Human LINE-1 retrotransposition complexes and genomic instability – RETROGENO

The last few years we have developed new molecular tools based on active LINE-1 elements. Using these tools combine with biochemistry, molecular and cellular techniques, we are willing to identify cellular partners of retrotransposition. We will also use next generation sequencing technology to adequately estimate the impact of LINE-1 insertion on chromatin.

With the use of newly developed bioinformatics tools, we have established that LINE-1 can mobilize small non-coding RNA through several mechanisms, some implicating a nuclear recruitment of the RNA by the retrotransposition complex. A larger analysis, with all mammalian genomes being part of a sequencing project, allowed us to highlight a wide variability of LINE-1 retrotransposition dynamic depending of the genome.
We tested several approaches to confer a specific target site to LINE-1. We also developed a new high throughput sequencing strategy to recover a large number of insertions. However, for the moment, we didn’t succeed to direct LINE-1 to a particular insertion site. Nevertheless, with the large number of insertion that we have characterized, we will be able to finely define the insertion sites at the chromatin level. By combining our data with databases from ENCODE, we will identify potential specific chromatin modifications enriched at the loci of LINE-1 insertions.
We have identified specific signature for the modification of the replication timing that could be correlated with the absence or presence of L1 elements during replicative stress associated to physiological or pathological aging.

The perspectives of the project are to transfer our knowledge on the impact of LINE-1 mobility on genomic instability and structural variations, to the potential use of induce pluripotent cells for cellular therapy. Indeed, global deregulations of mobile elements and genomic structural variations have been observed when cells are induced for pluripotency. Thus, it is of great importance to understand the mechanism involved in genomic instability.

1. Doucet AJ, Droc G, Siol O, Audoux J, Gilbert N. (2015). U6 snRNA Pseudogenes: Markers of Retrotransposition Dynamics in Mammals. Mol Biol Evol. 32(7):1815-32.
Genomic copies of snRNA are makers of genomic dynamics and variability associated with retrotransposition.
2. Doucet AJ, Basyuk E, and Gilbert N. Cellular localization of engineered human LINE-1 RNA and Proteins. Methods Molecular Biology, Submitted
Proctocols to visualize and follow L1 retrotransposition intermediates.
3. Juan Carlos Rivera-Mulia, Hélène Schwerer, Jiao Sima, Emilie Besnard, David M. Gilbert and Jean-Marc Lemaitre. Specific replication timing signatures of aging and Hutchinson–Gilford progeria syndrome can be reset. Submitted
Specific signature of the modification of the replication timing during replicative stress associated to physiological or pathological aging.
4. Haiqing Fu, Emilie Besnard, Romain Desprat, Michael Ryan, Malik Kahli, Jean-marc Lemaitre and Mirit Aladjem (2014). Mapping Replication Origin Sequences in Eukaryotic Chromosomes Curr. Protoc. Cell Biol.
Sequencing protocol for replication origins

Retrotransposition is a common molecular mechanism of all eukaryotes. It has largely contributed to the modelling and evolution of all genomes. In human, LINE-1 (Long Interspersed Nucleotidic Element-1) rétrotransposons represent about 17% of the genome. It is the only autonomous mobile element that remains active, and is responsible for the amplification of non-autonomous retrotransposons such as Alu sequenes and retropseudogenes. Thus, during evolution, LINE-1 has contributed to almost one third of the human genome.
Despite this contribution and the fact that these mobile elements have been already discovered for about forty years, little is known about the mechanisms implicated in LINE-1 amplification.
In this project, we are willing to elucidate several steps of the retrotransposition mechanism in human cells. More importantly we would like to identify cellular factors that are involved in the process. In recent work, we have developed new molecular tools that allowed us to identify the basal LINE-1 retrotransposition complex. This complex is composed of both LINE-1 proteins (ORF1p and ORF2p) and the LINE-1 RNA. In cell where LINE-1 is over expressed, this complex is accumulated in the cytoplasm and form cytoplasmic foci. We now envision to purify the LINE-1 ribonucleoproteic (RNP) complex in a non destructive manner and to identify cellular partners by mass spectrometry.
In a second aim, we want to characterize partners of the retrotransposition complex present in the nucleus. To achieve this part of the project, we will develop new molecular tools that will confer target site specificity to the LINE-1 retrotransposon. Being able to target a particular DNA sequence will allow us to study LINE-1 during the integration process.
Finally, to be transmitted to subsequent generations, retrotransposition has to occur in germ cells or during early human embryogenesis. We have thus decided to set up a retrotransposition assay in human embryonic stem cells (hESC) or in induced pluripotent embryonic cells (iPSC). This new system will allow the study of retrotransposition mechanisms in conditions close to the physiological conditions.
This project will tend to develop our knowledge of the major mechanism implicated in genomic instability which participates in structural and phenotypic variation and in some cases to cancer development. More importantly, it is of great importance for the development and the potential use of iPSC for cell therapy.