神經系統對動物至關重要，透過將信號傳遞到身體各個部位來調節不同的行為。大腦一旦受到傷害，會導致記憶力減退、運動平衡、認知功能障礙等等。與周邊神經系統不同，中樞神經系統表現出較差的再生能力。由於受影響神經元的內在再生潛力降低、髓鞘相關抑制分子增加和CNS損傷後觀察到的神經膠質瘢痕等等，以上都是導致中樞神經系統再生能力較差的原因。先前的研究指出，去除外在抑制分子不足以有效改善軸突再生。因此，我們把注意力放在可能會促進受損腦神經元再生的分子機制上。我們實驗室在先前的實驗已將WNT3A基因鑑定為有潛力神經再生相關基因。此外還預測了負責增加WNT3A表達的新穎增強子區域。鑑於高水平的增強子RNA（eRNA）傾向於從活性增強子轉錄，我們觀察了新穎增強子區域eRNA的表達。結果顯示在神經再生期間，來自新增強子區域（e7）的eRNA表現率增加。為了進一步研究調節WNT3A表達的機制，利用染色質免疫沉澱測定法(ChIP assay)分析受損皮質神經元在再生期間WNT3A增強子E7區域的組蛋白修飾（H3K4me1和H3K27ac）。eRNA已被證明是透過募集RNA聚合酶II和轉錄激活因子（例如p300）來驅動增強子 - 啟動子環化結構。因此，eRNA驅動的DNA環化通過組蛋白修飾而增強基因啟動子的活化並進一步增加基因表達。為此，我們計劃進行Clustered Regularly Interspaced Short Palindromic Repeats（CRISPR）實驗，以刪除目標增強子，看它是否影響WNT3A的表達。之後使用PC12細胞確認CRISPR的效率，並在皮層神經元中進行此實驗。

The nervous system coordinates animal behavior through transmitting signals to various part of the body. Injury of the brain causes memory loss, motor balance, and cognitive dysfunction. Unlike the peripheral nervous system, the central nervous system exhibits poor regeneration capacity due to decreased intrinsic regeneration potential of affected neurons, increased myelin-associated inhibitors and glial scar observed after CNS injury. Previous studies reveal that, removing the extrinsic inhibitory molecules is not enough for regeneration of long-distance axon. Thus, we focus on the intrinsic molecular mechanisms that may promote regeneration of injured brain neurons. Based on the preliminary results, our laboratory identified WNT3A gene as a promising regeneration associated gene. A novel enhancer region responsible for the increased expression of WNT3A was also predicted. Given that high level of enhancer RNAs (eRNAs) tend to be transcribed from active enhancers, we examined the expression of eRNAs transcribed from predicted novel enhancer region. Our results showed increased eRNAs from a sub-region (e7) of the novel enhancer during regeneration. To further study the mechanism that regulates the expression of WNT3A, histone modifications (H3K4me1 and H3K27ac) of the e7 region of WNT3A enhancer during regeneration of injured cortical neuron were analyzed using chromatin immunoprecipitation assays. eRNA is known to drive enhancer–promoter looping architecture through recruitment of RNA polymerase II and transcriptional activators (e.g. p300). eRNA-driven DNA looping would thus enhance the activation of gene promoters via histone modification and further increase gene expression. To this end, we plan to perform clustered regularly interspaced short palindromic repeats (CRISPR) assays to delete the target enhancer and see if it affects the expression of WNT3A. The deletion efficiency was confirmed using PC12 cells and will be confirmed in the primary cortical neurons.

目錄
摘要 2
Abstract 3
致謝 4
目錄 6
Introduction 8
Traumatic brain injury (TBI) 8
Regeneration of damaged Central nervous system (CNS) 9
WNT signaling pathway and WNT3A gene 10
Epigenetic regulation via DNA methylation and histone modification 12
Mechanism of enhancer regulation 15
Role of enhancer RNA (eRNA) in transcription 16
Materials and Methods 18
Reagents and antibodies 18
Experimental animals and primary cortical neurons culture 19
Cell culture 19
CRISPR/Cas9 constructs 20
Experimental injury assay 20
Total RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), Polymerase chain reaction (PCR) and real-time polymerase chain reaction(qPCR) 20
DNA Agarose Gel Electrophoresis and eRNA expression quantification 21
Chromatin immunoprecipitation (ChIP) assays 21
CRISPR/Cas editing 22
Statistical analysis 23
Results 24
Expression of eRNAs transcribed from the predicted enhancer for WNT3A gene 24
Histone modifications at e7 region during neuronal regeneration 25
Gene editing at the e7 enhancer region 26
Conclusion 27
Discussion 28
附圖 32
Table 1. List of primers sequences for PCR and qPCR 32
Table 2. List of primers sequences for ChIP assay qPCR 33
Table 3. List of sgRNAs sequences for CRISPR/Cas9 assay 34
Figure 1. Investigation of the expression of eRNAs that are derived from predicted enhancer for WNT3A gene 35
Figure 2. Histone modifications at e7 region during neuronal regeneration 38
Figure 3. Gene editing of the e7 enhancer region 41
Figure 4. Proposed enhancer regulation for WNT3A gene expression 44
Figure 5. Investigation of the expression of new e7 eRNAs that are derived from predicted enhancer for WNT3A gene 45
References 47

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