Applications of Next-Generation Sequencing Strategies for the Identification and Characterization of Enhancers in Plants Öffentlichkeit

Maher, Kelsey (Spring 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/p2676w55d?locale=de
Published

Abstract

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. This is due in part to the challenges presented by plant tissues, including a resilient cell wall, an abundance of extra-nuclear DNA, and the inability to maintain single-cell types via cell culture, making them less than ideal candidates for the latest next-generation sequencing technologies. Here we present two approaches to isolate nuclei with high purity from plant tissues for input into Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We used this method to delineate open chromatin regions and transcription factor (TF) binding sites across the Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa genomes. We find that the majority of open chromatin regions lie within 3 kb upstream of a transcription start site, and that TF-gene networks in the root tips are broadly conserved between the four species. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity on a global level, while TF motif analysis of differentially accessible chromatin regions identified a MYB-driven regulatory module unique to the hair cell’s fate and function. Finally, we generate single-cell type chromatin immunoprecipitation (ChIP-seq) datasets for the four histone modifications conserved across animal enhancers. Combined with our single-cell type chromatin accessibility data and available nascent transcript data, we compare the conservation of enhancer epigenetic characteristics between Arabidopsis thaliana, Homo sapiens, and Drosophila melanogaster. While animal promoters and enhancers show characteristic bimodal histone mark deposition and bidirectional transcription, this analysis revealed that Arabidopsis thaliana promoters and enhancers exclusively exhibit histone mark deposition and transcription in the sense direction, which may speak to a fundamental difference between transcriptional initiation in the plant and animal kingdoms. Together this work has interrogated the location, quantity, and characteristics of putative enhancers in plants on multiple levels, in multiple species, and in multiple cell types, and has generated new methodologies for the continued investigation of cis-regulatory elements in plants in the future.

Table of Contents

CHAPTER 1: INTRODUCTION                                                                                                        1

Gene Expression Regulation Begins with DNA                                                                        1

Enhancers                                                                                                                              6

Scope of the Dissertation                                                                                                      11

Figures                                                                                                                                16

   Figure 1.1                                                                                                                        16

   Figure 1.2                                                                                                                        18

   Figure 1.3                                                                                                                        20

Literature Cited                                                                                                                    22

CHAPTER 2: IDENTIFICATION OF OPEN CHROMATIN REGIONS IN PLANT GENOMES USING ATAC-SEQ     39

           Abstract                                                                                                                               39

           Introduction                                                                                                                         40

           Materials                                                                                                                             41

Methods                                                                                                                              45

Notes                                                                                                                                   50

Acknowledgements                                                                                                              54

Tables and Figures                                                                                                               56

   Figure 2.1                                                                                                                        56

   Figure 2.2                                                                                                                        58

   Table 2.1                                                                                                                          60

   Table 2.2                                                                                                                          61

   Table 2.3                                                                                                                          62

   Table 2.4                                                                                                                          63

   Table 2.5                                                                                                                          64

   Table 2.6                                                                                                                          65

Literature Cited                                                                                                                    66

CHAPTER 3: PROFILING OF ACCESSIBLE CHROMATIN REGIONS ACROSS MULTIPLE PLANT SPECIES AND CELL TYPES REVEALS COMMON GENE REGULATORY PRINCIPLES AND NEW CONTROL MODULES                     67

           Abstract                                                                                                                               67

           Introduction                                                                                                                         68

           Results and Discussion                                                                                                         71

           Summary and Conclusions                                                                                                    92

           Methods                                                                                                                              96

           Acknowledgements                                                                                                             102

           Author Contributions                                                                                                          102

Figures                                                                                                                               104

   Figure 3.1                                                                                                                       104

   Figure 3.2                                                                                                                       106

   Figure 3.3                                                                                                                       108

   Figure 3.4                                                                                                                       110

   Figure 3.5                                                                                                                       111

   Figure 3.6                                                                                                                       113

           Literature Cited                                                                                                                  115

CHAPTER 4: DIFFERENCES IN DIRECTIONALITY OF RNA POLYMERASE INITIATION UNDERLIE EPIGENOME DIFFERENCES BETWEEN PLANTS AND ANIMALS                                                                                                             127

           Abstract                                                                                                                             127

           Introduction                                                                                                                        128

           Results                                                                                                                               131

           Discussion                                                                                                                          140

           Acknowledgements                                                                                                             143

           Methods                                                                                                                            143

           Tables                                                                                                                                145

              Table 4.1                                                                                                                        145

              Table 4.2                                                                                                                        147

              Table 4.3                                                                                                                        148

Figures                                                                                                                               149

   Figure 4.1                                                                                                                       149

   Figure 4.2                                                                                                                       150

   Figure 4.3                                                                                                                       152

   Supplementary Figure 4.1                                                                                                154

   Supplementary Figure 4.2                                                                                                156

           Literature Cited                                                                                                                  157

CHAPTER 5: DISCUSSION – CONCLUSIONS AND FUTURE DIRECTIONS                               166

           The Distinction between Enhancers and Other Cis-Regulatory Elements is Ambiguous           167

           Histone Modifications in Plant Species                                                                                172

           Future Directions – Plant Enhancer Discovery with STARR-seq                                            175

           Figures                                                                                                                               179

              Figure 5.1                                                                                                                       179

              Figure 5.2                                                                                                                       180

           Literature Cited                                                                                                                  181

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