| 摘要: | DNA甲基化在生物發育的進程中扮演了許多重要角色，而在DNA上添加或移除甲基則主要是經由DNA甲基轉移酶的催化作用。舉凡X染色體的去活化、親代基因傳遞控制、維持基因的安定度及分裂中的細胞裡基因的分佈也是經由一系列的甲基化來調控。由於甲基化以及甲基轉移酶功能對於維持基因體完整十分重要，因此，甲基化和轉移酶的損害或失調可導致許多遺傳性疾病、發育異常和癌症的形成。 許多研究證明DNA甲基化及去甲基化對於神經發育十分重要，神經元之間的連結與訊息傳遞以及因為甲基化的調控所表現出來的行為模式已經小有研究成果，雖然DNMTs被認為在其中也扮演了重要角色，但是到目前為止尚無充分證據證明其作用機制。但由於DNA甲基轉移酶的基因缺失在哺乳類動物模型中無法存活，故本計畫建立斑馬魚dnmt3aa及dnmt3ab基因剔除品系，進行焦慮相關行為分析。同時，我們也將 進行人類dnmt3a基因載入，在斑馬魚身上過度表達且活化甲基酶以進行過度表達此基因的行為探討。之後將利用基因修改後的斑馬魚品系，在晝夜不同時間點下，進行全基因的萃取及甲基化基因譜的分析，藉由系統化的研究，搭配目標基因之蛋白質功能的驗證與統整分析，了解DNMT3A的甲基化功能基因缺失或過度活化在焦慮行為與晝夜調節的關係。
DNA methylation plays an important role on epigenetic regulations, such as X chromosome inactivation, imprinting, neuronal proliferation, learning ability, and memorization. Generally, DNA methyltransferases, DNMTs, are the writers of epigenome. Among the DNMTs, DNMT3 is the major de novo methyltransferase that can change the DNA methylation pattern, especially associated with environmental-stimulated animal behaviors. Based on previous studies in mouse model, ablation of mammalian Dnmt3a has been reported to induce anxiety behaviors; however, the tissue specific mutagenesis was insufficient to characterize the innate function of DNMT3a. Due to lethal of dnmt3a-/- in mice, therefore, we first aimed to establish a zebrafish model to unveil the function of dnmt3a on modulating fish behaviors. We applied various endpoint examines to evaluate the behavior of the wild type, dnmt3aa and dnmt3ab knockout zebrafish. We found that dnmt3ab knockout fish showed anxiety behaviors, appeared less interested in social activity. Most interestingly, the circadian rhythm was dysregulated in KO fish. Both knockout fish exhibited higher locomotion activity during night time compared to wild type fish. Therefore, we hypothesized that the mutated methylated pattern caused anxiety-induced circadian rhythm. In order to comprehensively dissect the function of dnmt3aa and dnmt3ab, our next goal will be to establish human dnmt3a overexpressing transgenic zebrafish. With comparison of wild type, dnmt deficiency, and gain-of-function transgenic model, a whole genomic epigenetic profiling will be conducted as our Aim 3. With the serial evaluation of methylation status, we try to identify the methylation network between dnmt and circadian-related genes. In addition, brain neurotransmitter levels and tissue protein expression will be detected in Aim 4 to validate DNMT network with protein level. Taken together, our proposal will not only establish a platform to examine anxious behaviors in zebrafish model, but also fully clarify DNMT3a function associated with circadian rhythm. With the exploration of DNMT3a methylation network involving in circadian control and stress-induced anxiety, our proposal may provide solid evidence for physiological research on social behaviors. |
