Establishment and Utilization of INTACT-ATAC-seq to Map Cell Type-Specific Gene Regulatory Networks in Plants Öffentlichkeit
Bajic, Marko (Fall 2019)
Published
Abstract
Differential transcription of protein-coding genes is the basis for cellular diversity and responses to environmental conditions. The precise control of timing and to what extent specific protein-coding genes are transcribed is regulated by transcription factors and chromatin organization. Nucleosomes, the fundamental units of chromatin compaction, can impede the ability of transcription factors (TFs) to bind to DNA. Chromatin regions where TFs bind to DNA are typically depleted of nucleosomes, either because TFs opportunistically bind to nucleosome-free DNA and reconfigure chromatin or because TFs bind nucleosomal DNA leading to nucleosomal repositioning or eviction. By virtue of this nucleosome depletion, TF binding sites can be identified by their increased sensitivity to nuclease cleavage or chemical modifications compared to the rest of the genome. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) utilizes a hyperactive Tn5 transposase to cleave DNA at these accessible sites and insert preloaded sequencing adapters into the cleaved DNA. The DNA fragments sequenced in ATAC-seq represent a genome-wide chromatin accessibility profile for the nuclei or cells that are assayed. By using nuclei isolated through Isolation of Nuclei TAgged in specific Cell Types (INTACT) for ATAC-seq, together referred to as INTACT-ATAC-seq, we demonstrated the ATAC-seq technique in Arabidopsis thaliana, which had not been done before. Additionally, we expanded INTACT-ATAC-seq for use in four other plant species, in 7 specific cell types, and during response to submergence stress. We found that ATAC-seq can be performed in plants with as little as 2000 nuclei as the starting material. In the diverse plant species examined, accessible chromatin sites were found predominantly within the 3 kb region upstream of the transcription start site, indicating the predominant compartmentalization of regulatory elements to those regions in plants, in contrast to their wide distribution in animal genomes. By coupling chromatin accessibility and transcriptome data, we built gene regulatory networks (GRNs) for two differentiated cell types of the Arabidopsis root epidermis, Arabidopsis shoot stem cells and multiple differentiating leaf cell types, as well as in the root tips of four diverse species under control conditions and during response to submergence stress. Of particular note, we identified 68 Submergence-UpRegulated gene Families (SURFs) that were upregulated during submergence stress in all four species analyzed, and we found that the same set of four TF families regulate these genes in all species. Interestingly, even though the four TFs and the 68 SURFs are all active in each of the plants during submergence, the connectivity between them varies among the plant species and is indicative of evolutionary adaption that allows rice to survive temporary submergence while the dryland-adapted tomato dies when flooded. In summary, I adapted ATAC-seq to multiple plant species, comprehensively profiled chromatin accessibility and transcription in cells of different plants and environmental conditions to build GRNs that connect chromatin accessibility with gene expression. These findings expand the research toolkit as well as our view of how chromatin is organized and gene regulation is maintained in plants, which is of biological significance for development and environmental stress response.
Table of Contents
CHAPTER 1: INTRODUCTION 1
Studying chromatin accessibility and TF binding in specific cell types 3
Scope of the dissertation 8
Figures 14
Literature Cited 18
CHAPTER 2: IDENTIFICATION OF OPEN CHROMATIN REGIONS IN PLANT GENOMES USING ATAC-SEQ 28
Abstract 28
Introduction 29
Materials 30
Methods 34
Notes 40
Acknowledgements 44
Tables and Figures 45
Literature Cited 55
CHAPTER 3: PROFILING OF ACCESSIBLE CHROMATIN REGIONS ACROSS MULTIPLE PLANT SPECIES AND CELL TYPES REVEALS COMMON GENE REGULATORY PRINCIPLES AND NEW CONTROL MODULES 56
Abstract 56
Introduction 57
Results and Discussion 60
Summary and Conclusions 81
Methods 84
Acknowledgements 91
Author Contributions 91
Figures 92
Literature Cited 103
CHAPTER 4: CHROMATIN ACCESSIBILITY CHANGES BETWEEN ARABIDOPSIS STEM CELLS AND MESOPHYLL CELLS ILLUMINATE CELL TYPE-SPECIFIC TRANSCRIPTION FACTOR NETWORKS 115
Summary 115
Introduction 116
Results 118
Discussion 128
Methods 132
Acknowledgements 139
Figures 141
Literature Cited 150
CHAPTER 5: EVOLUTIONARY FLEXIBILITY IN FLOODING RESPONSE CIRCUITRY IN ANGIOSPERMS 159
Abstract 159
Main Text 160
Acknowledgements 165
Materials and Methods 166
Figures 180
Literature Cited 187
CHAPTER 6: CHROMATIN ACCESSIBILITY CHANGES IN DIFFERENTIATING CELLS OF THE LEAF 195
Summary 195
Introduction 196
Results 198
Discussion 203
Methods 204
Tables and Figures 210
Literature Cited 232
CHAPTER 7: DISCUSSION – IMPLICATIONS AND FUTURE DIRECTIONS 237
Gene Regulatory Networks connect transcription factors and regulated gene targets 238
Technical improvements for INTACT-ATAC-seq 242
Final statements 244
Figures 246
Literature Cited 249
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