Non-coding regulation of transcription during plant development

Abstract

Eukaryotic gene transcription is a highly complex process with multiple overlapping modes of regulation, which has allowed for the development of highly varied body plans and flexibility in responding to changes in the environment. Plants, as sessile organisms, are especially under pressure to properly regulate transcription of their protein coding genes to time developmental transitions in coordination with environmental cues. As a result, the classical model of transcriptional regulation where transcription factors bind the promoters of genes to influence the activity of RNA polymerase II is insufficient, and consistently fails to wholly explain transcription dynamics. To better understand this process, additional layers of regulation must also be considered, including a host of non-coding and epigenetic mechanisms such as non-coding RNAs, chromatin accessibility, DNA methylation, and histone methylation. In recent years these processes have been extensively studied in plants, though our understanding of them remains largely incomplete, such as with regards to the role and extent of non-coding transcription in regulating protein coding gene expression. To further our understanding in this area, I comprehensively profiled non-coding transcription and chromatin accessibility dynamics during the seed-to-seedling transition of the plant Arabidopsis thaliana as a model system. Using highly sensitive molecular techniques such as capped-small RNA sequencing (csRNA-seq) and Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq), I revealed that the extent of non-coding transcription during this transition is an order of magnitude more prevalent than previously known. Additionally, many changes in protein coding transcription were associated with nearby regions showing evidence of non-coding RNA transcription and changes in chromatin accessibility, which were successfully experimentally validated. These regulatory regions likely serve as enhancers of protein coding expression, confirming a feature of plant transcriptional regulation that once was believed to be unique to metazoans. While this approach successfully identified thousands of potential active regulatory regions in Arabidopsis thaliana, the sampling of bulk tissues limits our understanding of the cell type-specific spatiotemporal regulation of plant genes by enhancers. To solve this limitation, I performed an in-depth exploration of the csRNA-seq method for the detection of transcriptionally active enhancers in plants and integrated it with single cell methods. Using Arabidopsis thaliana roots as a model system, I analyzed optimal parameters for detecting genome-wide non-coding transcription and matched the resulting data with a published single-nucleus Multiome (snMultiome; profiling both RNA and chromatin accessibility simultaneously) dataset of root tips. Starting from root-enriched pairs of associated non-coding regulatory elements and protein coding genes identified from csRNA-seq data, I showed that the extensin gene EXTENSIN3 and an associated upstream enhancer are specifically co-accessible in trichoblast cells, revealing a potential cell type-specific mode of regulation for this gene. Finally, my use of ATAC-seq data motivated me to create a specialized tool for quality control and filtering of aligned reads, as currently available solutions are computationally inefficient and do not account for plant-specific differences in the data. This tool, quaqc: Quick ATAC-seq Quality Control, is several times faster and requires much less memory than the current next best solution. It also natively handles both plant and non-plant data without needing to perform additional manipulations to the former. An accompanying R package provides functions for interactive use and plotting of quality control data, allowing for fast iterative testing of the effects of specific filtering parameters on data quality. In recent years the application of advanced molecular techniques in plants such as those presented in this thesis and by others have brought our understanding of gene regulation in plants closer to the level achieved in metazoans. Further application of these methods will provide the foundation for the successful engineering of gene expression in agronomically important plants.

Date of Award	12 Jul 2024
Original language	English
Supervisor	Julia Irene Questa (Director)