사람 타이로신 하이드록실레이즈 유전자의 promoter 내의 CpG부위에 관한 연구

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dc.contributor.advisor이명애-
dc.contributor.author양재원-
dc.date.accessioned2019-10-21T06:46:19Z-
dc.date.available2019-10-21T06:46:19Z-
dc.date.issued2007-02-
dc.identifier.other2160-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/16433-
dc.description학위논문(석사)--아주대학교 일반대학원 :신경과학기술과정,2007. 2-
dc.description.abstractTyrosine hydroxylase (TH) 는 카테콜아민 생합성 과정에서 첫 번째 속도 조절 효소이다. 우리는 기존 연구에서 TH 유전자의 발현 조절에 있어서 뇌신경세포 특이적 전사 저해를 일으키는 두 가지의 기작이 존재하고 이중 promoter 인접부위는 NRSF 의존적이나, 말단부위에서는 NRSF 비의존적인 강한 전사제한 요소가 존재한다는 것을 밝혔다. 또한, promoter 부위에 -2164~-1210bp 사이에서 강한 전사 저해요소가 존재한다는 것을 밝혀냈다. 본 연구에서는 -2164 ~ -1210bp 사이의 강한 전사 저해 요소를 밝히고자 하였다. 먼저, 이 부분을 3부분으로 나누어 X-1, X-2, X-3의 construct를 만들어 각각의 promoter 활성도를 측정해본 결과 실제적으로 X-1과 X-2부분에서 강한 전사저해가 일어남을 알 수 있었다, Sodium bisulfite DNA 서열분석 실험을 수행하여본 결과, 이 부분에서 강한 methylation이 일어나고 있음을 확인할 수가 있었다. 또한, ChIP assay 실험에서 methylation 된 부분에 MBDs단백질들이 결합되어있는 것을 확인할 수 있었다 이러한 결과는 신경줄기세포 내에서 -2164~-1210 bp 에서 특이적으로 DNA의 methylation이 일어나 MBDs 단백질들이 결합하여 효과적인 전사억제가 일어남을 암시 하고 있다. 또한 MBDs를 매개로 한 TH유전자의 전사 억제는 5-aza2`-deoxycytidine 의 처리에 의해 풀리는 것으로 보아, MBDs단백질에 의한 전사억제에 DNA의 methylation 이 필수적인 것을 알 수 있었다. 이로써, 사람 TH 유전자의 조직 특이적 발현 조절에 있어서 NRSF와 함께 DNA의 methylation 의 역할이 핵심적임을 알 수 있었다.-
dc.description.tableofcontentsⅠ. INTRODUCTION = 1 Ⅱ. MATERIAL AND METHODS = 3 1. Cell culture = 3 2. Plasmid constructs and mutagenesis = 3 3. cloning of -2164~-1210bp 5`-Flanking region of the human TH gene by PCR Ampification method = 4 4. Transfection and luciferase assay = 5 5. Western blot analysis = 6 6. RAN preparation and RT-PCR = 7 7. Chromatin immunoprecipitation assy = 7 8. Geonomic DNA isolation and Sodium bisulfite sequencing analysis = 9 Ⅲ. RESULTS = 11 Cell-specific expression pattern of hTH promoter constructs = 11 Transcriptional activation of the hTH promoter including mutant NRSE in hNSC = 12 De-repression of hTH promoter point mutants by introduction of dominant negative NRSF = 12 Cell-specific expression pattern of hTH promoter constructs (-2164 bp and -1210 in TH promoter) = 13 Analysis of CpG island promoter methylation = 14 Expression of MBDs and MBD occupancy in HBI.F3 and SH-SY5Y in vivo = 14 Effect of TSA and 5-aza-deoxycytidine epigenetic gene silencing modifiers on the expression of TH in human neural stem cells and SH-SY5Y = 15 Ⅳ. DISCUSSION = 17 Ⅴ. REFERENCE = 28 국문요약 = 32-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title사람 타이로신 하이드록실레이즈 유전자의 promoter 내의 CpG부위에 관한 연구-
dc.title.alternativeYang, Jae-Won-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameYang, Jae-Won-
dc.contributor.department일반대학원 신경과학기술과정-
dc.date.awarded2007. 2-
dc.description.degreeMaster-
dc.identifier.localId565669-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000002160-
dc.subject.keywordtyrosine hydroxylase-
dc.subject.keywordNRSF-
dc.subject.keywordneural stem cell-
dc.subject.keywordmethylation-
dc.subject.keywordMBDs-
dc.description.alternativeAbstractTyrosine hydroxylase (TH) is the first and rate-limiting enzyme in biosynthesis of catecholamines. In the CNS, neuronal restricted silencing factor NRSF plays a critical role as a key transcriptional repressor for neuron-specific genes in non neuronal cells. We have previously reported that two type of repression, including NRSE-II site/REST and region between -2164 and -1210bp, act as a regulatory element in TH gene silencing in human neural stem cells (NSCs). In this study, to investigate a silencing element(s) between -2164bp and -1210 in TH promoter, we divided 3 portion of region from -2164 and -1210bp into 3 part (X-1, X-2 and X-3), each containing NRSE-R, NRSE-I and NBRE site, respectively. While promoter activities of X1- and X2-luc reporter constructs decreased in NSCs, those of X-3 did not decrease. Many CpG rich regions, known to regulate tissue-specific gene silencing, were observed within this region. Using a bisulfite sequencing method, we found that a specific CpG site within CpG island around NRSE-R site was specifically methylated in human NSC, but not SH-SY5Y cells. Transcriptional repressors, methylated CpG binding proteins (MBDs), are expressed in NSCs, and MBDs actually bind to hightly methylated -2164 ~ -1210 bp of hTH promoter in NSC. Treatment of NSCs with demethylating agent 5-aza2`-deoxycytidine result in TH gene re-expression. All these results suggest that region-specific methylation and MBDs play an important role in the regulation of hTH gene in NSCs. How they interact with each other, and especially, how they interact during differentiation of NSC need further investigation.-
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Graduate School of Ajou University > Department of Neuroscience and Technology Course > 3. Theses(Master)
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