Regulation and impact of ongoing retrotransposition in the germline and soma: intersection with human disease

It is becoming increasingly evident that Transposable Elements (TEs) have been instrumental in shaping the structure/function of mammalian genomes. TEs are responsible for generating half of the human genome and a small group of active TEs from the retrotransposon class continue to impact our genome. Long INterspersed retroElements (LINEs) constitute 17% of our genomic mass and are the only autonomous active retrotransposon in humans, with ∼100 active LINEs per genome capable of mobilization (e.g., retrotransposition). Consequently, the ongoing mobilization of LINEs in the germline is responsible for sporadic cases of human disease; notably, others and us demonstrated that heritable LINE insertions in humans accumulate during early embryogenesis and that insertions in germ cells are rare. In fact, we recently characterized >80,000 de novo LINE insertions in human models and demonstrated that they insert randomly in our genome, explaining the wide array of disorders associated with de novo retrotransposition in the germline. Using pluripotent embryonic human cells, our lab established the first developmentally and physiologically relevant model to dissect LINE biology at a mechanistic level. These cellular models were/are instrumental to uncover mechanistic aspects of LINE retrotransposition, including spreading of epimutations by retrotransposition (past ERC-consolidator proposal), uncovering somatic retrotransposition in the human brain (see below), and demonstrating that LINE-retrotransposition drives genomic instability in Fanconi Anemia (FA) patients to cite a few. In sum, although LINE ret rotransposition has been and continues to be a major evolutionary force in the germline genome, we lack a complete understanding of LINE biology, LINE regulation, the impact of LINEs, and mechanist ic intersection/s with human disease, and this is a major Objective of our group. We will continue to exploit unique molecular genetics, biochemical, proteomic, and genomic approaches to better understand LINE activity and regulation in the germline, focusing in several sub-lines of research:

i) Mechanistic control of LINE retrotransposition by of Krab Zinc Finger (KZF) proteins in pluripotent and differentiated cells.

ii) Role of RNaseH2 and deregulation of retrotransposition in Aicardi-Goutieres Syndrome (AGS) patients.

iii) Genomic impact of retrotransposition in FA patients.

iv) Control of retrotransposition by DNA-repair processes in pluripotent cells, among others.

In sum, we posit that a mechanistic understanding of LINE biology is essential to elucidate the forces that contr ibute to human disease, human genet ic variation, and human evolution.

Active human retrotransposons, and in general any TE, represent a prototype of “selfish DNA”: DNA sequences that only replicate in genomes and have no known 63 STRATEGIC PLAN 2024-2027 cellular function. Indeed, the ongoing accumulation of de novo insertions in the germline, our heritable genome, has a clear evolutionary meaning for the retrotransposon. Surprisingly, others and us recently demonstrated that LINEs can also impact selected human somatic cells in the brain, including mature neurons, implying that each of our neurons (~109 cells) might contain a unique genome. Notably, we also found that retrotransposition in mesenchymal and hematopoietic stem is very rare or inexistent, suggesting that somatic retrotransposition in humans might be restricted to our brain. However, further research is needed to determine the extent of somatic LINE retrotransposition in human tissues. Thus, understanding the functional role of ret rotransposit ion in br ain biology and health is another major Objective of our lab. Although any role/impact for somatic retrotransposition remains to be discovered, the ongoing activity of LINEs in neurons imply that it is mathematically impossible to generate genetically identical brains, even in twins. To study retrotransposition in the human soma, we exploited our expertise in stem cell biology to analyse LINE expression and retrotransposition in isogeneic differentiated human cells and neuronal subtypes. However, to go beyond the current state of the art, we recently developed and validated an innovative, unique and robust in vivo model system to measure the impact of brain retrotransposition in real time, using populations of zebrafish (Danio rerio). Strikingly, the human and zebrafish genomes are very similar in their TE composition and presumed insertional activity. Remarkably, using our new in vivo model of retrotransposition, we demonstrated that brain LINE retrotransposition is conserved in vertebrates, and we are currently using our innovative zebrafish model to decipher key questions associated with LINE activity in the soma:

i) Are LINEs active in other somatic tissues?;

ii) What is the genomic impact of LINE activity in the brain?

iii) Which are the phenotypic consequences associated with LINE activity in brain?

iv) Is LINE activity in brain associated with common brain disorders (Schizophrenia, Rett syndrome, etc)?

While accumulating LINE insertions in somatic tissues has no evolutionary meaning for LINEs, deciphering the role of retrotransposition in brain biology is of paramount importance to understand this complex organ and a large number of human disorders.

It is remarkable that a single TE, LINE retrotransposons, have generated more than a third of our genome. Thanks to the “Next Generation DNA Sequencing (NGS) Revolution”, it is now undeniable that retrotransposons are a major force driving mammalian genome evolution. Indeed, de novo LINE insertions in the germline continue to generate inter- and intra-individual genetic diversity, and are sporadically implicated in a wide array of inborn genetic diseases, including hemophilia A, Duchenne muscular dystrophy, and Alport syndrome to cite a few. On the other hand, recent studies suggest that deregulation of LINE expression and the accumulation of byproducts of retrotransposition (RNAs, ssDNAs, RNA:cDNA hybrids, etc) in the germline and/or in the soma contributes to the pathophysiology 64 STRATEGIC PLAN 2024-2027 of a new group of rare (FA, AGS, Ataxia Telangiectasia, etc) and common human disorders (cancer, ageing, schizophrenia, etc). Strikingly, the role of LINEs to these disorders does not necessary imply accumulation of de novo insertions, adding a new layer of complexity to the impact of retrotransposition in human health. However, we know relatively little about the functional impact that a third of our genome exert in health and disease. We posit that a mechanist ic understanding of the impact of LINE ret rotransposition is essential to elucidate the forces that contribute to human disease, human genetic variation, and human evolution.

The research program in our lab is clearly transversal, robust, and innovative. Our lab is a world leader in studying LINE biology and has developed numerous reagents/models widely used by others. Often, the scientific and societal impacts of our research extend “LINE biology” and impact additional disciplines within Biomedicine, from Genome Evolution to Neuroscience, among others.

To strengthen our strategic research lines and international competitiveness, we will continue to pursue ambitious projects (i.e., ERC-Advanced) and will continue to collaborate with other leaders in international networks. In particular, we aim to continue our collaboration with industry (Centre for Drug Design and Discovery, Belgium), aimed to develop new and potent inhibitors of human retrotransposons. We will continue our mentoring activities and will promote the establishment of new PIs.