Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92152
標題: 一、 PAX3坐落於染色體之功能性探討 二、 PAX3位於DAPI密集區之功能性探討
1. Functional studies of PAX3 loading on mitotic chromosome 2. Functional studies of PAX3 locating on DAPI-dense foci
作者: Tsu-Fang Wu
吳祖方
關鍵字: 一、 有絲分裂
染色體
轉錄因子
瓦登伯革氏症候群
精胺酸甲基化修飾 二、 異質染色質
染色質複製時期
轉錄因子
1. Mitotic phase
mitotic chromosome
PAX3
Waardenburg Syndrome
PRMT5
Arginine methylation 2. Heterochromatin
S phase
DNA replication
PAX3
PARP1
DAPI-dense foci
pericentromeric heterochromatin
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摘要: 1. Functional studies of PAX3 loading on mitotic chromosome Mitosis is a stage where global chromatin structure is changed in the organization and nuclear architecture. During this stage, most transcription factors and co-factors exit from mitotic chromosome and transcription ceases. Although some sequence-sepcific transcription factors remain bound selective sites of the mitotic chromosome, which serves as bookmarking for rapidly gene regulation after mitosis, the details of transcription factors associating on mitotic chromosome are still unclear. Here, we identify global loading of paired box 3 (PAX3) on mitotic chromosome through its homeodomain binding. Waardenbug syndrome associated mis-sense mutations in the homeodomain of PAX3, result in the loss of the ability to load on mitotic chromosome. The dynamic association of PAX3 on mitotic chromosome is regulated through arginine methylation of PAX3 by Protein arginine methyltransferase 5 (PRMT5) and Jumonji domain-containing 6 (JMJD6). Loss of arginine methylation or mutation in the homeodomain of PAX3 disrupts mitotic chromosome association. Fluorescence resonance energy transfer (FRET) analysis determines conformation change of PAX3 from interphase to mitotic phase. Hence, PAX3 exiting mitotic chromosome leads to cell dividing abnormal and decreased cell proliferation. These might influence the body shape of larva in embryo development. 2. Functional studies of PAX3 locating on DAPI-dense foci Proper re-establishment of heterochromatin after each round of DNA replication is critical to the preservation of cell identity. It was found that paired box 3 (PAX3), a transcription factor important in embryo development, mediates the formation of pericentromeric heterochromatin. However, how PAX3 recognizes the heterochromatic environment and re-establishes it after DNA replication remains unclear. In this report, we found that PAX3 binds pericentromeric heterochromatin during middle-to-late S phase. Loading of PAX3 onto pericentromeric heterochromatin requires poly(ADP-ribose) polymerase 1 (PARP1). Furthermore, loss of PAX3 or PARP1 delays cell cycle progression through S phase. Our results reveal how PAX3 recognizes and maintains pericentromeric heterochromatin at S phase of the cell cycle.
一、 PAX3坐落於染色體之功能性探討 有絲分裂時期是染色質結構全面性改變的階段。在此時期,幾乎所有的轉錄因子和輔因子均會離開特定或非特定染色質區域而停止其作用。雖然仍有某些特殊轉錄因子部份坐落於有絲分裂染色體上,作為從有絲分裂之後使特定基因迅速表現的書籤角色,然而轉錄因子坐落於染色體的真實功能目前仍不清楚。 在本篇研究中,我們確定了PAX3坐落於整個染色體上是藉由其homeodomain的結合能力。測試與Waardenbug syndrome相關的missense突變的PAX3在有絲分裂時期的分佈,發現這些PAX3突變蛋白會喪失與染色體結合的能力。 而PAX3動態結合在染色體上是藉由PRMT5和JMJD6對PAX3上的arginine做甲基化修飾調控,PAX3的arginine失去甲基化修飾或其homeodomain突變會PAX3失去在染色體上的結合能力。螢光共振能量轉移 (FRET) 分析確定PAX3從細胞間期到有絲分裂期會產生結構性的改變。因此,我們認為PAX3失去與染色體的結合能力會導致細胞分裂異常、減少細胞增殖、並可能會影響胚胎發育導致形體失常。 二、 PAX3位於DAPI密集區之功能性探討 細胞分裂後,正確的異質染色質形成決定了子代細胞與母代細胞的相似性。而於染色質複製時期母股的遺傳訊息能否被忠實地複製於子股攸關異質染色質的正確形成與否。已知PAX3是早期胚胎發育中一個重要的轉錄因子,並與鄰近著絲點的異質染色質形成息息相關,然而參與此異質染色質形成的功能與確切的時間點是在於染色質複製時或之後,目前仍未明瞭。 在本篇報告中,我們發現PAX3於染色質複製的中與中後期接合在鄰近著絲點的異質染色體上,接合於異質染色體時需要PARP1的參與;功能性研究發現缺少PAX3和PARP1造成細胞週期延遲在染色質複製時期。 我們的研究闡明PAX3如何接合在鄰近著絲點的異質染色質,且影響鄰近著絲點的異質染色質形成是在染色質複製的中與中後期。
URI: http://hdl.handle.net/11455/92152
文章公開時間: 10000-01-01
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