Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - EVOLINE (Mobile elements: shuffling the regulome in development and disease.)

The genomes of all living organisms are dynamic entities that vary in small ways between individuals, thereby that constituting the basis for diversity and evolution. Deleterious, emerging genetic modifications are cleared away by processes like natural selection while new changes need to be introduced in every generation to keep a diverse and adaptable population, hence ensuring survival. Mobile genetic elements (MGEs) are a very powerful engine for introducing such modifications. These elements are small genetic units able to replicate and introduce new copies within the genome. The insertion of a new MGE copy in a given genomic location may affect the expression of nearby genes in a number of ways (reviewed in Macia et al. 2014). This insertional mutagenesis likely has more of an impact than regular substitutive mutagenesis due to the amount and strength of regulatory signals present within a new MGE copy in order to achieve its own replication through complex cellular physiology processes.
The defective replicative nature of many MGEs, together with the natural mutation of their sequences through replication cycles, attenuates the number of active MGE copies present in each genome. Nonetheless, evolution has diverted significant resources to restrict MGE activity in the germ line, in order to keep their activity under a limit that ensures a balance between diversity and genome integrity. As a result, a number of new MGE copies are present and segregating amongst the human population (so called polymorphic insertions) and a number of cases of human diseases have been ascribed to new MGE insertions. Remarkably, MGE activity during an organism’s development may also create diversity within its somatic cells by spreading new insertions in a subset of cells (so called somatic insertions), therefore contradicting a traditional dogma by which all the cells within an organism were thought to have an exact copy of the genome of the original fertilized egg. Epigenetic regulation plays a major role in controlling MGE activity, and is therefore present throughout the different cell types and tissues found within the body.
L1 is the only autonomous mobile element that remains active in the human genome. Around 17% of the human genome is estimated to be derived from L1 copies, and up to half from the activity of other still active MGEs, or those now inactive but active in the past (Lander et al. 2001). L1 elements contain certain specific features, including bidirectional transcriptional activity at both ends (Swergold 1990; Speek 2001; Faulkner et al. 2009) in order to achieve and maybe regulate its own mobilisation. However, at the same time, these could be affecting the regulatory expression of distant genes by long distance genomic interactions like the ones described between enhancers and promoters. L1 activity has been associated typically to the germline but also to certain somatic tissues like brain as well as certain cancer types. The activity in the brain has been estimated to be quite substantial by both genetic engineering-based experiments using transgenic models (Muotri et al. 2005; Coufal et al. 2009) and by direct high throughput sequencing of brain human tissue in order to detect somatic insertions underrepresented in cellular population (Baillie et al. 2011; Upton et al. 2015).
The aims of the project were to use genetic engineering to test if the L1 in mammals has elements that can perform long distance regulation of gene expression and, in that case, develop a high throughput sequencing-based technique to detect somatic insertions involved in interactions of this nature in both brain and cancer. However, during the development of the project, reports from several labs found contradicting, low levels of MGE mobilisation in the brain of human as well as other mammals and insects (Evrony et al. 2012 and 2015, Hazen et al. 2016, Erwin et al. 2016 and Treiber 2017). In order to resolve these contradictions, we performed a single-cell analysis combining three different techniques for somatic insertion screening in human hippocampal neurons: retrotransposon capture-sequencing (RC-seq, Upton et al. 2015); L1-sequencing (L1-Seq, Ewing et al. 2010; Evrony et al. 2012); and direct whole genome sequencing from amplified genome of single cells (Evrony et al 2015). From the combination of all of these methods, and a much more selective filtering, we concluded that the rate of L1 somatic mobilisation in the human brain may be lower than first thought, although this estimate is still heavily dependent on technical considerations, which would require a different approach to evaluate how somatic L1 insertions impact cellular phenotype.
Nonetheless we were able to identify a recurrently active polymorphic L1 insertion that had generated somatic mosaicism in the brain. This L1 copy is polymorphic in the human population, which means that only a subset of people carries it. Additionally, mutations within the copy have generated defective alleles. These alleles segregate like for normal genes, which means that only a subset of carriers are potentially susceptible of being impacted by somatic events arising from the source element (a system similarly described for other copies by Scott et al. 2016). Remarkably, after closer inspection we found that the donor element lacks an important transcription factor binding site due to a short 5’ truncation. Alterations on this site have been proved to not to be totally disabling of the transcriptional activity, although its intensity or accuracy for the starting point can be affected in different established cell lines (Athanikar et al. 2004).
We tested the status of epigenetic regulation for our donor L1, assessing the level of methylation of its DNA in order to find a basis for the recurrent activity of this L1 copy despite the crippling mutation it carries in its promoter. We found that this particular source L1 copy is not epigenetically repressed across different tissues and individuals. We also found that this L1 copy is very likely fully derepressed during embryonic development, in contrast to most L1 copies. After testing other L1 copies with similar mutations, we conclude that the lack of the aforementioned transcription factor binding site prevents repression at the cost of compromising the donor’s transcriptional activity. Additionally, the donor L1 was resistant to repression during in vitro differentiation to neurons. This could actually constitute a starting point for a new escapee family within the evolutionary arm race between host and L1, similar to other reported examples during L1 evolution (Jacobs et al. 2014, Castro-Diaz et al. 2014).
We developed a new strategy that couples the traditional way of studying methylation in non-repetitive loci within the genome to a high throughput analysis of locus-specific L1 copies. This allowed us to study this L1 copy within different tissues, individuals and cell lines, and also to study a higher number of specific L1 copies across all these samples. Also, optimisation of the DNA amplification allowed us to resolve the methylation pattern of a wider area within the L1 promoter in comparison to previous strategies (Scott et al. 2016, Tubio et al. 2014, Coufal et al. 2009). This technique has been already applied in other ongoing studies within the overseas laboratory of Prof. Faulkner in different samples of ovarian and hepatic cancers, and revealed differential repression of specific L1 copies between samples of normal tissue and cancer.
We have found that L1 somatic mobilisation in human brain may be lower than previously speculated, but also finding that this estimate is strongly dependent on technical considerations that remain unresolved. However, we have defined a subset of polymorphic elements that likely avoid cellular repression during embryonic stages and remain de-repressed to a variable extent in the cell population of mature tissues (e.g. healthy brain). Therefore, humans are a mosaic in terms of the activation/repression of these elements.

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Life Sciences
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