Regulation of R-loops for transcriptional control

The Dean group study the control of flowering time in the reference plant, thalecress (Arabidopsis thaliana). Several flowering pathways converge to regulate a gene called FLC and variation in expression of this gene contributes enormously to natural variation in flowering time of Arabidopsis types collected from around the world. One pathway regulating FLC involves so called non-coding RNAs - for FLC this is the RNA encoded from the opposite strand to the protein. This RNA has been called COOLAIR because it is up regulated by cold temperature. Analysis of the regulation of COOLAIR has recently led to the identification of a novel regulatory mechanism that involves an opening up of the DNA duplex, invasion by RNA and stabilization of this structure by a protein called AtNDX. This structure is called an R-loop and it inhibits production of RNA initiating in that region of the gene.

This proposal aims to further dissect this mechanism and identify other proteins that play a role. In the last few years similar non-coding RNAs have been found in many organisms but their regulation and function is generally not known. Advancing our understanding from a study such as this can provide concepts important to gene regulation across many genomes.

We are exploring different pathways involving RNA-mediated chromatin regulation of gene expression. The importance of these mechanisms has emerged through our work determining natural variation in flowering, an important adaptive trait in native plants and many crops. Multiple pathways regulate the Arabidopsis floral repressor FLC and these converge on co-transcriptional mechanisms involving antisense transcripts (named COOLAIR) and chromatin pathways. Regulation of COOLAIR transcription has been monitored using a COOLAIR:LUC reporter system. This enabled identification of a repressor of COOLAIR transcription that encoded an atypical homeodomain protein, which bound single-stranded DNA. Investigation of where single-stranded DNA might occur in vivo revealed the existence of an R-loop over the COOLAIR promoter. We intend to exploit this finding to identify and functionally analyse proteins that modulate the R-loop, which in turn influences transcription. We will build on preliminary data that identifies in vivo interactors of the homeodomain protein AtNDX and functionally analyse their action on the R-loop. We will also identify other regulators that repress COOLAIR transcription and investigate how their function intersects with R-loop formation, stabilization and resolution.

The project will have two overall objectives. The goal of the first will be the effective combination of genetic, molecular and proteomic approaches to define in vivo interactors of AtNDX and to understand how their function modulates the R-loop formation/stabilization/resolution thus influencing COOLAIR transcription. The goal of the second will be to clone four additional COOLAIR repressors and fully integrate their activities into the mechanism detailed in Objective 1. A mechanistic understanding generated from this study will detail the links between transcript processing, R-loop stabilization and RNA decay and is likely to be widely relevant.

Dissection of the mechanisms underlying RNA-mediated chromatin regulation is a very active area of biology at the moment. Important concepts relevant to gene expression generally are emerging from very different systems. The Dean lab has identified an excellent system in which to explore co-transcriptional mechanisms and the functioning of one class of non-coding RNAs, antisense transcripts. This has emerged through their work determining natural variation in flowering, an important adaptive trait in native plants and many crops. The chromatin mechanisms involved in flowering time control are likely to inform chromatin mechanisms relevant to many organisms. FLC regulation represents a system where different approaches can be combined to give a comprehensive view of the complexity and plasticity of gene regulation.

The work will also contribute fundamental information that provides a framework of understanding for dissection of flowering time control in less tractable but strategically important plants. The understanding emanating from analysis of the flowering network in Arabidopsis is a good paradigm for how fundamental information informs generally and influences experimental strategies of a wide community of plant biologists. The John Innes Centre is very unusual in combining basic biological research with crop-based studies. Groups interact on a daily basis, so results emerging from model species are quickly applied to crops such as Brassicas and cereals. Prof. Dean, collaboratively with Dr Judith Irwin (Dept. Crop Genetics, John Innes Centre) also has very good links with the plant breeding and biotechnology industries, and there are frequent visits in both directions. A clear understanding of the components regulating flowering will open up many avenues with impact in different areas.

A clearer understanding of the molecular basis of flowering time will inform strategies as to how to manipulate the timing of the transition to flowering, a key trait in breeding of many crops. The focus in the Dean/Irwin labs is manipulation of vernalization requirement and response - a key process in the production of many vegetable crops, broccoli, cauliflower, parsnips and carrots. Varieties of these vegetables are bred to ensure year round supply but the vagaries of winter temperatures tends to lead to gluts or shortages in production. Development of varieties less influenced by temperature but still producing in different seasons of the year would considerably reduce waste, potentially open up new production areas and increase efficiency of delivery. Ongoing funded collaborations with breeding companies aim to translate this understanding into practical benefits.