Periodic Reporting for period 1 - EvoPWO (Evolution of plant PWO proteins and their function: a synthetic biology approach)
Reporting period: 2021-04-01 to 2023-03-31
Polycomb Group (PcG) proteins are responsible for maintaining cellular identity in multicellular organisms, including plants. Among the PcG proteins, the Polycomb Repressive Complex 2 (PRC2) plays a significant role in controlling the expression of crucial developmental genes by establishing the repressive epigenetic mark on specific target genes. The proper functioning of PRC2, along with its associated accessory proteins, is crucial for orchestrating gene expression patterns that govern various aspects of plant growth and development. Among the accessory proteins, PWWP DOMAIN INTERACTOR OF POLYCOMBS 1 (PWO1) serves as a recruiter and regulator of the PRC2 complex, highlighting the functional importance of PWO proteins in plant development. Furthermore, the loss of PWO proteins in Arabidopsis leads to seedling lethality and the arrest of shoot and root meristems, emphasizing the necessity of investigating the proper function of PWO in plant evolution. We began our research by establishing that PRC2 orthologs are highly conserved from unicellular algae to modern plants. However, we noted that the evolutionary pattern of PWO-like proteins differs. These proteins exclusively arise at a specific time in plant evolution, coinciding with the colonization of land and the emergence of vascular systems. This raises the question of why an essential and conserved PcG protein complex requires the addition of an accessory protein such as PWOs to fulfill its function at a specific time during plant evolution. This intriguing observation forms the fundamental hypothesis of the EvoPWO project. To address this, a synthetic biology approach was employed with the aim to assess the impact of ancestral PWOs functions in modern plants. Simultaneously, the fundamental question regarding the involvement of PWOs proteins in the PcG complex throughout plant evolution is also being investigated. Altogether, the EvoPWO project aimed to address the evolution and overall function of PWOs proteins across different species. The overall objective of the EvoPWO project was to analyse the function of ancestral PWOs in PWO-less species as well as in modern plants and investigate the interaction of ancestral PWOs to Arabidopsis PRC2 and CRWN1 proteins. Lastly, to identify regulatory proteins that interact with ancestral PWOs to regulate plant growth and development.
To gain insights into the evolution and function of PWO proteins associated with the PRC2 complex across various species, we performed a phylogeny analysis and identified the sudden emergence of PWO proteins in the Lycophytes. Structure prediction of PWOs from different plant species revealed highly conserved PWWP domains and a C-terminal tail (C-tail). We observed that the identified PWO proteins were separated into four major clades - PWO1, PWO2/3, Polypodiopsida-specific clade, and Ancestral PWO clade. We also conducted experiments to characterize the localization and interactions of PWO proteins. The Selaginella moellendorffii (Sm)-PWOa and SmPWOb, belonging to the ancestral PWO clade, were found to form nuclear speckles, similar to Arabidopsis thaliana (At)PWO1. However, SmPWOb predominantly localized to the nucleolus in plant nuclei. We demonstrated that SmPWOa interacts with PRC2 subunits from different species, such as A. thaliana, S. moellendorffii, and Physcomitrium patens, and tethers AtCLF to nuclear speckles. On the other hand, SmPWOb recruits AtCLF to the nucleolus in N. benthamiana. Moreover, we generated transgenic Arabidopsis lines overexpressing SmPWOa, which resulted in an increase in nuclear size. This observation suggests that SmPWOa may influence the nuclear structure of these lines. Collectively, our findings indicate that the PWO-PRC2 interaction exists in plants from the very beginning of PWOs emergence, highlighting the functional significance of PWOs in epigenome regulation during land plant evolution.
During the MSCA project, two secondments were realised. MSCA secondment 1: 19 January – 08 February 2023, Dr. Sara Farrona Group, University of Galway, Ireland; MSCA secondment 2: 20 February – 06 March 2023, Prof. Daniel Schubert Group, Freie Universität Berlin, Germany.
