A Study on the Role of Arabidopsis Histone Acetyltransferase HAG1 in Developmental Transition and Plant Cell Totipotency
애기장대 히스톤 아세틸화 효소 HAG1의 발달전이 및 식물세포 전형성능 관련 기능에 대한 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 8. 노유선.-
dc.description.abstractEvery cell in an organism carries an identical genome. However, multicellular eukaryotes are composed of different cell types that show different morphology and carry out diverse functions. Each cell types are characterized by distinct gene expression patterns that are generated during development and stably maintained. The main mechanism by which the information stored in the genome can be selectively controlled is based on epigenetics. Epigenetic regulation is referred to the stable control of gene expression caused by mechanisms that are not dependent on underlying DNA sequence. Epigenetic mechanisms include DNA methylation, chromatin remodeling by ATP-dependent remodelers or histone modifications, nuclear positioning, histone variant incorporation, and regulation by noncoding RNAs.
Histone acetylation is an epigenetic modification associated with euchromatin and is catalyzed by histone acetyltransferases (HATs). HATs transfer the acetyl group from acetyl-CoA to lysine residues within the N-terminal histone tails. The negative charge of the acetyl group (CH3COO-) neutralizes the positive charges of histones, reducing their affinity to negatively charged DNA. This renders DNA more accessible to the transcriptional machinery or generates binding sites for acetyl lysine-binding proteins to spread the modification, leading to transcriptional activation.
In this study, I address two distinct roles HAG1/AtGCN5, a member of the Arabidopsis Gcn5-related N-acetyltransferase (GNAT) family of HATs. In the first part, I show that the HAG1 and PRZ1/ADA2b-containing SAGA-like histone acetyltransferase (HAT) complex controls the juvenile-to-adult phase transition. In Arabidopsis, vegetative phase transitions are regulated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) factors that are post-transcriptionally regulated by miR156 in an age-dependent manner. I demonstrate that HAG1 and PRZ1 directly controls the transcription of SPLs by catalyzing H3Ac of their chromatin, and thus determines the time for juvenile-to-adult phase transition. Furthermore, this epigenetic mechanism is also crucial for miR156-independent induction of SPLs and acceleration of phase transition by light and photoperiod or during post-embryonic growth.
In the second part, I address the role of HAG1 in the acquisition of pluripotency in callus during de novo organogenesis. For decades, callus was considered as an unorganized mass of undifferentiated cells. However, recent studies have provided evidences that callus is formed by the proliferation of a subset of existing stem cell-like pericycle cells adjacent to the xylem poles. These studies have also shown that callus cells have gene-expression profiles similar to those of root primordia. I demonstrate that HAG1 protein is induced in developing calli and plays a pivotal role in the activation of root meristem genes. Upon callus induction, histone acetylation at the root meristem genes, namely WOX5/WOX14, SCARECROW (SCR), PLETHORA 1 (PLT1), and PLT2, is increased in a HAG1-dependent manner. This allows a high transcriptional output of the root meristem genes, and then the transcription factors encoded by the root meristem genes act as potency factors conferring competence for regeneration that is necessary for de novo shoot formation.
dc.description.tableofcontentsChapter I. General Introduction 1
1.1 Epigenetic regulation of gene expression 2
1.1.1 DNA methylation 3
1.1.2 Chromatin remodeling 5
1.1.3 Noncoding RNA-mediated silencing 13
1.2 Plant life cycle 16
1.2.1 The life cycle of Arabidopsis 16
1.2.2 Vegetative transition 18
1.2.3 MiR156-SPL module 19
1.3 Plant-cell pluripotency and de novo shoot regeneration 22
1.3.1 Step 1: Callus formation 24
1.3.2 Step 2. De novo shoot formation 29
1.3.3 Molecular link between callus induction and de novo shoot regeneration 30
1.3.4 Epigenetic control of callus formation and de novo shoot regeneration 31

