Applications of Next-Generation Sequencing Strategies for the Identification and Characterization of Enhancers in Plants Público
Maher, Kelsey (Spring 2020)
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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