探討DNA甲基化在Aeolosoma viride的前端再生過程中扮演的角色

Abstract

DNA甲基化是表觀遺傳中進行轉錄調控的重要因子，可以在不改變基因序列的情況下調控基因的表現。在細胞不同的分化過程中，DNA甲基化可以調控特定基因的表現，進而在一些生物中進行生命現象的調節，例如幹細胞自我更新與去分化等過程。瓢體蟲(Aeolosoma viride)是一種具有很強再生能力的環節動物，本研究用它來了解DNA甲基化與再生的關聯，本論文已於瓢體蟲的transcriptome database及體內找到 DNA甲基轉移酶1 (dnmt1)、DNA甲基轉移酶2 (dnmt2)和甲基CpG結合域(Methyl-CpG-binding domain, mbd)的序列，並且針對open reading frame完成cloning。接著利用ELISA及超高效液相層析法(UPLC) 測量瓢體蟲的DNA甲基化程度，根據實驗結果，於瓢體蟲體內確定有低DNA甲基化存在，另外，也利用定量聚合酶鍊反應(qPCR)檢測出Avi-dnmt1、 Avi-dnmt2、 和 Avi-mbd在瓢體蟲再生過程中第48小時具有最高的基因表現量，也藉由全胚體原位雜交技術（Whole-mount in situ hybridization, WISH）發現Avi-dnmt1於前端再生過程中會在再生端表現。此外，瓢體蟲的再生顯著地受到DNA甲基化抑制劑5-Azacytidine影響，檢測其前端再生的甲基化程度時，可以看到再生前期的甲基化程度相較於未再生個體有下降的表現。因此瓢體蟲的再生是會受到DNA甲基化的調控。

DNA methylation is an essential epigenetic factor for transcriptional regulation. It can regulate gene expression without altering the genetic sequences. At different developmental stages, DNA methylation can potentially regulate the expression of specific genes, and further modulate biological process such as the renewal and dedifferentiation in stem cells. Aeolosoma viride, an annelid with strong regeneration capacity was used to study the relationship of DNA methylation and regeneration. In this study, DNA methyltransferase 1 (dnmt1), DNA methyltransferase 2 (dmnt 2) and Methyl-CpG-binding domain (mbd) in A. viride have been identified and cloned. To examine the level of DNA methylation in the genome, DNA methylation level was detected by using both ELISA and Ultra-Performance Liquid Chromatography (UPLC). Accordingly, DNA methylation did exist in A. viride. Furthermore, quantitative polymerase chain reaction (qPCR) was used to detect and found that Avi-dnmt1, Avi-dnmt2, and Avi-mbd all had the highest gene expression level at 48 hour-post-amputation (hpa) in the regeneration process. By WISH result, the gene expression of Avi-dnmt1 clearly showed at regeneration side during anterior regeneration. In addition, regeneration of A. viride was significantly affected by 5-azacytidine, a DNA methylation inhibitor treatment. Therefore, DNA methylation is involved in the anterior regeneration of A. viride.