Periodic Reporting for period 1 - InvADeRS (Investigating the Activity of transposon Derived Regulatory Sequences in the placenta) Reporting period: 2019-05-01 to 2021-04-30 Summary of the context and overall objectives of the project The placenta is crucial for the initiation and maintenance of pregnancy. Common complications of human pregnancy, such as preeclampsia, preterm birth, recurrent miscarriage, and growth restriction, affect ~20 % pregnancies and are each associated with problems with the placenta. The in utero environment also profoundly affects the future health of the baby, and is linked to adult conditions such as heart disease, diabetes and hypertension. Despite this, the placenta itself is a vastly understudied organ, and the causes of its success or failure are often unknown. The development of the placenta, although heavily influenced by the environment, is ultimately controlled by its genetics and those of the mother. Understanding of the role of genetics in placental development are therefore vital to understanding complications of human pregnancy, as well as adult diseases that stem from early development. The InvADeRS research project sought to define the role of a poorly understood and as-yet mostly unexplored class of genetic sequences, known as transposons, in human placental development and disease. Transposon sequences make up around 50 % of the human genome. They are relics of viral infections that have occurred throughout evolution, and most transposon sequences are silent and likely inert. However, a subset of transposons have recently been shown to regulate genes by increasing or decreasing gene activity, resulting in changes in cellular function and organism physiology. In one intriguing example, a transposon sequence was shown to extend the length of pregnancy through upregulation of a single gene.The idea that transposons may regulate genes in the human placenta is an exciting one, and based on several lines of evidence. Firstly, in the placenta, transposon DNA tends to be in an active state. This is in contrast to other organs and tissues of the body, where transposons are silenced and have a restrictive DNA conformation. In this way, their ability to regulate genes seems increased specifically in the placenta, although as yet we do not know why. Secondly, the placenta forms a diverse array of structures and shapes in different mammals. Transposons are highly variable between different species, even closely related ones, and so this variety could very well have contributed to the rapid evolution and structural variation of the placenta. Finally, transposons in other mammals, such as mice, have been shown to regulate hundreds of genes in the placenta. The overall objectives of the InvADeRS project were to evaluate the role of transposons in gene regulation in the human placenta, and measure their potential impact on complications of pregnancy. We found that several families of primate-specific transposons harbour the potential for regulating placental development. Our candidate transposon families positively impact placental gene expression overall, and are enriched in binding sites for molecules important for placental invasion. We identified key genes that are directly regulated by transposons in human placenta, including several required for placental invasion, demonstrating that transposon regulation is an important part of placental development in humans, and therefore their deregulation could contribute to complications of pregnancy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far By analysing of the structure of DNA in human placental stem cells (derived from the fetal side of the placenta), as well as first trimester and term placentas, the InvADeRS project has identified transposon sequences with the potential to act as gene regulators in the human placenta. Overall, we identified an increase in placental gene expression for genes located close to transposons (and thus potentially regulated by them) compared to the rest of the genome, indicating that transposons have a positive impact on gene expression in the human placenta. Next, by examining genes likely to be regulated by candidate transposons, as well as signalling molecules predicted to interact with candidate transposons, we found that many transposons were linked to genes and pathways that control the invasion of maternal tissues by the placenta. The transposon sequences we identified to potentially regulate placental development were all primate-specific. This is consistent with previous studies that show that the majority of primate-specific regulatory DNA, (i.e. regulatory sequences not present in species other than primates), is transposon derived. To irrevocably prove that transposons could regulate placental gene expression, we used a genetic cutting method to remove candidate transposons from the genome of placental cells in culture. We then measured the effect on nearby genes, identifying five cases where transposons acted as gene ‘enhancers’, increasing gene expression.Our results have been disseminated through talks and posters at the following national and international conferences and seminars: In 2019 - The EpiGeneSys Conference, Francis Crick Institute, UK; The Centre for Trophoblast Research Conference, Cambridge, UK, the Epigenetics Gordon Research Conference, Holderness School, NH, USA, and The Physiological Society Departmental Seminar Scheme, University of Southampton. In 2020 - the Cold Spring Harbour Laboratory virtual transposable elements meeting. In 2021 - the Medical and Molecular Genetics virtual seminar series, King’s College London, UK and the FASEB Virtual Mobile DNA Conference. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The candidate transposons we found were primate specific. This is particularly interesting because placental invasion in humans and other higher primates, particularly great apes, is very extensive when compared to other mammals. The placenta invades very far into maternal tissues and causes major remodeling of her uterine arteries, maximising blood flow to the baby. Therefore, the primate specific transposons we have identified are excellent candidates for the potential regulation of this process.In addition, we identified a number of genes that were regulated by transposons that are linked to placental invasion. Our candidate transposons were also predicted to bind signalling molecules important in invasion of maternal tissues. This is important because the extent of placental invasion is a principal factor in the health of a pregnancy. If the invasion process is aberrant, or incomplete, the baby may not be supplied with enough blood, leading to growth restriction, preeclampsia and/or preterm birth, or the embryo may fail to implant altogether, resulting in miscarriage. In this way, our findings contribute a novel set of candidate human genetic sequences that may be important for proper placental development and normal pregnancy. Further investigation of the genetics and regulation of these transposons in the population will improve future research into pregnancy complications, providing new avenues for research, screening of at-risk couples, diagnosis, and therapy.