Fanconi anaemia and LINE-1 retrotransposition in the mammalian genome

Abstract

Transposable elements (TEs) are discrete, repetitive sequences of DNA that mobilise within genomes. For decades, TEs were dismissed as “junk DNA”, however, it is now clear that these elements have the potential to trigger genome instability, cause disease and shape the course of genome evolution. L1 elements constitute the only autonomous elements which remain active in the human genome and comprises approximately 17% of human DNA. As a retrotransposon, L1 canonically mobilises through a “cut and paste” mechanism called target primed reverse transcription (TPRT). Due to the deleterious impacts of L1 activity, mammalian cells have evolved a range of mechanisms to supress the mobilisation of these elements. The interactions between L1 elements and the host factors which regulate them are therefore an area of active research. Several DNA repair genes have shown potential as regulators of L1 activity. Moreover, in cell lines deficient in non-homologous end-joining, L1 has shown the potential to retrotranspose without its ORF2p endonuclease, which is usually a requirement for canonical TPRT. This retrotransposition has been termed endonuclease independent (ENi) retrotransposition, and takes place at unrepaired double stranded breaks in the DNA. Interestingly, several DNA repair factors have also been identified as potential regulators of L1 retrotransposition (both positive and negative), including a number of proteins from the Fanconi Anaemia pathway. The relationship between these factors and L1 has yet to be fully characterised, and it remains to be seen whether L1 can exploit other DNA lesions in the way that it utilises DSBs in ENi retrotransposition. This thesis aims to further investigate the relationship between L1 retrotransposition in the mammalian genome and DNA repair factors, particularly those comprising the Fanconi Anaemia pathway. Using cultured cell retrotransposition assays, I systematically tested a battery of mutant element in cells deficient in different proteins of the FANC pathway. In this way, I establish that ENi retrotransposition can be observed in a FANC background. I also demonstrate that FANC A deficient cells support retrotransposition of several L1 mutants which are immobile in parental cell lines. This includes elements with severe ORF1p mutations, mutations in the ORF2p endonuclease domain and mutations in the ORF2p PIP box. Despite testing a range of cell lines deficient in different DNA repair factors, including cells deficient in a range of FANC proteins, the retrotransposition of ORF1p, PIP and mutants appears to be unique to FANC A. My results are potentially indicative of a unique mechanism of retrotransposition in FANC A cells, a phenomena which has precedence in the ENi pathway of retrotransposition. Mass spectrometry of immunoprecipitated T7-tagged ORF1p, both in FANC A and parental cells, demonstrated that a different selection of host factors interact with ORF1p in the two cell lines. Several of these have not been previously identified as L1 interactors, including YTHDF2, a protein which binds and destabilises m6A-containing RNA. Previous reports suggest that YTHDF2 regulates the stability of RNA:DNA hybrids in vivo, and associates with R loop containing loci. Through co-immunoprecipitation of YTHDF2 with ORF1p, I confirm that the protein interacts with L1 elements in vitro