TFII-I isoforms act as a co-regulator potentiating Nurr1-induced human TH expression during dopaminergic neurogenesis
TFII-I isoforms act as a co-regulator potentiating Nurr1-induced human TH expression during dopaminergic neurogenesis
- 주제(키워드) Parkinson disease , dopaminergic neurogenesis , transcriptional regulation , tyrosine hydroxylase , Nurr1 , TFII-I , SUMOylation
- 발행기관 아주대학교
- 지도교수 Myung Ae Lee
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025626
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Nuclear receptor related 1 protein (Nurr1) plays a vital role in development and maintenance of midbrain dopaminergic (mDA) neurons. Our previous study showed that Nurr1 actively represses human tyrosine hydroxylase (hTH) transcription in human neural stem cells (hNSCs), while it activates hTH expression via Nurr1 binding element (NBRE-A) in dopaminergic (DA) neuronal cells. To identify the interacting protein partners of Nurr1 to regulate hTH expression during DA neurogenesis, we performed DNA pulldown assay and identified TFII-I, a multifunctional transcription factor having four spliced isoforms. Polymerase chain reaction analysis of midbrain of embryonic mice from E9.5 to E13.5 showed that TFII-I expression switched from TFII-I∆ to TFII-Iγ isoform. TFII-IΔ preferentially interacts with SUMOylated Nurr1 and occupies hTH promoter in hNSCs, which resulted in repression of TH promoter activity. In contrast, TFII-Iγ interacts with Nurr1 on hTH promoter and enhanced hTH promoter activity in DA cells. Two TFII-I binding sites, an enhancer box (E-Box) and an Initiator element (Inr) flanking upstream and downstream of the NBRE-A respectively are present in conserved region among human, mouse and rat. In addition, ELM analysis and immunoprecipitation showed that only TFII-I∆ modified by SUMO1 at two position, K221 and K240 in putative motifs. So, we investigated the possibility whether Nurr1 and TFII-I∆ could form a repressor complex around NBRE-A on hTH promoter in hNSC-specific manner. TFII-I∆ majorly localized in nucleus while TFII-Iγ in cytoplasm of hNSCs. In addition, SUMO modified TFII-I∆ in a transcriptional complex with SUMOylated Nurr1 on the NBRE-A element, makes a complex in functional synergic control (SC) motifs and represses hTH promoter activity in hNSCs. Furthermore, SUMO deficient K221R, K240R and K221/240R forms of TFII-I Δ showed enhanced binding and resulted in hTH activation in hNSCs only. Mutation of E-box and Inr showed that TFII-I∆ represses hTH activity via Inr. Collectively, our data shows that TFII-I∆ isoform is modified by SUMO1 at SUMO consensus motifs overlapping SC motif exhibiting a strong contact with SUMO-2 modified Nurr1 leads to recruitment of synergic control factors (SCFs) and corepressors to hTH promoter resulting in repressed hTH activity in hNSCs. We further showed that de-SUMOylation did not interfere with nuclear-cytoplasmic transport rather exhibited enhanced affinity to hTH promoter and thus promoted its transactivation. Our findings contribute to understanding of specific expression of TFII-I isoforms in midbrain of mouse during embryonic development. Importantly our data established opposing role of TFII-I∆ and γ accountable for Nurr1 mediated repression and activation of hTH respectively during dopaminergic neurogenesis. Notably SUMO modified TFII-I∆ act as a critical corepressor of Nurr1 mediated hTH repression likely leading to loss of DA phenotype.
more목차
I. INTRODUCTION 1
A. DA neurogenesis, Nurr1 and TH 2
B. Human Tyrosine Hydroxylase regulation by Nurr1 4
C. An interplay of Nurr1 with other transcription factors 4
D. General Transcription factor II-I (TFII-I) and Nurr1 6
E. Posttranslational modification of TFII-I to regulate gene transcription 7
F. Aims of thesis research 9
II. MATERIALS AND METHODS 10
1. Cell lines 10
2. Antibodies and plasmids 10
3. Transfection and luciferase assay 11
4. Reverse transcription-polymerase chain reaction (RT-PCR) 11
5. Western Blot 12
6. Biotinylated Oligonucleotide-Streptavidin pull-down Assay 13
7. Co-immunoprecipitation and Western Blotting 14
8. Immunocytochemistry 15
9. Chromatin Immunoprecipitation assay 15
10. SUMO mutant’s construction 16
11. Quantitative image Analysis and Statistical Analysis 17
III. RESULTS 20
1. Dual role of Nurr1 in hTH gene regulation during dopaminergic neurogenesis 20
2. Identification of Nurr1 interacting partners at NBRE-A site 24
3. Expression pattern analysis of TFII-I isoforms 28
4. Distinct physical interaction between TFII-I isoforms and Nurr1 31
5. Opposing regulatory functions of TFII-IΔ and TFII-Iγ on hTH expression. 33
6. TFII-IΔ and TFII-Iγ exhibit differential cellular localization and binding affinity on hTH promoter 35
7. Characterization of SUMOylation and SCM of TFII-I 38
8. Construction of SUMO deficient mutants and confirmation of SUMO
modification of TFII-IΔ and γ in HB1.F3 cells 42
9. SUMOylation of TFII-IΔ facilitates repression of hTH expression in hNSCs 44
10. SUMO deficient TFII-IΔ does not interfere with its nuclear localization
but enhances DNA-binding on hTH promoter hNSCs 46
11. Identification of TFII-I binding sites on hTH promoter using MatInspector 48
12. TFII-IΔ regulates Nurr1-mediated transcriptional activity of the TH promoter via Inr element 50
IV. DISCUSSION 52
V. SUMMARY AND CONCLUSION 63
REFERENCES 65
