 當 SUMO protein fusion 在 HP1α 的羧基端時,會增加與 H3K9me3 coloczlize 的比率。4. HP1α K159R 會提高微核的比例,表示 HP1α 失去 SUMOylation 將可能降低染色體的穩定性。

Chromatin is differentiated into euchromatin and heterochromatin. The structure of heterochromatin is highly condensed, which can maintain genome stability and gene silencing. Previous study showed that HP1α binds H3K9me3 to facilitate heterochromatin formation. In the process, SUMOylation promotes HP1α targeting to heterochromatin regions. SUMO protein can specifically conjugate on target protein by SUMO E3 ligase. It has been shown that SUV39H1 and PIASy promote HP1α SUMO1 SUMOylation. However, the mechanism remains unknown. In our lab, we found that SUMO2, and SUMO3 were colocalized with DAPI foci, suggesting that SUMOylation is important for heterochromatin formation. In this study, I demonstrate the mechanism of PIASy and SUV39H1 promoting HP1α SUMOylation (SUMO1, SUMO2, SUMO3) and the importance of HP1α SUMOylation locating on heterochromatin. First, I observed PIASy and SUV39H1 promoted HP1α SUMO2/3 SUMOylation by conjugation assay. To determine if PIASy act as SUMO E3 ligase for HP1α SUMOylation, I found that HP1α interact with PIASy through chromoshadow domain (CSD) by co-immunoprecipitation (Co-IP). PIASy RINGmt, which lost SUMO E3 ligase enzyme activity, failed to promote HP1α SUMOylation. Moreover, PIASy RINGmt blocked wild type PIASy promoting HP1α SUMOylation, suggesting PIASy was SUMO E3 ligase for HP1α SUMOylation. However, SUV39H1, without SUMO E3 ligase enzyme activity, interacted with PIASy. I further found PIASy RINGmt blocked SUV39H1 promoting HP1α SUMOylation. These results indicated that SUV39H1 promoted HP1α SUMOylation through PIASy. In addition, I found all of the HP1α domains could be SUMOylated. This result showed there was more than one SUMOylation site on HP1α. I mutated HP1α lysine (K) residues, which might be SUMOylation sites, to arginine (R). The SUMOylation of HP1α K159R was decreased, indicating HP1α K159 was one of the SUMOylation sites. Furthermore, I observed the colocalization of HP1α and H3K9me3 was increased while SUMO protein was fused on the HP1α C-terminal region, suggesting SUMOylation was important for HP1α locating on heterochromatin. Finally, to investigate if HP1α SUMOylation affected heterochromatin stability, I found HP1α K159R induced micronucleus. This result supported that loss of HP1α SUMOylation decreased heterochromatin stability. In summary, PIASy is an HP1α SUMO E3 ligase and promotes HP1α SUMOylation ,which SUV39H1 may be involved in. There is more than one SUMOylation sites on HP1α, one of which is HP1α K159 on CSD. The colocalization of HP1α and H3K9me3 is increased while SUMO protein fused on HP1α C-terminal region. HP1α K159R induces micronuclei, suggesting loss of HP1α SUMOylation decreases heterochromatin stability.
URI: http://hdl.handle.net/11455/97554
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