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미분화 신경줄기세포에서 BTK와 Nurr1의 상호작용에 관한 연구

BTK functions as a corepressor of Nurr1 in human neural stem cells

  • 박명선 (Park, Myung-Sun, The Graduate School, Ajou University)

초록/요약

파킨슨 병은 주로 중뇌의 substantia nigra에서 도파민 신경세포의 소멸로 인해 발병된다. 카테콜아민 생합성 과정에서 속도 조절 효소인 tyrosine hydroxylase (TH) 유전자의 발현은 신경세포의 발달과정동안 전사과정과 다양한 환경적인 자극에 의해 조절된다. 또한 Orphan nuclear receptor 중 하나인 Nurr1은발달 과정 중 중뇌의 substantia nigra와 ventral tegmental 부위에서 발현되고,도파민 신경 세포 전구체가 완전한 도파민 신경세포로 분화하는데 중요한 역할을 한다. Nurr1은 발달 과정 동안 TH 유전자의 발현을 유도하는데 중요한 전사인자라고 생각된다. 우리는 TH 유전자의 발현에서 세포특이적인 전사 조절을 하고, Nurr1에 의한활성에 가장 크게 관여하는 NBRE-A 부분에서 Nurr1과 함께 복합체를 이루어TH 발현을 위해 전사 조절에 관여하는 인자들을 찾아 동정하고자 하였다. 이에DNA pull-down assay를 이용하여 NBRE-A 부분에 결합하여 전사 조절 인자들을 살펴보았다. 이 결과 Nurr1과 결합는 후보 단백질인 PARP, TOPO, BTK를 선택할 수 있었고, 이 각각의 단백질이 Nurr1과 결합하여 TH 유전자의 발현을 조절하는지를 확인하였다. 이 중 BTK는 Nurr1과 결합하여 사람 줄기세포주인 F3 세포에서 TH유전자의 발현을 억제시키는 것을 확인 할 수 있었다. 이 결과는 향후 TH 유전자의 발현 정도에 따라 나타나는 퇴행성 뇌질환의 연구분야에 큰 공헌을 할 것으로 기대된다.

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초록/요약

Nurr1, a member of the orphan nuclear receptor superfamily, was 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 ratelimiting enzyme of dopamine biosynthesis, is not well understood. In our previous study, we found 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 TH 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, SHSY5Y. In order to identify the transcription factors that mediate these contradictory functions in each of the cell types, we conducted biotin-labeled oligonucleotide of Nurr1-binding element (NBRE) and streptavidin-agarose beads pull-down assays. We identified three proteins, bruton tyrosine kinase-associated protein (BTK) and topoisomerase II β (TOPO II β) in F3 and poly (ADP-ribosyl) transferase (PARP) in SH-SY5Y. To know the interaction of these candidate proteins with NBRE-A site, we performed by immunoprecipitation and chromatin immunoprecipitation. BTK and TOPOII interact specifically with Nurr1 in the F3 cells, PARP interacts with SH-SY5Y cells. And to know these proteins regulate TH promoter activity, we performed the Luciferase assays. We found BTK repressed TH promoter activities but TOPO and PARP did not. Also when BTK was knockdown, TH promoter activity in F3 cells was activated. These results showed that these Nurr1 binding proteins perform a crucial role expression of the TH gene.

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목차

I. INTRODUCTION 1
II. MATERIAL AND METHOD 4
1. Cell culture 4
2. Plasmid constructs and mutagenesis 4
3. Transient transfection 5
4. Luciferase Assay 6
5. Biotinylated Oligonucleotide-Streptavidin Pull-Down 6
6. Immunoprecipitation assay and Western blotting 7
7. Chromatin immunoprecipitations (ChIP) 8
8. Construction of siBTK Duplexes and Transfection 8
III. RESULT 10
1. Cell type-specific expression patterns of human TH promoter constructs through
NBRE-A motif 10
2. Purification of Nurr1 and recruitment cofactor complex in NBRE-A region 11
3. Conformation BTK, TOPO IIβ and PARP co-immunoprecipitate with Nurr1 in F3 and
SH-SY5Y cells 13
4. Physical interaction both identified proteins and NERE-A motif of Nurr1 14
5. BTK, TOPO II and PARP are co-regulators of Nurr1 in the F3 and SH-SY5Y cells 15
IV. Discussion 22
V. CONCLUSION 24
VI. REFERENCE 25
국문요약 33

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