Investigation of the pluripotency-specific response to LINE1 transposons in early development
File(s)
Author(s)
Garcia Llagostera, Ferran
Type
Thesis or dissertation
Abstract
Long interspersed nuclear element (LINE1) retrotransposons comprise 17% of the human genome and some of them remain capable of retrotransposition. LINE1 is expressed in two different contexts: early development/pluripotency and pathogenic somatic contexts like senescence. In senescent cells, LINE1 expression consolidates the senescent phenotype through the retrotranscriptase and endonuclease enzymatic activities of its encoded protein, ORF2p. In a somatic context, cytosolic single-stranded DNA (ssDNA) intermediates generated by LINE1 trigger an innate immune response that establishes a state of chronic aseptic inflammation. On the other hand, LINE1 expression during pluripotency does not trigger any detrimental effect and new retrotransposition events are very rare. Moreover, LINE1 RNA is necessary for embryonic development progression. These different responses to LINE1 expression suggest an evolutionary pressure exerted by LINE1 to prevent its detrimental potential in pluripotency and allow the cell to tolerate its expression.
I have confirmed the deficiency of ssDNA sensing in mESCs via the cGAS/STING DNA sensing pathway. Expression analyses suggest that the lack of signalling seems to be caused by lack of expression in pluripotency of the DNA sensor cGAS and the innate immunity adapter STING, making mESCs incapable of producing IFN. Complementary to this, I also found that despite an attenuated response to type I IFN, activation of IRF3, the downstream effector of the cGAS/STING pathway, is enough to mount an antiviral response and drive defects in the self-renewal and pluripotency expression network of mESCs.
In parallel, I characterized the endogenous LINE1 ORF1p interactome in pluripotency. This has allowed for the description of new potential LINE1 interactors specific to this developmental stage. To identify potential repressors, I developed a screening platform, consisting of an homozygous LINE1 EGFP reporter insertion in the Rosa26 locus of mESCs. This allowed me to identify a group of potential novel repressor candidates of LINE1 in pluripotency.
I have confirmed the deficiency of ssDNA sensing in mESCs via the cGAS/STING DNA sensing pathway. Expression analyses suggest that the lack of signalling seems to be caused by lack of expression in pluripotency of the DNA sensor cGAS and the innate immunity adapter STING, making mESCs incapable of producing IFN. Complementary to this, I also found that despite an attenuated response to type I IFN, activation of IRF3, the downstream effector of the cGAS/STING pathway, is enough to mount an antiviral response and drive defects in the self-renewal and pluripotency expression network of mESCs.
In parallel, I characterized the endogenous LINE1 ORF1p interactome in pluripotency. This has allowed for the description of new potential LINE1 interactors specific to this developmental stage. To identify potential repressors, I developed a screening platform, consisting of an homozygous LINE1 EGFP reporter insertion in the Rosa26 locus of mESCs. This allowed me to identify a group of potential novel repressor candidates of LINE1 in pluripotency.
Version
Open Access
Date Issued
2023-12-24
Date Awarded
2024-10-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Percharde, Michelle
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)