Dissecting the molecular mechanisms of lncRNA function in X chromosome inactivation across mammalian gestation evolution

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

Background:
In eutherian mammals, X-chromosome inactivation (XCI) is required in females to ensure equal transcription levels of most X-linked genes for both males and females. In female (XX) preimplantation embryos, both X-chromosomes are transcriptionally active from embryonic genome activation until a long non-coding RNA (X-inactive specific transcript: XIST) mediates the inactivation of one. We
know that species with different types of placental development have substantial diversity in the timing and regulation of XCI initiation but we do not know how this process works.

Objectives:
1-To determine the RNA, DNA and protein components that XIST interacts with in embryos from different placental phenotypes.
2-To integrate interaction data and extract the mechanism/s of function of XIST as it relates to placental evolution.
3-To identify evolutionary conserved and divergent molecular mechanisms in the XIST functional complex across different placental and embryo phenotypes.

Novelty:
This cutting edge project combines reproductive biology, RNA biology and computational evolutionary biology to address the fundamental questions of how the lncRNA XIST regulates XCI and the evolution of the mechanism across different placental types.

Timeliness:
This will be the first time the functional evolution of lncRNAs will be addressed in vivo. The in vivo pull-down methods are at the cutting edge of transcriptomic and proteomic technologies. Our recently
founded LeedsOmics will provide the bioinformatics training required.

Experimental Approaches:
Embryos from mouse, bovine, sheep and pig (i.e. different placenta types) will be cultured with sex-sorted semen where possible. Lysates will be generated from the female embryos and then XIST RNA will be pulled-down with antisense biotinylated oligos (in vivo). The components of these XIST complexes will be identified by nano-LC MS/MS (proteins), RNA-seq (RNA) and ChIP-Seq(DNA). Resulting NGS data will be analysed computationally and will be compared to high quality mammal genomes to identify regions of conservation and variation.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1941968 Studentship BB/M011151/1 30/09/2017 30/10/2021 Ioannis Tsagakis
 
Description Maintaining the activity of the correct number of genes across sexes is vital for proper development of placental mammals. Failure to do so results in death early in development. The long non-coding RNA (lncRNA) Xist maintains the balance of gene activity from sex chromosomes via X chromosome inactivation (XCI) of one of the two X chromosomes in female placental mammals. Perturbances of this regulator can also lead to X-linked diseases.

The Xist lncRNA can be found throughout placental mammals, contrary to the trend of weak conservation across most lncRNAs. Nevertheless, most studies to date have focused on its role in mice or human stem cells. However, there are numerous differences in early pregnancy events of distinct placental mammals which could influence the implementation of XCI during embryogenesis. These include the timing of embryonic genome activation, blastocyst formation and implantation, timing and nature of XCI (imprinted vs random), pregnancy duration and placenta shape.

We set out to to reveal whether protein partners of mouse Xist are also present and interacting with XIST from other placental mammals with divergent early pregnancy events such as human, cow and pig.

Our work highlighted that Xist's protein partners in mice exhibit >70% amino acid identity across humans, mice, cows and pigs, and are co-expressed with Xist in the endometrium of these species. We have used RNA immunoprecipitations to show a direct interaction between human Xist and 4 protein partners in a human endometrial cell line.

Overall, my work so far underscores that some protein partners of the mouse Xist lncRNA are retained in humans, which could mean that human XIST lncRNA employs a similar network of interactors to achieve XCI whereas it is plausible a different protein roster governs this process in cows. In the future, it would be interesting to examine how these interactions are maintained by an evolving lncRNA.
Exploitation Route This project is of major significance because it addresses several gaps in our knowledge of lncRNA evolution and more importantly of their evolutionary relationships with interacting partners. Elucidating how these interactions of Xist with its binding partners evolve together in four model organisms, each with diverse early pregnancy morphologies, will provide a better understanding of the XCI molecular mechanisms and dosage compensation processes. This in turn will be beneficial in driving more targeted approaches for candidate screening in X-linked disease treatment.