The results of the EvoPWO project are currently being prepared for publication, and the manuscript is yet to be published. We expect that a peer-reviewed publication will be available in early 2024, accessible through Open Access. However, the project's findings have already been shared and presented at various international conferences and seminars. These include poster presentation at two international conferences such as ‘7th European Workshop on Plant Chromatin held in Prague, Czech Republic from May 18-20, 2022’ and ‘9th Plant Genomics and Gene Editing Congress organized by Global Engage Ltd in Raleigh, North Carolina, USA from October 24-25, 2022’. Additionally, the project's findings were also presented orally at the University of Galway in Ireland on February 3, 2023, at Freie Universität in Berlin, Germany on February 23, 2023, and at the Plant Sciences Seminars held at the Biology Centre CAS in České Budějovice, Czech Republic on March 30, 2023. In addition, the results were also disseminated and discussed six times at regular lab meetings of Iva Mozgová Group at Biology Centre (BC) CAS. I also contributed to make MSCA program more visible and attractive for other postdoctoral researchers at BC, https://www.bc.cas.cz/en/news/news-detail/6772-msca-fellowships-are-of-great-benefit-to-the-biology-centre-cas/(opens in new window)
During the MSCA project, two secondments were realised. MSCA secondment 1: 19 January – 08 February 2023, Dr. Sara Farrona Group, University of Galway, Ireland; MSCA secondment 2: 20 February – 06 March 2023, Prof. Daniel Schubert Group, Freie Universität Berlin, Germany.
The results of the EvoPWO project are currently being prepared for publication, and the manuscript is yet to be published. We expect that a peer-reviewed publication will be available in early 2024, accessible through Open Access. However, the project's findings have already been shared and presented at various international conferences and seminars. These include poster presentation at two international conferences such as ‘7th European Workshop on Plant Chromatin held in Prague, Czech Republic from May 18-20, 2022’ and ‘9th Plant Genomics and Gene Editing Congress organized by Global Engage Ltd in Raleigh, North Carolina, USA from October 24-25, 2022’. Additionally, the project's findings were also presented orally at the University of Galway in Ireland on February 3, 2023, at Freie Universität in Berlin, Germany on February 23, 2023, and at the Plant Sciences Seminars held at the Biology Centre CAS in České Budějovice, Czech Republic on March 30, 2023. In addition, the results were also disseminated and discussed six times at regular lab meetings of Iva Mozgová Group at Biology Centre (BC) CAS. I also contributed to make MSCA program more visible and attractive for other postdoctoral researchers at BC, https://www.bc.cas.cz/en/news/news-detail/6772-msca-fellowships-are-of-great-benefit-to-the-biology-centre-cas/(opens in new window)
The project has generated novel insights into the evolution of PWOs and the function of PWO-PRC2 in plant evolution. The conducted phylogenetic analysis in this study has provided valuable information regarding the evolutionary patterns of PWO proteins across different species. By the end of its duration, the EvoPWO project achieved several key outcomes. Firstly, it aimed to unravel the molecular activities and functions of ancestral PWO proteins in modern plants. This investigation provides information on the specific roles and mechanisms through which PWO proteins contribute to plant development and gene regulation. Additionally, the project helps in characterizing the interaction between ancestral PWO proteins and Arabidopsis PRC2 subunits. By elucidating the nature of this interaction, we can gain a deeper understanding of the regulatory processes involving PWOs and PRC2 in plant systems. The findings of the EvoPWO project have significant socio-economic implications. The obtained insights into the intricate molecular interactions and regulatory mechanisms of PWOs function open up new avenues to understand the PWO-PRC2 function. This information can be utilized to investigate possible mechanisms and develop innovative applications for Biotechnology and Crop Improvement. Moreover, the outcome of the project has contributed to the scientific community through conference presentations and seminars, aiding in the dissemination of knowledge and fostering scientific exchange. By sharing project findings and engaging in discussions, we contribute to the broader societal implications of advancing our understanding of plant biology. This knowledge can ultimately benefit various sectors, including agricultural sciences.