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Cellular response against expression of Arabidopsis DEMETER DNA demethylase in animal cells

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dc.contributor.advisor허진회-
dc.contributor.author목영근-
dc.date.accessioned2018-05-28T16:37:00Z-
dc.date.available2019-04-18-
dc.date.issued2018-02-
dc.identifier.other000000150548-
dc.identifier.urihttps://hdl.handle.net/10371/140815-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 협동과정 농업생물공학전공, 2018. 2. 허진회.-
dc.description.abstractEpigenetics refers to heritable changes of phenotype or gene expression without the modification of DNA sequence. DNA methylation, a prominent epigenetic marks, is associated with gene expression, X chromosome inactivation, gene imprinting and developmental process. DNA methylation occurs by DNA methyltransferase to add methyl group at the 5th carbon of a cytosine to produce 5-methylcytosine (5mC), which is achieved by a similar mechanism in both the plants and animals. However, DNA demethylation pathways are fundamentally different in plant and animal. In animals, DNA demethylation is achieved by several enzymatic pathways, where TET1 catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and successive oxidation of 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), and deamination by AID/APOBEC. More efficient DNA demethylation is accomplished in plants by the DEMETER 5-methylcytosine DNA glycosylase, which mediates direct excision of 5mC from double strand DNA.
In order to see if this plant DME is working properly in animal systems, Arabidopsis DME gene was introduced into human HEK-293T cells. DME was a successfully implemented to remove 5mC and it led to dysregulation of cell proliferation, cell cycle arrest at S phase, and severe DNA damage. Gene expression profile showed that diverse genes were differentially expressed in DME expressing cells such as cell cycle component genes, heat shock proteins, and interferon stimulated genes (ISGs). Interestingly, DME expressing cells seem to induce antiviral response through the dsRNA generated from activation of retrotransposons. Furthermore, DME could be a novel candidate for epigenome editing via the successful excision of 5mC in animal cells. DME appears to activate specific endogenous gene expression in combination with TALE module and nuclease deficient CRISPR/Cas9 and, which induced DNA demethylation at the specific locus.
These works demonstrated that plant DNA demethylase catalyzed DNA demethylation and the interferon signaling played a crucial role to relieve genotoxic stresses in this DME-induced DNA demethylation processes in mammalian cells. Furthermore, DME can be utilized for specific gene regulation in DNA methylation associated disease and may shed light on the role of DNA methylation in human disease.
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dc.description.tableofcontentsLITERATURE REVIEW 1
INTRODUCTION 2
EPIGENETICS 3
DNA METHYLATION 4
DNA METHYLTRANSFERASE 5
DNA METHYLTRANSFERASE IN MAMMAMLS 6
DNA METHYLTRANSFERASE IN PLANTS 7
DNA DEMETHYLATION 9
ACTIVE DNA DEMETHYLATIN IN MAMMAMLS 10
ACTIVE DNA DEMETHYLATIN IN PLANTS 12
GENOME EDITING 14
ZINC FINGER DOMAIN 15
TRANSCRIPTION ACTIVATOR-LIKE EFFECTORS (TALEs) 16
CRISPR/Cas9 17
BEYOND GENOME EDITING 19
REFERENCES 21
CHAPTER I DEMETER plant DNA demethylase induce antiviral response by interferon signaling in
animals cells 38
ABSTRACT 39
INTRODUCTION 40
MATERIALS AND METHODS 45
Cloning 45
Cell culture 46
Cell Proliferation 46
GFP positive cells sorting 46
in vitro 5-methylcytosine (5mC) glycosylase activity assay 47
Cell cycle analysis and PI staining 47
Immunofluorescence and FACS analysis of TUNEL Assay 48
Western blot 50
Microarray analysis 51
Quantitative real time PCR analysis 51
Methylation array 52
Locus specific bisulfite sequencing 53
Northern blot 53
5-azacytidine treatment 54
polyinosinic:polycytidylic acid (poly (I:C)) transfection 54
TAG aided sense/antisense transcript detection (TASA-TD) PCR 55
Small RNA sequencing 55
RESULTES 59
In vitro activity of DME expressed in mammalian cells 59
DMEΔ is detrimental to cell proliferation in mammalian cells 65
Direct 5mC excision induces DNA damage 67
Excessive excision of 5mC generate cell death in mammalian cells 69
Gene expression profile analysis of DMEΔ-expressing cells 71
The DNA methylation levels were maintained in 293T-DMEΔ cells 74
Upregulation of heat shock proteins 77
DMEΔ induced a cell cycle arrest at S phase 79
Interferon stimulated genes were elevated by DME 83
Type I interferons were elevated in 293T-DMEΔ cells 86
Antiviral response activated by dsRNA from TEs 90
Passive DNA demethylation leads to a cellular response similar to active DNA demethylation 94

DISCUSSION 96
REFERENCES 103
CHAPTER II Reactivation of silenced gene through sequence-specific DNA demethylation 114
ABSTRACT 115
INTRODUCTION 116
MATERIALS AND METHODS 122
Construction of TALE-DME fusion proteins 122
Construction of Cas9 for in vivo DNA cleavage assay 123
Construction of nCas9-DMEs and dCas9-DMEs fusion proteins 123
Cell culture and Transfection 125
Quantitative real time PCR analysis 125
Locus-specific bisulfite sequencing 126
Constructs of Cas9 and TALE-DME for protein purification 126
Protein expression and purification 126
DNA Binding Assays 127
DNA cleavage assay in vivo and in vitro 129
RESULTES 133
Expression level of OCT4 was slightly activated by TALE-DME 133
KL-1 TALE-DME induced KLF4-targeted DNA demethylation 139
dCas9- and nCas9-DME activated RANKL expression 145
DISCUSSION 153
REFERENCES 158
ABSTRACT IN KOREAN 164
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dc.formatapplication/pdf-
dc.format.extent35452837 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectDEMETER-
dc.subjectDNA methylation-
dc.subjectDNA demethylation-
dc.subjectCRISPR/Cas9-
dc.subject.ddc660.6-
dc.titleCellular response against expression of Arabidopsis DEMETER DNA demethylase in animal cells-
dc.typeThesis-
dc.description.degreeDoctor-
dc.contributor.affiliation농업생명과학대학 협동과정 농업생물공학전공-
dc.date.awarded2018-02-
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College of Agriculture and Life Sciences (농업생명과학대학)Program in Agricultural Biotechnology (협동과정-농업생물공학전공)Theses (Ph.D. / Sc.D._협동과정-농업생물공학전공)
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