Additionally, pushing our knowledge on the molecular details of XCI further will implicitly extend our perception regarding sexual dimorphisms. This pertains to variable disease penetrance as well as severity of phenotypic manifestation in the clinic. Illuminating the misty landscape of cellular differentiation by studying the role of Xist at the onset and maintenance stages of XCI can help steer stem cell biology in entering a path towards more reliable representations of pluripotency in the in vitro setting.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Biochemical Society travel grant (general meeting)
Amount £600 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2020 
End 01/2020
 
Description Junior Scientist Conference Grant, non-GS Meeting
Amount £750 (GBP)
Organisation The Genetics Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 10/2019
 
Description White Rose RNA Salon / Club 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Together with two of my supervisors at Leeds, myself and them reached out to colleagues in Sheffield and York to establish an RNA community via applying for a grant from the RNA Society to hold an RNA Salon. I wrote part of the application, co-organised a mini-symposium day at Leeds and wrote the feedback form to return to the RNA Society together with my supervisors. Additionally, I collected and collated attendance statistics to report to the RNA Society as well as used these to help colleagues with another application for the same scheme the year after, which resulted in them receiving the funding to sustain and continue holding these events. Our "White Rose" RNA salon were centered at 3 Universities in Yorkshire, UK; Leeds, Sheffield and York. Activities were focused over 3 days, one at each of the 3 institutions. Each day will have a specific RNA theme to represent and attract RNA researchers; 1) RNA-binding proteins, 2) non-coding RNAs and 3) RNA in disease and development. These are broad to include the communities but will enable each day to have a specific emphasis. Each of three days will include; short talks, keynote, poster session (lunch/coffee breaks) and workshop session. Short talks were chosen from abstracts submitted by members of the communities, and we specifically encouraged submissions from PhD students/postdocs. Abstracts not selected for talks were presented at the poster session. We also encouraged posters from masters students and technicians. Keynote speakers selected, represented a range of PI career stages and both genders. Workshops; having surveyed the communities, there was a great of deal of interest in sharing technical expertise and career development opportunities. Another workshop focused on RNA techniques. The content related to the theme of that day and the keynote speaker eg RNA pull-downs. Activities were organised by a steering committee that included representative from across 3 universities, different career stages of group leaders and junior researchers.
Collaborator Contribution Our "White Rose" RNA salon were centered at 3 Universities in Yorkshire, UK; Leeds, Sheffield and York. Activities were focused over 3 days, one at each of the 3 institutions. Workshops; having surveyed the communities, there was a great of deal of interest in sharing technical expertise and career development opportunities. One event included a presentation on potential career options for our young scientists and networking opportunities to help develop career development relationships for the future. We had several scientists who have recently made the transition to independence on fellowship/group leader positions talking about their experiences. Activities were organised by a steering committee that included representative from across 3 universities, different career stages of group leaders and junior researchers.
Impact The RNA Salon has had a substantial impact of the Yorkshire RNA community, both within the three universities and across the three: 1) It has been an excellent opportunity for students to give their first talks in a local supportive environment. 2) Prizes have encouraged early career researchers and given several students extra confidence 3) Early career organisers have gained valuable experience in organising meetings and improved confidence in chairing sessions and asking questions 4) Our knowledge, as a community, of some specific approaches has increased as a result of the RNA techniques workshop 5) Early career researchers across the Yorkshire RNA community have developed networks to discuss experiments and share experiences. 6) The profile of new groups has been raised by encouraging talks from new PIs 7) New collaborations have already developed e.g. on neuronal lncRNA-ribosome interactions between groups in Leeds and Sheffield. The White Rose Specialised ribosome network that started this year has also gained additional members as a result of discussions at the RNA Salon events. 8) Encouraging early career researchers to ask questions during the Salon events has increased confidence. 9) It has provided an ideal environment for research technicians to present their work for the first time (posters) as part of their career development. 10) Increased understanding of research and technical expertise across the White Rose Network will, in the longer-term help stimulate grant applications and joint PhD studentship (discussions already taking place as a result of Salon). 11) Because the events have moved between the 3 universities, those at each university whose main interest is not RNA have also been able to engage with the RNA community, helping engagement with local RNA networks. 12) Organising the White Rose Salon has stimulated the local RNA communities through the team building that indirectly happens. 13) The opportunity for early careers researchers to present their work to the RNA community
Start Year 2018
 
