Establishment and Utilization of INTACT-ATAC-seq to Map Cell Type-Specific Gene Regulatory Networks in Plants Restricted; Files Only

Bajic, Marko (Fall 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/7d278v06f?locale=es
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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