CORDIS

• What was the problem/issue being addressed?
Every organism faces the challenge to respond favorably to changes in their environment. The blueprint for these responses is their DNA. Cutting-edge genomic technologies show that transcriptional activity predominantly acts on sequences that lack functional annotation. Transcription changes in response to the environment largely occur in non-protein coding DNA regions instead. This suggests that non-coding regions may be part of the response architecture to environmental change. Understanding the role of coding and non-coding transcription dynamics represents a key question in modern biology. By studying the act of transcription, my project gave unprecedented resolution of environmental sensing at the molecular level.

• Why is it important for society?
The cultivars produced by plant breeders have a desired trait, for example more suited to changing environmental conditions. However, the molecular mechanisms of how genomic material helps the adaptation are often unclear. The project drew from cutting-edge studies of transcription dynamics to bridge the gap and elucidate these mechanisms. Thus, the project enabled a potentially more targeted approach to deal with changing environments. There is often a gap of understanding between researchers, the public, industry and policy-makers. To bridge this gap and ensure relevant results are shared with relevant audiences, the project pursued an active communication strategy to make the research visible and openly accessible. Interesting research results was re-written as popular science pieces and published.

• What were the overall objectives?
Objective 1: Develop NET-seq to Arabidopsis to map nascent transcription.
The project aimed to adapt Nascent Elongation Transcript sequencing (NET-seq) to plants (plaNET-seq). With plaNET-seq, we could study the act of transcription in the model plant Arabidopsis thaliana.
Objective 2: Identify environmental responding non-coding RNAs in Arabidopsis with plaNET-seq.
The project used plaNET-seq to map the nascent transcriptome during the response to environmental changes, generating temporal transcription maps of compensatory regulation.
Objective 3: Study the dynamics of transcription in key genomic regions in response to low temperature.
The project looked at specific regulons, important for plant cold-acclimation with a focus on long non-coding RNA function in addition to transcription dynamics (e.g. stalling and release) in response to low temperature.

• What are the conclusions?
The project succeeded in adapting plaNET-seq to Arabidopsis. To achieve the objectives of the action, plaNET-seq was implemented in the early stages of cold-acclimation (3 and 12h of 4°C). Our main conclusions are:
• Transcriptional stalling is greatly affected by cold temperatures. All stages of the transcriptional cycle (initiation, elongation and termination) are affected.
• Generally, the changes in stalling are rapidly adapted between 3h and 12h of treatment to 4°C. This indicates that plants quickly respond and change the chromatin environment and associated transcriptional regulation to cold temperatures.
• With plaNET-seq, close to 10000 novel transcripts were found. This was largely due to their rapid degradation which makes it challenging to detect them with steady-state RNA detection techniques (RNA-seq).
• Many novel non-coding transcripts were found in close proximity to coding genes, predominantly on the antisense strand. plaNET-seq identified for the first time in plants divergent and convergent transcripts. These transcripts are common in yeast and human cells but their function is still largely unknown.
• The characterization of a non-coding transcript (SVALKA) revealed a novel repressive mechanism of gene regulation. SVALKA is transcribed on the antisense strand of the CBF1 gene, a gene important for cold-acclimation in Arabidopsis. Transcription of SVALKA results in RNA Poly

As specified in the DoA, plaNET-seq was adapted to Arabidopsis using a epitope-tagged version of the main RNA Polymerase in the eukaryotic cell (RNAPII). The tagged version of RNAPII covers a lethal mutation ensuring that all RNAPII complexes in the cell are tagged and I generated a stable line of this construct. I used the tag to immuno precipitate RNAPII complexes followed by isolation of attached RNA (i.e. actively transcribed RNA). The RNA was then used to generate a library and deep-sequenced. The bioinformatic pipeline required for analysis was created within the host group by Dr. Maxim Ivanov. The main results from the study will soon be re-submitted to Nucleic Acid Research. This work also includes the characterization of transcription dynamics in response to cold temperatures.
An objective for the action was to identify long non-coding RNA transcripts that are important for the acclimation to cold temperatures. With plaNET-seq we identified close to 10000 novel non-coding transcripts. Two of those transcripts were further characterized. The first one, which we named SVALKA, is transcribed antisense to an important gene in Arabidopsis cold-acclimation and negatively regulates this gene. The main results from this work are published in Nature Communications. The other non-coding transcript is also transcribed antisense to a key gene in cold-acclimation. Here, transcription of the non-coding transcripts is positively regulating the gene. This work is under preparation and will be submitted for peer-review soon.

Overview:
Objective 1: plaNET-seq has been adapted to Arabidopsis.
- Results are ready for resubmission to Nucleic Acid Research.
Objective 2: Interesting non-coding regions in cold-acclimation were identified and characterized.
- Results published in Nature Communications.
- Additional results are being prepared for submission.
Objective 3: Transcription dynamics were studies with plaNET-seq.
- Part of manuscript to be resubmitted to Nucleic Acid Research.

In addition to the objectives outlined in the DoA, I have been involved in a project concerning transcription start sites in Arabidopsis. This work was published in PLOS Genetics in 2019.
Any societal implication of this project is hard to measure due to its fundamental research basis. There are clear future potential for the results from this action, but this has not been pursued due to time limitations.