Description White Rose RNA Salon / Club 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Together with two of my supervisors at Leeds, myself and them reached out to colleagues in Sheffield and York to establish an RNA community via applying for a grant from the RNA Society to hold an RNA Salon. I wrote part of the application, co-organised a mini-symposium day at Leeds and wrote the feedback form to return to the RNA Society together with my supervisors. Additionally, I collected and collated attendance statistics to report to the RNA Society as well as used these to help colleagues with another application for the same scheme the year after, which resulted in them receiving the funding to sustain and continue holding these events. Our "White Rose" RNA salon were centered at 3 Universities in Yorkshire, UK; Leeds, Sheffield and York. Activities were focused over 3 days, one at each of the 3 institutions. Each day will have a specific RNA theme to represent and attract RNA researchers; 1) RNA-binding proteins, 2) non-coding RNAs and 3) RNA in disease and development. These are broad to include the communities but will enable each day to have a specific emphasis. Each of three days will include; short talks, keynote, poster session (lunch/coffee breaks) and workshop session. Short talks were chosen from abstracts submitted by members of the communities, and we specifically encouraged submissions from PhD students/postdocs. Abstracts not selected for talks were presented at the poster session. We also encouraged posters from masters students and technicians. Keynote speakers selected, represented a range of PI career stages and both genders. Workshops; having surveyed the communities, there was a great of deal of interest in sharing technical expertise and career development opportunities. Another workshop focused on RNA techniques. The content related to the theme of that day and the keynote speaker eg RNA pull-downs. Activities were organised by a steering committee that included representative from across 3 universities, different career stages of group leaders and junior researchers.
Collaborator Contribution Our "White Rose" RNA salon were centered at 3 Universities in Yorkshire, UK; Leeds, Sheffield and York. Activities were focused over 3 days, one at each of the 3 institutions. Workshops; having surveyed the communities, there was a great of deal of interest in sharing technical expertise and career development opportunities. One event included a presentation on potential career options for our young scientists and networking opportunities to help develop career development relationships for the future. We had several scientists who have recently made the transition to independence on fellowship/group leader positions talking about their experiences. Activities were organised by a steering committee that included representative from across 3 universities, different career stages of group leaders and junior researchers.
Impact The RNA Salon has had a substantial impact of the Yorkshire RNA community, both within the three universities and across the three: 1) It has been an excellent opportunity for students to give their first talks in a local supportive environment. 2) Prizes have encouraged early career researchers and given several students extra confidence 3) Early career organisers have gained valuable experience in organising meetings and improved confidence in chairing sessions and asking questions 4) Our knowledge, as a community, of some specific approaches has increased as a result of the RNA techniques workshop 5) Early career researchers across the Yorkshire RNA community have developed networks to discuss experiments and share experiences. 6) The profile of new groups has been raised by encouraging talks from new PIs 7) New collaborations have already developed e.g. on neuronal lncRNA-ribosome interactions between groups in Leeds and Sheffield. The White Rose Specialised ribosome network that started this year has also gained additional members as a result of discussions at the RNA Salon events. 8) Encouraging early career researchers to ask questions during the Salon events has increased confidence. 9) It has provided an ideal environment for research technicians to present their work for the first time (posters) as part of their career development. 10) Increased understanding of research and technical expertise across the White Rose Network will, in the longer-term help stimulate grant applications and joint PhD studentship (discussions already taking place as a result of Salon). 11) Because the events have moved between the 3 universities, those at each university whose main interest is not RNA have also been able to engage with the RNA community, helping engagement with local RNA networks. 12) Organising the White Rose Salon has stimulated the local RNA communities through the team building that indirectly happens. 13) The opportunity for early careers researchers to present their work to the RNA community
Start Year 2018
 
Description Athena SWAN Discovery Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Science fair with activities at stalls aimed at 8 to 14-year olds. Described the route from DNA to protein through making of 'codon' bracelets for proteins of different animals while highlighting diverse scientists and their contributions
Year(s) Of Engagement Activity 2018
 
Description • 2019 BeCurious, University of Leeds 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Science fair with activities at stalls aimed at all ages. Helped run activities on the topics of embryo development and implantation.
Year(s) Of Engagement Activity 2019
 
Description • 2019 Pint of Science Leeds 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Science fair with limited activities at stalls and seminar series aimed at all ages. Live-tweeted talks to reach a bigger audience, helped run activities and quizzes on the topics of embryo development while also communicating my research
Year(s) Of Engagement Activity 2019