Chapter II. Epigenetic control of juvenile-to-adult phase transition by the Arabidopsis SAGA-like complex 39
2.1 Abstract 40
2.2 Introduction 41
2.3 Materials and methods 43
2.3.1 Plant materials and growth conditions 43
2.3.2 Constructs and plant transformation 45
2.3.3 RT-PCR or RT-qPCR analysis 46
2.3.4 ChIP assay 52
2.3.5 Northern blot analysis 56
2.3.6 Histochemical GUS assay 56
2.4 Results 57
2.4.1 Mutations in HAG1 strongly delay juvenile-to-adult phase transition in Arabidopsis 57
2.4.2 Transcript levels of a group of SPL-family genes are reduced in hag 1 mutants 58
2.4.3 Mutations in HAG1 result in delayed flowering and reduced expression of SPL-target genes 58
2.4.4 miR156-independent transcriptional control of SPL3 and SPL9 by HAG1 59
2.4.5 Histone acetylation at the SPL3 and SPL9 loci is controlled by the HAG1- and PRZ1-containing HAT complex 60
2.4.6 HAG1 plays a major role in juvenile-to-adult phase transition and SPL activation among Arabidopsis HATs 62
2.4.7 HAG1-mediated H3Ac is important for miR156-independent light and photoperiodic induction of SPL9 63
2.4.8 HAG1-mediated H3Ac is important for miR156-independent induction of SPL3 during germination 63
2.5 Figures 65
2.6 Discussion 85

Chapter III. HAG1-mediated epigenetic reprogramming is essential for the acquisition of pluripotency in Arabidopsis 87
3.1 Abstract 88
3.2 Introduction 89
3.3 Materials and methods 91
3.3.1 Plant materials and growth conditions 91
3.3.2 Constructs and plant transformation 91
3.3.3 Culture conditions 93
3.3.4 Microscopy 93
3.3.5 Immunoblot analysis 94
3.3.6 Histochemical GUS assay 94
3.3.7 RNA sequencing analysis 94
3.3.8 RT-qPCR analysis 95
3.3.9 ChIP-qPCR analysis 97
3.3.10 ChIP sequencing analysis 100
3.4 Result 101
3.4.1 Mutations in HAG1 cause severely defective de novo shoot regeneration 101
3.4.2 Global gene expression profiling reveals distinct expression dynamics in hag1 101
3.4.3 HAG1 plays a pivotal role on CIM 103
3.4.4 WOX5 expression in calli is severely reduced in hag1 103
3.4.5 SCR expression in calli is largely affected by hag1 mutation 104
3.4.6 PLT1 and PLT2 transcript levels are severely reduced in hag1 105
3.4.7 Genome-wide H3Ac profile of WT and hag1 calli 105
3.4.8 H3Ac within WOX5, SCR, PLT1, and PLT2 chromatin is directly controlled by HAG1 106
3.4.9 WOX5 and WOX14 act redundantly in the control of de novo shoot regeneration 107
3.4.10 WOX5 and SCR have additive roles in de novo shoot regeneration 108
3.4.11 Activation of WOX5 on CIM partially rescues the shoot regeneration defect of hag1 109
3.5 Figures 110
3.6 Discussion 138

References 140

Abstract in Korean 165
dc.format.extent14739745 bytes-
dc.publisher서울대학교 대학원-
dc.subjectde novo shoot regeneration-
dc.subjecthistone acetylation-
dc.subjectJuvenile-to-adult transition-
dc.titleA Study on the Role of Arabidopsis Histone Acetyltransferase HAG1 in Developmental Transition and Plant Cell Totipotency-
dc.title.alternative애기장대 히스톤 아세틸화 효소 HAG1의 발달전이 및 식물세포 전형성능 관련 기능에 대한 연구-
dc.contributor.affiliation자연과학대학 생명과학부-
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College of Natural Sciences (자연과학대학)Dept. of Biological Sciences (생명과학부)Theses (Ph.D. / Sc.D._생명과학부)
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