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研究生：	許家豪 Hsu, Chia-Hao
論文名稱：	GTPase相關基因參與NRF-1所調控的神經突生長功能 GTPase related genes mediate the function of NRF-1 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：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	60
中文關鍵詞：	神經突生長、轉錄因子
外文關鍵詞：	NRF-1, RHOA, SMAP1, RAPGEF6, neurite outgrowth, transcription factors
相關次數：	點閱：158 下載：2
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Nuclear respiratory factor 1（簡稱NRF-1）是人類基因組中重要的轉錄因子。近年來，我們發現NRF-1具有調控神經突生長的新功能。藉由生物資訊方法，我們發現有2,100多個基因其啟動子區域含有預測的NRF-1 responsive element（簡稱NRE），其中30個基因其NRE序列在人與小鼠兩物種完全相同。在這30個基因當中，RHOA，SMAP1和RAPGEF6三者為GTPase相關的基因。因此我們假設這3個基因為NRF-1的下游基因而且參與NRF-1所調控的神經突生長功能。我們利用electrophoretic mobility shift assay 和chromatin immunoprecipitation分別證明在試管內以及細胞內這3個基因之NRE會和NRF-1有所鍵結。在人類神經纖維母細胞株IMR-32中，調降NRF-1的表現會造成RHOA和SMAP1兩基因mRNA表現量增加，但造成RAPGEF6 mRNA表現量減少。調降RHOA的表現本身可促進神經突生長，但是不會進一步促進NRF-1所誘發的神經突生長。調降SMAP1的表現本身亦可促進神經突生長，但是也會進一步促進NRF-1所誘發的神經突生長。反之，調降RAPGEF6的表現本身可減少神經突生長，並進一步降低NRF-1所誘發的神經突生長。以上結果指出RHOA，SMAP1和RAPGEF6此三個GTPase相關基因均為NRF-1所調控的下游基因，但是只有SMAP1和RAPGEF6參與在NRF-1所調控的神經突生長。

Nuclear respiratory factor 1 (NRF-1) is one of the major transcription factors in the human genome. Recently, our lab has indentified a novel function of NRF-1 in neurite outgrowth. Using bioinformatic analyses, we have found that ~2,100 genes have putative NRF-1 response element (NRE) in their promoter regions and 30 of them have identical NRE between the human and mouse. GTPase related genes, RhoA, SMAP1 (small ArfGAP1) and RAPGEF6, are 3 of these 30 genes. Therefore, we proposed that human RhoA, SMAP1 and RAPGEF6 are downstream from NRF-1 and mediate the function of NRF-1 in neurite outgrowth. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that NREs of RhoA, SMAP1 and RAPGEF6 are bound by NRF-1 in vitro and in vivo. In neuroblastoma IMR-32 cells, knockdown of NRF-1 positively regulates RhoA and SMAP1 mRNA levels but negatively regulates the RAPGEF6 mRNA level. Knockdown of RhoA alone in IMR-32 cells increases neurite outgrowth, but does not further increase NRF-1 induced neurite outgrowth. Knockdown of SMAP1 alone increases neurite outgrowth, and further increase NRF-1 induced neurite outgrowth. However, knockdown of RAPGEF6 alone decreases neurite outgrowth and further decreases NRF-1 induced neurite outgrowth. These results suggest that human RHOA, SMAP1 and RAPGEF6 are genes downstream from NRF-1 and only SMAP1 and RAPGEF6 involve in NRF-1-mediated neurite outgrowth.

