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How retrotransposons remodel the genome during early development and reprogramming

Periodic Reporting for period 2 - TransposonsReprogram (How retrotransposons remodel the genome during early development and reprogramming)

Reporting period: 2018-06-01 to 2019-12-31

How retrotransposons remodel the genome during early development and reprogramming

The overall goal, objectives and why this research is important for society

Around half of the DNA within the human genome is derived from ancient viruses or “transposons,” most of which use a copy-and-paste mechanism to make new copies of their own DNA to insert into our genome, allowing for their stable inheritance. This part of our genome of viral origin is often referred to as “dark matter” because little is known about its function. The overall goal of our ERC starting grant is to understand how DNA-sequences of viral origin have been beneficially repurposed in mammals to allow normal progression through development. Understanding these novel mechanisms is important for society because it will help us to direct which type of cell a pluripotent cell will ultimately become and how to reprogram committed cells back into pluripotent cells, which can then be used to form new cell types. Such research is relevant to making more effective stem cell therapies. More broadly, though, this research will help us to understand how genetic mutations within non-coding regions can lead to developmental diseases in which transposons are implicated, such as Repeat Expansion Disorders or Aicardi Goutières Syndrome. We hypothesize that ancient transposon-derived DNA sequences play essential roles in cell fate transitions in early development and during reprogramming. We think this because viruses are ideal tools that the cell can repurpose as regulatory hubs because they contain compact promoters, enhancers and silencers within a short long terminal repeat (LTR) sequence, for example, and mounting evidence shows these ancient viruses to be switched on in early development. In contrast, the bulk of most viral coding sequences of transposons may have been lost through genetic drift and recombination. The key objectives of this ERC starting grant are to identify the functions of several types of transposons during early development and reprogramming and assess the mechanisms of how they remodel the genome to control cell fate. Our approach is to employ two novel zinc finger proteins that bind to transposon DNA as tools to explore the function of these transposons.

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We are exploring how retrotransposons remodel the genome during early development and reprogramming. We are using the term “remodel the genome” to refer to changes in enhancer and promoter activity and changes to chromatin structure that ultimately lead to cell fate transitions. (1) This diagram shows how chromatin can be remodelled from an inactive state (left) to an active state where a network of genes becomes switched on (right). (2) Little is known to date on how transposon-derived DNA sequences can direct cell fate and we are addressing this question by focusing on zinc finger proteins (ZFPs) specific for transposon-derived DNA. ZFPs recruit the epigenetic complexes stated here to mediate gene silencing. We have performed targeted CRISPR/Cas9-mediated knockout of these ZFPs in a developmental model as a tool to address the roles of their cognate transposons in regulating cell fate. (3) The model that we are using is naïve mouse embryonic stem cells, which are representative of early embryos that we differentiate in vitro into neural progenitor cells (NPCs) and neurons. We hypothesize that although these ZFPs may have evolved to block the spread of transposons, they now may function to regulate cell fate transitions and our results to date support this notion. (4) The concepts we discover in our mouse model of development with also be relevant for understanding human gene regulation and the importance of non-coding DNA in developmental and neurodevelopmental diseases.
We previously identified two KRAB-domain-containing zinc finger proteins that regulate expression of transposons in mouse embryonic stem cells. Our goal was to use these two zinc finger proteins as tools to dissect the function of transposons in an in vitro neural differentiation assay. Our first aim (Aim 1) is concerned with mapping the regulatory landscape of these two ZFPs and their cognate transposons and the first step was to perform chromatin-immunoprecipitation (ChIP) experiments to determine the binding profile of these ZFPs. We carried this out by overexpressing triple HA-tagged versions of the ZFPs in mouse embryonic stem cells (mESCs) and performing ChIP-seq using a HA antibody (see objectives 1A and 1C). Our binding data reveal these two ZFPs to exhibit binding profiles to distinct classes of transposons. Furthermore, we have performed CRISPR/Cas9-mediated genome-editing of these ZFPs in mESCs and selected four independent clones for follow-up as well as appropriate Cas9 control clones and wildtype controls. RNA was extracted from these clones and used for total RNA-sequencing (see objective 1C). ChIP-sequencing and RNA-sequencing data were then analysed and integrated together including with public data on histone marks, to obtain an overview of the regulatory landscape of these ZFPs. Our landmark achievement to date, resulting from these data, is to discover that both these ZFPs regulate distinct stage-specific gene expression programmes through binding to transposon-derived sequences. Furthermore, we have successfully set up our in vitro model of neural differentiation (see objective 1B) using a Rex1-GFP mESC reporter cell line (kind gift from Austin Smith). We have been able to differentiate mESCs into anterior (forebrain) Pax6 positive neural progenitors (with advice from James Briscoe) using N2B27 media + basic fibroblast growth factor, growing monolayers of cells on laminin-coated plates. We can demonstrate that upon differentiation, cells downregulate expression of the Rex1-GFP reporter, as expected, and upregulate Pax6, which we can detect with a Pax6-PerCP-cy5.5 antibody using an intracellular flow cytometry staining protocol (see objective 1B). Setting up this system has been paramount to us obtaining another key finding that one of these ZFPs appears essential for neural differentiation (see objective 2A).
Poppy Gould (one of our two ERC-funded postdocs) was selected to present her work both Nationally and Internationally this year, and through these opportunities we have built two exciting collaborations that will enhance the importance, impact and dissemination of our work. Firstly, Poppy was selected to present her work as a poster at a recent Royal Society funded conference entitled: Crossroads between transposons and gene regulation (May 2019, UK). At this conference, we met Frank Jacobs (University of Amsterdam) who employs brain organoids to assess the functions of ZFPs in development, and we set up a collaboration whereby Poppy will employ his brain organoid assay in his lab to assess the role of the ZFPs and transposons we are studying in organoid development. Secondly, Poppy was selected for a talk at a FASEB conference entitled: The Mobile DNA Conference: 25 Years of Discussion and Research (June 2019, California, USA). Through this conference, she met Arian Smit (Institute for Systems Biology, Seattle), who works on annotation and evolutionary history of transposons in the genome and he is now collaborating with us to track the evolutionary history of the transposons that we work on here. This additional data will further enhance our ERC work and our understanding of how mammalian genomes are regulated and how they continually evolve and are remodelled by transposons. Other exciting experiments that we have planned are ATAC-sequencing and chromatin topology experiments using chromosome conformation capture technologies (see objective 1C). This is because our metadata analyses (objective 1D) suggest that the mechanism by which these transposons may control cell fate is through structural changes to chromatin. Furthermore, we have identified a collaborator for aim 3A (George Kassiotis, based at the Francis Crick Institute, UK) and have planned experiments where we will test the effect of ZFP-genome editing on blastocyst development in vitro (see objective 3A). Finally, Rocio (one of our two ERC funded postdocs) has pioneered new methods to better analyse expression of transposons using RNA-sequencing data and is using her approaches to apply to our data obtained in this project and public data. Overall, we plan to consolidate and extend our current impactful findings and prepare our work for publication and dissemination.
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