POU3F2 mediated transcriptional regulation of human NTF3 gene

神經滋養因子三號Neurotrophin-3(NTF3) 是神經發育過程中重要的生長因子之一。神經發育過程中細胞表現NTF3並藉由結合上酪氨酸受器激酶C (TrkC receptor) 去調控並且維持神經細胞的功能，包括細胞生長存活、分化、 突觸的形成以及維持神經突觸可塑性 (synaptic plasticity) 。先前研究報告指出在神經系統中(central nerves system) ， NTF3會影響許旺氏細胞髓鞘化、軸突的成熟，也和記憶調控、神經修復、阿茲罕默症、帕金森氏症等神經退化的疾病有關。目前許多的研究都針對NTF3在神經系統中的影響作探討,對於NTF3本身所受到調控相關的研究相對較少。轉錄調控(Transcriptional regulation)是參與基因表現的重要步驟，不同的細胞由特定的轉錄因子調控，轉錄因子藉由結合DNA上特定的序列進而調控下游的基因表現，以調控細胞應有的特性。我們藉由生物資訊的方法去預測NTF3啟動子上(promoter)是否有與神經細胞相關的轉錄因子調控序列，並發現NTF3啟動子序列上有一個重要的神經轉錄因子”POU3F2”的調控序列。POU3F2因子大量表現於中樞神經系統，並且在神經發育和神經元的分化路徑裡扮演很重要的角色。目前已知POU3F2轉錄因子的功能影響神經系統的層面包含：許旺氏細胞髓鞘化、神經前趨細胞的分化刺激、小鼠腦部神經元區化訊號、刺激纖維母細胞轉換成神經元...等。為證實POU3F2在神經系統中會參與調控NTF3基因表現及其在神經發育中的影響，我們選擇了幾株內緣性表現POU3F2的細胞，並利用NT2/D1細胞株建立一神經分化的細胞模型進行相關實驗。我們首先利用啟動子分析(promoter assay)實驗分析NTF3啟動子各片段之活性，發現在不同的細胞株中，包含POU3F2調控序列的片段皆有增加啟動子活性之能力。電泳遷移率實驗(EMSA)更進一步證實POU3F2會與我們預測的調控序列結合。我們也在NT2/D1神經細胞中偵測不同分化時間點中NTF3及POU3F2的表現量。綜合以上結果，我們的研究發現了一個新的NTF3轉錄因子POU3F2，這些資訊將幫助我們更瞭解NTF3的轉錄調控，並有助於後續神經分化調控之相關研究。Neurotrophin-3 (NTF3) is a member of neurotrophin factors, a family of growth factors involved in nervous system and regulating diverse functions of central nervous system (CNS). NTF3 affects development of neurons in the embryo when it is expressed in human placenta and produced to maintain the adult nervous system functions, survival, differentiation, growth and synaptic plasticity. NTF3 also contributes in facilitating memory and implicated engaging in the pathophysiology of a wide variety of neurodegenerative and psychiatric disorders. The transcriptional regulation of NTF3 is poorly known. Here we hypothesize that POU domain, class 3, transcription factor 2 (POU3F2) may involves in the transcriptional regulation of NTF3 through bioinformatic prediction. POU3F2 is an important transcription factor that specifically expressed in central nervous system (CNS) and neuronal cell lineages and engaged in early stage of differentiation. POU3F2 regulates terminal hypothalamic neuronal differentiation and neuronal phenotypes, including activation of neuron specific gene expression, axonal excitation, and maintenance of neuronal viability. In current study, we try to validate the transcriptional regulation of NTF3 is modulated by POU3F2 and further to investigate its effect in neuron. At first, we establish an inducible neuron differentiation model by NT2/D1 cell line and confirm the characteristics of the differentiated neuron. Promoter-luciferase reporter assay is applied to examine transcriptional regulation role of POU3F2 on the promoter of NTF3 gene. The DNA-protein binding assay of NTF3 promoter and POU3F2 are also performed by EMSA assay. Furthermore, alteration of NTF3 and POU3F2 expression level during neuron differentiation process is also been measured. Results from this study will contribute to the understanding of POU3F2-mediated NTF3 regulation in CNS and provide important insights in related neuropsychiatric diseases.Abstract 1 中文摘要 2 1. INTRODUCTION 7 1.1. Neuronal development 7 1.1.1. Neural Stem Cell and Neurogenesis 7 1.1.2. Transcriptional regulation of neuron 8 1.2. Neurotrophin 9 1.3. Neurotrophin-3 (NTF3) 10 1.3.1. Molecular property of human NTF3 10 1.3.2. The biological function of NTF3 10 1.3.3 Pathogenic role of NTF3 11 1.4. POU (Pit-Oct-Unc) family of transcription factors 12 1.5. POU domain, class III, transcription factor 2 (POU3F2) 14 1.5.1. The function of POU3F2 14 1.5.2. POU3F2 regulation in neuron 15 1.6. Research objective 16 2. MATERIAL AND METHOD 17 2.1. Cell culture 17 2.2. Neuron induction 17 2.3. RNA isolation and Reverse transcription 17 2.4. Semi-Quantitative Polymerase Chain Reaction 18 2.5. Real-time Quantitative Polymerase Chain Reaction 18 2.6. Western Blot 19 2.7. Promoter assay construct and plasmid DNA extraction 19 2.8. Cell transfection and Luciferase Assay 20 2.9. Electrophoretic mobility shift assay (EMSA) 20 2.10. Statistical analysis 21 3. Result 22 3.1. Bioinformatic prediction on Neutrophin-3 promoter 22 3.2. Establishment of Ntera2/D1 derived neuron cell model 22 3.3. POU3F2 expression in neuron- related cell lines 23 3.4. Construction and Characterization of the human NTF3 promoter activity 24 3.5. Validation of the POU3F2 binding in NTF3 promoter region 25 3.6. Alteration of POU3F2 expression level under neuron differential 25 3.7. Alteration of NTF3 expression level under neuron differential 25 4. Discussion 26 4.1. The transcribes of NTF3 26 4.2. Transcriotional regulation of NTF3 27 4.3. POU3F2 associated Transcription factors in neural progenitor cells 28 5. Conclusion 29 6. Reference 30 Figure 1. NTF3 gene structure and promoter prediction 40 Figure 2. The mRNA expression level of neuronal marker during NT2/D1 cell differentiation 41 Figure 3. Morphological changes during NT2/D1 cell differentiation 42 Figure 4. POU3F2 protein is expression in neuron-related cell lines. 43 Figure 5. All constructs used in NTF3 promoter analysis. 44 Figure 6. Promoter reporter assay of NTF3 performed in three cell lines 46 Figure 7. EMSA of POU3F2-DNA binding complex 47 Figure 8. POU3F2 and NTF3 expression level during neuronal differentiation 48 Table 1. Oligonucleotides used in this study 5