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研究生：	童志偉 Tong, Chih-Wei
論文名稱：	藉由核內呼吸因子結合位的基因組分析定出參與神經突生長的新基因 Genome-Wide Analysis of Nuclear Respiratory Factor-1 Binding Sites Identifying Novel Genes Involved in Neurite Outgrowth
指導教授：	黃阿敏 Huang, A-Min
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	58
中文關鍵詞：	NRF-1反應片段、人類基因、啟動子、核酸干擾、神經突生長
外文關鍵詞：	NRF-1 response element, human genes, promoter, RNA interference, neurite outgrowth
相關次數：	點閱：187 下載：2
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核內呼吸因子(簡稱NRF-1)是調控粒腺體能量代謝相關基因的轉錄因子。近年來我們發現NRF-1在神經突生長的新功能；然而，有多少NRF-1的下游基因參與在這樣的多基因型性狀並不清楚。本篇研究利用隱馬爾可夫模型為基礎的全基因組分析方法來找出NRF-1的下游基因。我們利用電泳遷移率改變分析技術訂出一個錄用分數並且找出在人類神經母細胞瘤細胞株中有916個人類基因的啟動子上具有NRF-1反應片段(簡稱NRE)。其中，有691個基因是屬於已註解並且可分類成29種生物程序。我們挑選五個基因(MAPRE3、NPDC1、RAB3IP、TRAPPC3與SMAD5)並進行他們在神經突生長的功能分析。電泳遷移率改變分析技術、染色質免疫沉澱技術與點突變法的結果證實此五個基因的NRE對於NRF-1的結合是很重要的。啟動子活性分析與核醣核酸干擾技術更顯示了MAPRE3、NPDC1、RAB3IP與SMAD5的表現受NRF-1所調控。利用過表現候選基因來探討他們的功能，發現在NRF-1減少表現的細胞中，MAPRE3、NPDC1與SMAD5可以修復神經突長度的短少。因此，此篇研究顯示基因組型的生物資訊分析與生物性實驗的結合是一種可助於找出新NRF-1下游基因，並且參與神經突生長的有效方法。

Nuclear respiratory factor (NRF-1) is an important transcription factor known to regulate gene expression involved in mitochondrial biogenesis. We recently discovered a novel function of NRF-1 in neurite outgrowth; however, the number of NRF-1 target genes involved in this polygenetic phenotype remains unknown. This study employs a hidden Markov model-based genome-wide analysis approach to identify NRF-targeted genes. A total of 916 human genes containing the NRF-1 response element (NRE) in their promoter region in human neuroblastoma cells were identified using a cutoff score determined by results from electrophoretic mobility shift assays. Among them, 691 genes were annotated and categorized into 29 biological processes. Five genes, MAPRE3, NPDC1, RAB3IP, TRAPPC3, and SMAD5, were selected for functional analysis of their roles in neurite outgrowth. Results from electrophoretic mobility shift assays, chromatin immunoprecipitation, and site-directed mutagenesis confirmed that all NREs of these five genes are critical for NRF-1 binding. Promoter activity and RNA interference assays further demonstrated that expression of MAPRE3, NPDC1, RAB3IP, and SMAD5 were regulated by NRF-1. Functional studies using overexpressed full-length cDNA of candidate genes demonstrated that MAPRE3, NPDC1, and SMAD5 can rescue neurite outgrowth in NRF-1-knockdown cells. Overall, our data show that the combination of genome-wide bioinformatic analysis and biological experiments is a powerful approach to identify novel NRF-1-regulated genes, which play important roles in neurite outgrowth.

摘要	1
Abstract	2
致謝	3
Table of contents	4
I. Introduction	8
1-1. Physiological importance of neurite outgrowth	8
1-2. Molecules involved in regulation of neurite outgrowth	8
1-3. Nuclear transcription factor 1	9
1-4. Neural functions of NRF-1	10
1-5. Hypothesis	11
1-6. Specific aims	11
II. Materials and Methods	12
2-1. Genome-wide searching for NREs in human promoter sequences	12
2-2. Biological classification of genes containing putative NRE	12
2-3. Cell Culture	13
2-4. Electrophoretic mobility shift and supershift assays (EMSA)	13
2-5. Chromatin immunoprecipitation assay (ChIP)	14
2-6. Plasmid constructs	14
2-7. Transient transfection	15
2-8. Dual-luciferase assay	15
2-9. Lentiviral production and infection	16
2-10. Quantitative reverse transcription polymerase chain reaction (qRT-PCR)	16
2-11. Measurement of longest neurite length	17
2-12. Statistical analysis	17
III. Results	18
3-1. Genome-wide identification of human genes containing putative NRE in the promoter regions	18
3-2. Selection of candidate genes for biological confirmation	19
3-3. Interaction of putative NREs of five candidate genes with NRF-1 in vitro and in vivo	19
3-4. Putative NREs critical for regulating promoter activity	20
3-5. The mRNA levels of MAPRE3, NPDC1, RAB3IP, and SMAD5 regulated by NRF-1 silencing	21
3-6. MAPRE3, NPDC1, and SMAD5, but not RAP3IP mediate NRF-1-regulated neurite outgrowth in IMR-32 cells	21
IV. Discussion	23
4-1. Major findings	23
4-2. Putative NRF-1 targeted genes in the human genome	23
4-3. NRF-1 targeted genes are involved in different biological processes	24
4-4. Estimation of the number of NRF-1 regulated genes involved in neurite outgrowth in IMR-32 cells	25
4-5. NRF-1 regulates MAPRE3, NPDC1, RAB3IP, and SMAD5, but not TRAPPC3	26
4-6. The possible mechanisms of candidate genes in NRF-1-mediated neurite outgrowth	27
V. Conclusion	29
VI. References	30
VII. Tables	36
Table 1. List of 19 known human NREs	36
Table 2. Primers for NRF-1 binding experiments	37
Table 3. Primers for promter activtiy experiments	38
Table 4. Primers for construction of gene cDNA	39
Table 5. Primers for qRT-PCR	40
Table 6. Putative NRF-1 downstream genes (n = 691) categorized into 29 groups based on biological process.	41
Table 7. Information of 73 genes containing putative NRE whose core element conserved between human and mouse	42
Table 8. Alignment of partial promoter sequences of candidates in human (h) and mouse (m).	48
Table 9. Five putative NRF-1 downstream geness	49
Table10. Summary of this study	50
VIII. Figures	51
Figure 1. Genome-wide searching for NRF-1 target genes in the human genome and selection of candidate genes for biological confirmation.	51
Figure 2. Interaction of NRF-1 with putative NREs of different HMMER scores revealed by electrophoretic mobility shift assays.	52
Figure 3. The distribution of NREs on promoters of 691 annotated genes.	53
Figure 4. NRF-1 binding to NREs of MAPRE3, NPDC1, RAB3IP, TRAPPC3, and SMAD5 in vitro and in vivo.	54
Figure 5. Impairment of the promoter activity of MAPRE3, NPDC1, RAB3IP, TRAPPC3, and SMAD5 genes in IMR-32 cells by site-directed mutagenesis of the putative NRE.	55
Figure 6. Promoter activities of MAPRE3, NPDC1, RAB3IP, and SMAD5, but not TRAPPC3, regulated by NRF-1.	56
Figure 7. Decrease in the mRNA levels of MAPRE3, NPDC1, RAB3IP, and SMAD5 genes by knockdown of NRF-1 in IMR-32 cells.	57
Figure 8. Mediation of NRF-1-regulated neurite outgrowth in IMR-32 cells by MAPRE3, NPDC1, and SMAD5, but not RAB3IP.	58

                                    

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