Mapping and Dynamics of Regulatory DNA and Transcription Factor Networks in A. thaliana

Abstract

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. One way in which cis-regulatory elements can be delineated is by their characteristic hypersensitivity to the endonuclease DNase I. In this dissertation I present the development and application of a DNase I-based assay for A. thaliana. I describe the development of a DNase I-seq method that can be used to map genome-wide chromatin accessibility with nucleotide resolution and cell-type specificity. I then use this method to map DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and use genomic footprinting to delineate ~700,000 sites of in vivo transcription factor (TF) occupancy. I reveal general properties of DHSs in A. thaliana are novel, including evidence that highly significant GWAS variants are enriched within DHSs and that widespread TF binding within exons may have shaped codon usage patterns. I also show that the architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. I then explore chromatin dynamics in response to environmental stimuli by mapping the chromatin landscape during heat shock and photomorphogenesis, disclosing thousands of environmentally-sensitive elements and the TFs that bind them. Next, I continue exploring chromatin dynamics, this time looking at developmentally dynamic DHSs during the maturation of seed-coat epidermal cells during the transition from growth to mucilage secretion. I show that differentially expressed genes are associated with dynamic DHSs and implicate new TFs and candidate genes involved in seed coat epidermal differentiation. Finally, I investigate the natural variation in chromatin accessibility through the examination of chromatin landscapes of 5 diverse A. thaliana ecotypes. I show that variable DHSs are more polymorphic than static DHSs across the accessions. I also show that deletions account for 15% of variable DHSs, suggesting they are a powerful force in shaping diverse patterns of gene regulation in the ecotypes. In the appendices I present supplemental figures and methods for Chapter 2, as well as a project summary and general information on phenotypic robustness, which is affected by cis-regulatory elements.