SOMATIC_MOSAICISM · LINE-1 retrotransposition in human somatic cells
FP7 — People (Marie Curie Actions)
- Duration
- 2013-06-12 → 2015-06-11
- EU contribution
- €168,896
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
LINE-1 retrotransposition in human somatic cells
In the project SOMATIC MOSAICISM, we are developing a mouse model to get insight into a major recent discovery in human biology: due to the activity of Transposable Elements (TE), the genome of our brain (or: of all somatic cells of our body) is a mosaic. The host lab and others recently demonstrated that some types of TEs are active in the human brain, generating variation in neuronal genomes. LINE elements are expressed in human Neuronal Stem Cells (NSCs) and their activity impact the functioning of NSCs genomes1. However, it is not known if the somatic activity of LINEs is restricted to NSCs or if it also occurs in other somatic cells. Recent results indicate that most LINE activity occurs in pluripotent stem cells during early embryogenesis. Thus, to deeply understand the impact of the somatic activity of LINE elements, we are developing and we will use a model organism to answer the following questions: 1) Are LINEs active in all three germ layers?; 2) Are LINEs differentially regulated, depending on the germ layer?; 3) What’s the impact of LINE activity in the somatic tissues?. In order to answer the first aim, we successfully carried out retrotransposition assays in different (tissue) stem cells, like hESCs, NPCs, MSCs, HSCs, etc. We found high levels of retrotransposition only in NPCs and NPCs in vitro differentiated to mature neurons. This is a remarkable result, as it shows true somatic retrotransposition in non-dividing neurons. In a second approach, we will use a construct allowing inducible LINE-1 retrotransposition. We are currently generating hESC cell lines with one stable insertion of this vector in characterized loci in the genome. Once we have obtained and characterized these cell lines, we will differentiate them in vitro and in vivo (in mice) into the three germ layers. Switching on retrotransposition will allow us to determine its relative rate depending on the germ layer. We have furthermore characterized the endogenous expression of LINEs on the RNA and protein level in the different germ layers, with the result that NPCs moderately express LINE-1 RNA and protein, while the other germ layers show much lower levels. This could explain the potential of NPCs to sustain high levels of somatic retrotransposition. Finally, we have been able to successfully map several new LINE-1 insertions in hESCs with a new mapping protocol. This will allow us to detect hot spots of insertion depending on each germ layer. In parallel, using other funds of the host lab and with the help of a talented PhD student, we have been developing a similar approach using an inducible LINE element in zebrafish (see attached report on the zebrafish project). The comparison of both model organisms will allow us to draw conclusions on whether somatic LINE activity is evolutionary conserved and what is its impact in vertebrates.
Data: CORDIS, © European Union
Project objective
The human genome is largely composed of non-coding DNA, only 3-5% codes for exons. The majority of the non-coding DNA is however fundamental for the correct functioning and regulation of the genome. Transposable elements (TEs) like LINEs generated up to 50% of the human genome during evolution. They can mobilize, causing mutations but also conferring genomic plasticity. The generation of new insertions in the germ line led to the concept of TEs as ‘selfish’ DNA. However, inconsistent with their hereditary transmission, it was recently shown by the host lab and others that most of the action of TEs occurs in somatic cells during early embryogenesis.Thus, to deeply understand the impact of the somatic activity of LINE elements, we propose to develop and use an in vivo mouse model for LINE transposition. We will use human embryonic stem cells (hESCs) and tissue-specific iPSCs, containing an eGFP-marked LINE reporter cassette. Once injected into immuno-suppressed mice these cells will develop teratomas containing tissues of the 3 germ layers (endo, meso, ectoderm). For the proof of principle, the host lab has injected human embryonic carcinoma cells (hECs). With this model, we aim to answer the following: 1) Are LINEs active in all three germ layers?; 2) Are LINEs differentially regulated, depending on the germ layer?; 3) What’s the impact of LINE activity in the somatic tissues?To answer these questions, we will FACS-sort parts of the teratoma into ecto-, meso-, and endoderm-like cells and map the site of new LINE insertions during development, using deep sequencing. We aim to detect hot spots of LINE integration, allowing the analysis of possible genetic and phenotypic consequences. Imaging of the rest of the teratoma will further show us cell types where transposition occurs with higher frequency and possible phenotypic alterations. We therefore hope to detect new principles of genomic plasticity that could regulate gene expression.
Original text from CORDIS.
Participants
- FUNDACION PUBLICA ANDALUZA PROGRESO Y SALUD M.P. · SevillaCoordinatorSpain
Links
Data: CORDIS, © European Union