Investigating the dynamics of transcription initiation and nucleosome turnover in Arabidopsis thaliana Público
Willett, Courtney (Spring 2024)
Published
Abstract
The process of transcription is regulated by a variety of factors, such as RNA Polymerase II (RNA Pol II) initiation, histone post-translational modifications (PTMs) and cis-regulatory elements (CREs). Previous analysis of nascent RNA and chromatin patterns has revealed distinctions between plant and animal models. In plant models, nascent RNA transcripts and transcription-associated histone PTMs accumulate mainly in the gene body, suggesting that RNA Pol II does not initiate in the upstream direction of genes, as compared to animal models where RNA Pol II initiation can initiate bidirectionally. We wanted to investigate these patterns further at transcription start sites (TSSs) and at CREs to identify epigenetic and transcriptional differences between plant and animal models. Using a cross-species analysis of accessible chromatin, histone PTMs and nascent RNA data, we concluded that plants prefer strictly unidirectional RNA Pol II initiation both at the TSS and CREs, whereas Drosophila and Homo sapiens both show bidirectional transcriptional at either the TSS or CREs. Moreover, transcription can be highly disruptive to chromatin structure as the nucleosome must be at least partially disassembled to allow for RNA Polymerase II to transcribe the DNA. This disassembly and subsequent reassembly of the nucleosome can result in the loss of the original histone components and incorporation of non-canonical histone variants, which have a profound effect on epigenetic and transcriptional states. In particular, histone variant H3.3 is often incorporated into the nucleosome during transcription in a transcription-coupled manner and is associated with the maintenance of euchromatic, active regions of the genome. Knockdown of H3.3 in plant, yeast, and animal models predominantly affects environmental response genes; loss of H3.3 or its chaperone HIRA results in transcriptional defects but has a limited effects on global transcriptional outputs. Moreover, while is H3.3 incorporated across gene bodies in both plants and animals, there is a distinct enrichment preferentially at the 3’ end of genes in plants, further underscoring potential differences in nucleosome assembly between plants and animals. We developed an inducible system to measure incorporation of H3.3-GFP into chromatin. We observed a positive correlation between enrichment of H3.3-GFP incorporation and transcript levels, wherein the highest transcribed genes had the greatest H3.3-GFP incorporation at the 3’ end of gene bodies over the span of 24 hours of induction, thus creating a useful system for measuring H3.3-GFP turnover dynamics relative to transcription activity. Overall, these studies highlight key differences in how plants regulate transcription and provide a novel tool for measuring nucleosome dynamics in Arabidopsis.
Table of Contents
I. Chapter 1: Introduction ……………………………………………………………… 8-20
II. Chapter 2: Differences in transcription initiation directionality underlie distinctions between plants and animals in chromatin modification patterns at genes and cis-regulatory elements ………………………………………………………………… 21-57
III. Chapter 3: Measuring genome-wide dynamics of H3.3 turnover in Arabidopsis
thaliana ……………………………………………………………………………. 58-81
IV. Chapter 4: Discussion ………………………………………………………………. 82-88
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