Table of contents
Abstract	1
摘要	2
致謝	3
Table of contents	5
I. Introduction
1-1. Physiological importance of neurite outgrowth	9
1-2. Molecules involved in the regulation of neurite outgrowth	10
1-3. Nuclear transcription factor 1	12
1-4. Neural functions of NRF-1	14
1-5. Using Bioinformatic approach to search for genes that are regulated by  NRF-1	15
1-6. GTPase related genes in regulation of neurite outgrowth	16
1-7. Hypothesis	17
1-8. Specific aims	17
II. Materials and methods
2-1. Cell culture	18
2-2. Preparation of nuclear extracts	18
2-3. Gel electrophoretic mobility shift assay (EMSA)	19
2-4. Chromatin immunoprecipitation (ChIP)	20
2-5. RNA isolation and Reverse Transcription (RT)-PCR	21
2-6. Real-time PCR	21
2-7. Recombinant Lenti-virus production	22
2-8. Recombinant Lenti-virus infection	22
2-9. Transient transfection	23
2-10. Neurite outgrowth assay	23
2-11. Statistical analysis	24
III. Results
3-1. NRF-1 binds to the putative NREs of human RHOA, SMAP1 and RAPGEF6 in vitro	25
3-2. NRF-1 binds to the putative NREs of human RHOA, SMAP1 and RAPGEF6 in vivo	26
3-3. NRF-1 regulates mRNA levels of RHOA, SMAP1 and RAPGEF6 in IMR-32 cells	26
3-4. Knockdown of RHOA increases neurite outgrowth but does not further increases NRF-1 induced neurite outgrowth	27
3-5. Knockdown of SMAP1 increases neurite outgrowth in IMR-32 cells as well as increases NRF-1 induced neurite outgrowth	28
3-6. Knockdown of RAPGEF6 decreases neurite outgrowth in IMR-32 cells as well as decreases NRF-1 induced neurite outgrowth	28
IV. Discussion
4-1. Major findings	30
4-2. Conservation of promoter sequences between human and mouse RHOA, SMAP1 and RAPGEF6	30
4-3. Expression of RHOA and its function in neurite outgrowth	31
4-4. Expression of SMAP1 and its function in neurite outgrowth	32
4-5. Expression of RAPGEF6 and its function in neurite outgrowth	33
4-6. Working hypothesis for the mediation of RHOA, SMAP1 and RAPGEF6 in NRF-1 induced neurite outgrowth	35
4-7. Conclusion	35
V. References	37
VI. Figures
Figure 1. The Bioinformatic strategy for searching for human genes that have putative NRF-1 response element (NRE) in their promoter regions	42
Figure 2. The time points for neurite outgrowth assay	43
Figure 3. NRF-1 binds to putative NREs of human RHOA, SMAP1 and RAPGEF6 in vitro as revealed by gel electrophoresis mobility shift assay	44
Figure 4. NRF-1 binds to putative NREs of human RHOA, SMAP1 and RAPGEF6 in vivo as revealed by chromatin Immunoprecipitation assay	45
Figure 5. Knockdown of NRF-1 up-regulates RHOA and SMAP1 but down-regulates RAPGEF6 mRNA level in IMR-32 cells	46
Figure 6. Knockdown of RHOA increases neurite outgrowth but does not mediate NRF-1 induced neurite outgrowth	48
Figure 7. Knockdown of SMAP1 increases neurite outgrowth and further increases NRF-1 induced neurite outgrowth	49
Figure 8. Knockdown of RAPGEF6 decreases neurite outgrowth as well as NRF-1 induced neurite outgrowth.	50
Figure 9. Working hypothesis for the mediation of RHOA, SMAP1 and RAPGEF6 in NRF-1 induced neurite outgrowth	51
Figure 10. Conservation of the NRF-1 binding elements in RHOA, SMAP1, and RAPGEF6 promoter regions between human and mouse	53
VII. Appendix
Table 1. NRF-1 downstream genes involve in mitochondrial biogenesis	54
Table 2. NRF-1 and E2F downstream genes that are cell cycle related and implicated in neuronal differentiation	55
Table 3. NRF-1 downstream genes in different function and implicated in neuronal differentiation	56
Table 4. Primer sequences for EMSA	57
Table 5. Primer sequences for ChIP	58
Table 6. The primer sequences for semi-quantitative or real-time RT-PCR.	59
Table 7. The shRNA clones for RHOA, SMAP1 and RAPGEF6	60

                                    

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