사람 tyrosine hydroxylase 유전자의 발현에서의 Nurr1에의한 세포 특이적인 조절작용
Differential regulation of transcriptional activity by Nurr1 in expression of human tyrosine hydroxylase gene
- 주제(키워드) Nurr1 , human TH , Human NSCs
- 발행기관 아주대학교
- 지도교수 이명애
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 석사
- 학과 및 전공 일반대학원 신경과학기술과정
- 본문언어 영어
초록/요약
Nuclear receptors represent a large family of transcriptional factors involved in development, differentiation, homeostasis, and cancer. Nurr1, a member of the orphan nuclear receptor superfamily, was recently demonstrated to be of critical importance in the developing central nervous system, where it is required for the generation of midbrain dopaminergic cell. Although its role during dopaminergic neurogenesis has been extensively studied, its transcriptional regulation of TH, the rate-limiting enzyme of dopamine biosynthesis, is not well understood. Here we show that Nurr1 directly mediated transcriptional activation or repression of TH gene through NBRE-A site and a key regulating complex of TH expression, that may be formed in NBRE-A site during dopaminergic neurogenesis. In recent years, a growing number of cofactor has been discovered that participate in the regulation of the transcriptional activity of promoter. So, we present the identification of a cofactors binding in and around NBRE-A, which differentially regulates the transcriptional activity of the human TH promoter in human neural stem cell, F3, and human neuroblastoma cell, SH-SY5Y, we are preformed 2-D gel electrophoresis and LC peptide sequencing analysis. Together, these data suggests that Nurr1 and cofactors may regulate differentially transcription activity of TH promoter in neural stem cell and dopaminergic neurons. Keywords: tyrosine hydroxylase, Nurr1, doapminergic neuron, neural stem cell, neuroblastoma cell.
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TABLE OF CONTENTS
ABSTRACT ………………………………………………………………… 4
TABLE OF CONTENTS …………………………………………………..... 5
LIST OF FIGURES ……………………………………………………….... 7
I. INTRODUCTION …………………………………………………….…. 8
II. MATERIAL AND METHODS ……….…………………………….…… 11
1. Cell culture ……………………...….…………………………….......... 11
2. Plasmid constructs ............................………………………………….. 11
3. Transient Transfection …………….…………….…………..………… 12
4. Luciferase assay ………………….…………………….…...………… 13
5. Biotinylated Oligonucleotide-streptavidin Pull –Down …..….….…..... 13
6. Western blot analysis …………….………….……………………….... 14
7. Two-dimensional PAGE ………………………………………………. 15
8. Protein visualization and image analysis ………………………...…… 16
9. In-gel digestion ……..............………………………………......…….. 17
10. Mass spectrometry and database search …………………..………....... 18
III. RESULTS ……………………….…………………………..……….... 19
1. Differential regulation of human TH gene by Nurr1 in cell-type
dependent Manner ………….………………………...………………… 19
2. The upstream promoter region of the hTH gene, mediating full
responsiveness to transcriptional regulation by Nurr1, contains
multiple NBRE-like sequence motif ……..…………………………….. 21
3. NBRE-A region is critical for transregulation of the TH promoter
Activity by Nurr1 …………………………………………….…………. 22
4. HDAC- independent inhibition of Nurr1-mediated hTH promoter
transcriptional repression in F3 cell line ……..…………………………. 24
5. Purification of Nurr1 and recruitment cofactor complex in
NBRE-A region ………..……………………………..………..…….... 25
6. Protein differentially expressed in human neural stem cells and
human neuroblastoma cells by Nurr1 ………………………………...…. 26
IV. DICUSSION ………….……………………...………………………… 28
V. REFERENCES ….……………………..……………………………….. 42
국문요약 …………………………………….……………………………. 47
Acknowledgement …………………………………………………………. 48
