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The autonomous floral pathway: a WIndow to Study the tight link between non-coDing RNA and chrOMatin regulation

Periodic Reporting for period 1 - WISDOM (The autonomous floral pathway: a WIndow to Study the tight link between non-coDing RNA and chrOMatin regulation)

Reporting period: 2018-04-01 to 2020-03-31

Problem being addressed:
RNA-mediated chromatin regulation is central to gene expression in many organisms. However, the mechanisms by which RNA influences the local chromatin environment are still poorly understood. The work supported by my MSCA fellowship showed how RNA 3’ processing factors promote proximal polyadenylation of an antisense transcript and how this physically links with chromatin modifiers FLD/LD/SDG26.

Why important for society:
Both long and short non-coding chromatin-associated RNA transcripts have emerged as key regulators of the chromatin environment. Detailed mechanisms of how 21-24 nucleotide RNAs initiate and maintain heterochromatin have been elucidated. However, less is understood about the mechanisms linking long non-coding RNA, chromatin regulation and transcription. The work supported by my MSCA fellowship will reveal any further parallels between COOLAIR and Xist function, thus elaborating the evolutionary understanding of RNA-mediated chromatin silencing by the research community.

Overall objectives:
This fellowship addressed the following four interconnected questions:
1. Does the FCA/FY-mediated repression of FLC depend on SDG26?
2. What are the primary chromatin changes delivered by FLD/SDG26?
3. How does the chromatin environment delivered by FLD/SDG26 repress transcription?
4. What is the link between FCA and FLD/SDG26?
"1.2.1 Overview of the results and their exploitation and dissemination:
All proposed tasks have been fully completed and the results were exploited and disseminated as planned.
Currently, I have published part of my work sponsored by this fellowship in Nature (Fang et al., 2019 Nature 569, 265-269) and PNAS (Fang et al., 2020 PNAS doi.org/10.1073/pnas.2007268117). I also wrote a review article to better disseminate my research results (Wu et al., 2020 Plant Physiology 182, 27-37).
The publications were promoted via social media:
https://twitter.com/CarolineDeanLab/status/1123634927864561671?s=19
https://twitter.com/CarolineDeanLab/status/1272740437967724545?s=19
https://www.jic.ac.uk/news/physical-link-between-rna-processing-and-epigenetic-silencing-discovered/
and archived with open access in Zenodo (https://zenodo.org) for public availability:
(Nature) https://zenodo.org/record/3897415#.XuollS3My1s
(PNAS) https://zenodo.org/record/3898386#.XuomAC3My1s
(Plant Physiology) https://zenodo.org/record/3898400#.XuomyC3My1s

Objective 1. Does the FCA/FY-mediated repression of FLC depend on SDG26?
The interaction between FLD and SDG26 was confirmed by co-immunoprecipitation (co-IP) (Task 1-3; Deliverable D1.2). To test whether SDG26 behaves in the same way as FLD, I first combined sdg26 with fca and found no additivity compared to fca with respect to flowering time or FLC expression (Task 1-2). Combination of a 35S-FCA transgene with sdg26 mutations then showed that sdg26 mutation compromised the effect of overexpressed FCA on FLC (Task 1-1; Deliverable D1.1).

Objective 2. What are the primary chromatin changes delivered by FLD/SDG26?
I analysed the effect of FLD and SDG26 mutations on H3K4me1 and H3K4me2 levels on FLC using Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR). The results showed a major role of the FLD/SDG26 complex in inhibiting H3K4me1 accumulation through the demethylase activity of FLD. (Task 2-3). However, I failed to detect activity of SDG26 with in vitro assays (Task 2-1; Task 2-2; Deliverable D2.1).
I next sought to determine the association of FLD and SDG26 on FLC locus. However, despite enormous effort, I didn’t manage to find any ChIP signal of both proteins over FLC locus (Task 2-4). Given that no ChIP signal was observed in wildtype background, I didn’t perform the ChIP experiments in fca, fy, cstf64, prp8 mutant backgrounds (Task 2-5).

Objective 3. How does the chromatin environment delivered by FLD/SDG26 repress transcription?
The interaction between SDG26 and DDB1 was not reproducible in later IP-MS experiments, even when crosslinking was first performed to capture dynamic or transient interactions (Task 3-1). No further actions will be planned on DDB1 (Deliverable D3.2; Task 3-2).

Objective 4. What is the link between FCA and FLD/SDG26?
The strong genetic interactions between FLD/LD/SDG26 and FCA raised the question of how FCA function is molecularly linked to FLD. No in vivo physical interactions of FCA with 3’ processing factors or chromatin regulators had been found until our recent analysis using a technique termed crosslinked nuclear immunoprecipitation and mass spectrometry (CLNIP–MS) (Deliverable D4.1 and D4.2) (Task 4-1) (Fang et al., 2019). I found FCA interacted with both RNA and a range of proteins and in vivo localises to nuclear condensates that are highly dynamic (Fang et al., 2019). I applied CLNIP–MS to SDG26 (Deliverable D3.1). Surprisingly, I found some 3’ RNA processing factors, including FCA, FPA, FY and CPSF160, were detected in the SDG26 immunoprecipitation after crosslinking. These data suggest that the interactions between the FLD/LD/SDG26-containing complex and 3’ processing factors provide a physical link (Task 4-1)."
1.3.1 The knowledge transfer between me and my host: I was supported by the bioimaging and proteomics facilities from JIC on how to perform imaging and mass spectrometry analysis, which was a sizeable part of my Fellowship. In return, I developed the CLNIP-MS protocol, which is widely used by the host lab as well as colleagues across the JIC site. I generated many genetic materials that will be useful resources for my host lab in the future. I also gave CDB departmental seminars (201809 and 202003) to disseminate my results, which stimulated very useful discussions, thoughts and constructive feedback.

1.3.2 Improved my scientific competencies: except for all the information described in the DoA, I acquired techniques in bio-imaging, genetics; developed new methods for IP-MS to capture transient and dynamic interactions; produced three high-impact open-access publications; and more importantly, found a faculty position as an assistant professor in School of Life Sciences in Tsinghua University, Beijing, China. In addition, I attended the grant-writing course run by Prof. Mark Buttner (JIC) and the training workshops on lab-management skills run on site, which will help me enormously to run my own lab in the near future.

1.3.3 Built up my collaboration network: in addition to the collaborations described in the DoA, I also collaborated with Dr. Pilong Li at Tsinghua University in Beijing to test the in vitro phase separation of FCA. I also attended conferences and workshops, which broadened my collaboration network for my future career as an independent researcher.
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