중추신경계 재생 및 가소성에 관여하는 유전자 발현 및 조절
Expression and regulation of genes associated with CNS regeneration and plasticity
- 주제(키워드) Axon regeneration , conditioning injury , RAGs , epigenetics
- 발행기관 아주대학교
- 지도교수 김병곤
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021963
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. Axonal injury in the CNS leads to functional impairment and disability in several neurologic disorders, such as stroke, and spinal cord injury. Failure of axonal regeneration is attributed to a growth-inhibiting environment at the injury site and an age-dependent decline in the intrinsic growth potential of axon. While axonal regeneration is highly restricted in the CNS, spontaneous axonal regeneration is possible in the peripheral nervous system (PNS). Compared to the CNS neurons, the neurons in PNS possess heightened intrinsic capacity to regenerate axons. However, the dorsal root ganglia (DRG) neurons, which extend one axonal branch into the PNS and the other into the CNS, cannot regenerate axons when their central branch is severed. It has been known that when peripheral branch is injured before the severance of central one, the DRG axons can mount successful regeneration responses accompanied by robust upregulation of regeneration associated genes (RAGs) such as Gap-43, Sprr1a, Npy, Lgals1, alpha 1 tubulin, etc. However it is not fully understood how the preconditioning injury can orchestrate the coordinated gene expression of a group of RAGs in DRG neurons. There are several epigenetic mechanisms such as DNA methylation and histone modification that can regulate the gene expression level. Especially, DNA methylation is involved in stable epigenetic regulation lasting a long time. Several previous studies suggested involvement of histone modifications in the transcriptional activation of RAGs in the CI-induced regeneration model. However, it has not been addressed whether DNA methylation can contribute to the orchestration of RAG upregulations. My thesis research focused on the role of DNA methylation the regulation of RAGs in an animal model of condition injury (CI). To analyze the DNA methylation in DRGs, methylated DNA immunoprecipitation (MeDIP-seq) was performed using DNA samples from L4-6 DRGs followed after CI by next generation sequencing (NGS). The MeDIP-seq data revealed that approximately 2000 genes were hypermethylated and a similar number of genes were demethylated after CI. The total level of 5-methylcytosine was not significantly changed, suggesting CI induces both hypermethylation and demethylation to a similar extent. However, the extent of changes in the level of DNA methylation was not correlated with gene expression levels measured by RNA-seq in most genes, including the RAGs. Intriguingly, pharmacological inhibition or activation of methylation showed significant decrease of neurite outgrowth potentials in preconditioned DRG sensory neurons, suggesting that DNA methylation may play a role in axon regeneration in conditioning effect. Further studies will be required to elucidate how DNA methylation changes contribute to enhanced neurite outgrowth capacity following CI. Having found that most RAGs did not show significant changes in DNA methylation, I examined DNA methylation of the transcription factor genes that can potentially interact with the promoters of RAGs. The bioinformatics analysis yielded candidate transcription factor genes of which methylation changes correlated with changes in gene expression levels. Bisulfite sequencing validated robust demethylation of c-Myc gene as early as 4 hours after injury, correlated with upregulation of mRNA expressions. Chromatin immunoprecipitation assay showed increased binding of c-Myc after CI to the promoters of a group of RAGs such as c-Jun, Atf3, and Sprr1a, but not to those of Gap43, Npy, and Galanin. AAV-mediated overexpression of c-Myc resulted in an increase in the extent of c-Myc occupancy on the promoters of c-Jun, Atf3, and Sprr1a. Intraganglionic injection of AAV5-c-Myc enhanced neurite outgrowth potential compared to control AAV5-GFP. Furthermore, overexpression of c-Myc significantly prevented axonal retraction from the injury site in in vivo spinal cord injury model. These results suggest that DNA methylation of c-Myc precedes and triggers transcription of a group of crucial RAGs that regulated intrinsic regeneration capacity. I also found that DNA methylation of c-Myc was accompanied by various key histone modifications with regards to the c-Myc regulation following preconditioning injury. The increase of acetyl of histone 3 lysine 9 (H3K9ac) was paralleled by a reduction of methylation of demethylation of histone 3 lysine 9(H3K9me2) binding after CI. These results suggest that expression of c-Myc is regulated by sophisticated epigenetic mechanism following CI and that the epigenetic regulation of c-Myc may be one of the crucial signals to orchestrate the coordinated RAG expression. Follow-up studies will reveal detailed epigenetic changes leading to c-Myc DNA demethylation and how the injury signal is linked to the c-Myc demethylation process. Together, my thesis research provides evidence that DNA methylation process is implicated in RAG upregulation and resultant enhancement of intrinsic regenerative potentials in DRG neurons following preconditioning peripheral nerve injury. I believe that the results from the current research will provide novel insights on how the coordinated RAG upregulation is regulated at an epigenetic level. Further studies will examine if or to what extent c-Myc demethylation is necessary for the enhancement of regenerative capacity of preconditioned DRG neurons. Molecular mechanism linking injury signals and epigenetic changes of the key genes that drive upregulation of RAGs will be an important line of researches to identify strategies to promote intrinsic regenerative potentials following CNS injury.
more목차
I. INTRODUCTION 1
A. Spinal cord injury pathophysiology 1
B. Factors for the failure of CNS axon regeneration 2
C. Conditioning injury (CI)-induced axon regeneration 3
D. Regeneration associated genes (RAGs) 4
E. Epigenetics 5
F. Neuroepigenetics in CNS injury 8
G. Next generation sequencing (NGS) in epigenomic studies 10
H. Aims of this study 11
II. Materials and methods 13
1. Animals and surgical procedures 13
2. In vivo 5-aza and SAM administration 13
3. Primary culture of dissociated adult DRG neurons 14
4. Genomic DNA extraction for MeDIP-seq 14
5. Methylated DNA immunoprecipitation and sequencing 15
6. Bioinformatics analysis 15
7. Bisulfite sequencing 17
8. Chromatin immunoprecipitation assay 18
9. ChIP-PCR primers 19
10. qRT-PCR primers 20
11. Methylation assay 21
12. Tissue processing and immunohistochemistry 21
13. Quantification 22
14. Production of recombinant adeno-associated virus (AAV) 23
15. Statistical Analysis 23
III. RESULTS 25
Part A. Changes in DNA methylation landscape in DRG sensory neurons after conditioning injury 25
1. Changes in global DNA methylation landscape following conditioning injury 25
2. Correlation between DNA methylation and RNA transcription 34
3. Functional significance of DNA methylation 37
Part B. Demethylation of c-Myc gene triggers transcription of a group of RAGs in DRG sensory neurons following conditioning injury 42
1. Transcription factor gene selection using Oreganno-analysis 42
2. c-Myc expression is upregulated in DRGs in response to CI 44
3. The effects of c-Myc overexpression by AAV5 without CI 49
4. Histone modifications involvement in the regulation of c-Myc after CI 57
IV. DISCUSSION 60
V. SUMMARY AND CONCLUSION 68
REFERENCES 69
국문요약 85
