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標題: | 探討後轉錄因子WDHD1和ADAR1在細胞生長及分化中之角色 Epigenetic regulation of cell growth and differentiation by post-transcriptional regulators WDHD1 and ADAR1 |
作者: | Chia-Ling Hsieh 謝佳玲 |
指導教授: | 呂勝春 |
關鍵字: | 表關遺傳,肌生成作用, WDHD1,ADAR1,epigentics,myogenesis, |
出版年 : | 2012 |
學位: | 博士 |
摘要: | WDHD1調控著絲點沉默路徑之後轉錄階段
著絲點是一個高度特化的染色體要素,對於染色體在有絲分裂時的分離扮演很重要的角色。我們新近的努力發現一個具有WD40-domain和 HMG-domain蛋白, WDHD1, 是個不可缺的與著絲點異染色質(centromeric heterochromatin)結合之蛋白。我們的研究也發現這個蛋白和著絲點的沉默(centromere silencing) 以及異染色質結構的穩定有關。抑制WDHD1 蛋白表現會改變與這個染色質區域相關的表觀基因標誌(epigenetic marker)之表達模式。因此,這樣的結果會破壞著絲點上之異染色質狀態, 進一步造成有絲分裂的損壞。更進一步的,我們也探討了WDHD1在其中所扮演的機制,結果發現WDHD1對於著絲點區域之DNA衛星重複序列(satellite repeats)所製造出來的非編碼小RNAs (non-coding RNAs) 的生成扮演非常重要的角色。這樣的過程可能是藉由穩定Dicer和著絲點RNA的結合,而經由後轉錄模式的調控來逹成。因此,這些結論表明了WDHD1可能是個參與在經由RNA調控表觀遺傳的機制中之重要因子,使維持著絲點完整結構的。 ADAR1調控C2C12成肌細胞之肌生成作用 腺苷脫氨酶1(ADAR1)會催化雙股RNA上的腺苷(adenosine) (A)去胺基成為次黄嘌呤核苷(inosine) (I)。已知ADAR1具有兩個都有RNA編輯功能的同工型(isofroms),一個是分子量150kDa的干擾素(interferon)誘導表現的ADAR1 p150,另一個是持續表現的ADAR1 p110。兩者都可以位在細胞核,只有p150能位在細胞質。A變成I的RNA編輯作用不只影響了RNA轉譯成蛋白質的密碼子或是其結構,也調控了反轉錄轉位子(retrotransposons)和基因的沉默(gene silencing)。在這個研究中,我們確認在骨骼肌成肌作用之調控中ADAR1的新穎生物作用。我們觀察骨骼肌成肌細胞C2C12在由成肌細胞變成肌小管(myotube)的分化過程中ADAR1的表現。在細胞聚滿(cell confluence)和分化初期ADAR p150會短暫的增加,並且兩種ADAR1的表現在隨後形成肌小管時都減少。然而,我們發現分化過程中ADAR1 p150的短暫增加是由於干擾素調控之反應片段Interferon stimulated response element (ISRE)所影響的干擾素訊息傳導路徑造成,並且隨後在分化的過程中由MyoD抑制其表現。有趣地,抑制ADAR1或是過量表現其起催化作用的區域(catalytic domain)突變的蛋白質造成成肌作用中的指標蛋白質myogenin (MyoG)和myosin heavy chain (MHC)的表現減少,說明了在細胞核中ADAR1 p110所調控的RNA編輯作用在早期成肌作用中的重要性。另外,ADAR1 p150在分化早期的作用可能是參與調節PKR所影響的細胞程式凋亡(apoptosis)。然後我們證實在分化後期ADAR1蛋白質表現減少是由於miR-1/206所引起的抑制作用。然而,在分化後期異常的ADAR1過度表現則會造成不正常的肌小管形成,更進一步指出在成肌過程中ADAR1的階段性特殊作用。總而言之,我的們結果指出在這過程中ADAR1所參與的調控,可能藉由編輯未知的轉錄本,此轉錄本對於預定的骨骼肌成肌作用之發展是必需的。 WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1a localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1’s involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability. ADAR1 regulates the myogenic program in C2C12 myoblast ADAR1 (adenosine deaminase acting on RNA 1) catalyzes the deamination of adenosine to inosine on RNA molecules of double stranded structure. Two isofroms of ADAR1 are known, both of which possess RNA-editing activity: an interferon (IFN) inducible 150 kDa protein (p150) and a constitutively expressed amino-terminally truncated 110 kDa protein (p110). The p150 isoform is found in both the cytoplasm and nucleus, while the p110 protein is localized predominantly in the nucleus. A-to-I RNA editing not only affects targeted transcripts by altering the sequence and/or structure of the encoded products, but also serves to regulate retrotransposons and gene silencing. In this study, we identified a novel aspect of biological function of ADAR1 in the regulation of skeletal myogenesis. We have investigated ADAR1 expression during myoblasts–myotubes differentiation in myoblast cell line C2C12: ADAR1 p150 expression transiently increased during cell confluence and initial differentiation process, whereas the expression of both ADAR1 forms dropped in the differentiated myotubes. Furthermore, we discovered that ADRA1 p150 transient up-regulation in early differentiation is mediated by the IFN signaling pathway-associated interferon stimulated response element (ISRE) and subsequently repressed by the myogenic transcription factor MyoD. Intriguingly, knockdown of ADAR1 or overexpression of a catalytic mutant reduced the expression of myogenesis-associated markers, demonstrating the significance of ADAR1 p110-mediated nuclear RNA editing in the early myogenic program. On the other hand, the function of ADAR1 p150 in early stage of differentiation possibly lies in the regulation of PKR-mediated apoptosis. Next, we demonstrated that in the late stage of myogenesis, miR-1/206-mediated inhibition facilitated the down-regulation of ADAR1 proteins. However, ectopic ADAR1 over-expression at this stage caused abnormal myotube formation, further implying stage-specific functions of ADAR1 in the myogenic process. Taken together, our results pointed to the scenario that ARAR1-mediated regulation, possibly through editing of as yet unknown transcripts, is essential for scheduled progression of skeletal myogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16522 |
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