6 research outputs found


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    La sindrome di Aicardi-Gouti\ue8res (AGS) \ue8 una rara malattia infantile di origine genetica, i pazienti AGS sono caratterizzati da: atrofia cerebrale, calcificazioni intracraniche ed elevati livelli di interferone-alfa nel liquido cerebro-spinale. Questa inappropriata produzione di IFN\u3b1 pu\uf2 diventare deleteria, promuovendo l\u2019innesco di una risposta autoinfiammatoria nel paziente. AGS \ue8 causata da mutazioni in alcuni geni che codificano per enzimi che metabolizzano o rilevano la presenza di acidi nucleici: TREX1, ognuna delle tre subunit\ue0 (RNASIH2A, RNASIH2B, e RNASIH2C) del complesso enzimatico RNasi H2, SAMHD1, ADAR1 e MDA5. Molteplici evidenze sperimentali suggeriscono che gli acidi nucleici siano responsabili dell\u2019attivazione di sensori dell\u2019immunit\ue0 innata. Pertanto, l\u2019ipotesi pi\uf9 accreditata \ue8 che un accumulo di acidi nucleici endogeni non processati possa essere la principale causa della patologia, inducendo gli elevati livelli di IFN\u3b1 nel liquido cerebro-spinale. Tuttavia, quale sia la natura e il meccanismo di accumulo di questi acidi nucleici resta ancora ignoto. Tra tutti i geni responsabili della sindrome di Aicardi-Gouti\ue8res, \ue8 stato dimostrato che TREX1, ADAR1 e SAMHD1 possono interferire nel metabolismo dei retroelementi LINE1, sequenze di DNA che possono muoversi nel genoma tramite un intermedio a RNA. Un\u2019ipotesi plausibile \ue8 quindi che sia proprio l\u2019accumulo di alcuni intermedi di retrotrasposizione una delle cause scatenanti della iperattivazione dell\u2019immunit\ue0 innata. Caratterizzare un possibile coinvolgimento del metabolismo dei retroelementi e in particolare di LINE1, nella sindrome di Aicardi-Gouti\ue8res \ue8 l\u2019obbiettivo a lungo termine di questo progetto.Aicardi-Gouti\ue8res syndrome (AGS) is a rare genetically heterogeneous disease that typically affects newborns and infants and AGS patients are characterized by cerebral atrophy, intracranial calcifications and elevated levels of IFN\u3b1 in the cerebrospinal fluid (CSF). Such inappropriate activation of type I IFN can be detrimental to the host by promoting autoinflammatory responses. AGS is caused by mutations in several genes encoding nucleic acids sensor or metabolizing enzymes: the 3\u2032 exonuclease 1 (TREX1), any of the three subunits (RNASEH2A, RNASEH2B, and RNASEH2C) of the ribonuclease H2 (RNase H2) enzyme complex, the triphosphohydrolase encoded by SAMHD1, the adenosine deaminase acting on RNA 1 (ADAR1), or the RNA sensor melanoma differentiation associated protein 5 (MDA5). Strong evidence suggest that the accumulation of nucleic acids is responsible for triggering sensors of the innate immunity. Therefore, it is generally believed that an accumulation of unprocessed, endogenous nucleic acids could be the main pathogenic trigger of AGS, increased CSF IFN\u3b1 levels. Among all the AGS-causing genes, TREX1, ADAR1 and SAMHD1 could affect the correct metabolism of LINE1 retroelements, DNA sequences able to move in the genome through an RNA intermediate. Then, a conceivable cause of innate immunity hyperactivation could be retrotransposition intermediates accumulation. To characterize a possible involvement of retroelements metabolism in the AGS pathogenesis is the long term goal of this project

    Fanconi anaemia and LINE-1 retrotransposition in the mammalian genome

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    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

    A Mechanistic Examination of APOBEC3-Mediated LINE-1 Inhibition.

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    Protein-coding genes account for ~3% of the human genome, and a typical gene resides permanently at a discrete chromosomal address. The human genome, however, is not simply a static catalogue of genes; in many ways, it is an ever-changing entity. One dynamic component of the human genome is transposable elements (TEs), or “jumping genes”. Long Interspersed Element-1 (LINE-1 or L1) is a TE whose sequences make up ~17% of human DNA. Although most L1s are inactive, a few retain the ability to mobilize by a process called retrotransposition. L1 is often regarded as a molecular parasite that can be damaging to the host. In fact, ~95 cases of human disease have been attributed to L1-mediated retrotransposition events, and it stands to reason that humans have evolved ways to curtail L1 mobility. The human APOBEC3 (A3) family of cytidine deaminases represents a component of innate immunity hypothesized to have evolved to restrict TE mobility, yet mechanisms by which A3 proteins combat exogenous and endogenous threats are the subject of ongoing study. The focus of my thesis has been a mechanistic examination of A3-mediated L1 inhibition. I have elucidated a mechanism of L1 inhibition by APOBEC3A (A3A), in which A3A deaminates cytidines to uracil in transiently exposed single-stranded DNA during L1 target-site primed reverse transcription (TPRT). In concert with the action of cellular DNA repair factors, which recognize uracil in DNA as damage and degrade deaminated TPRT intermediates, this editing is at least partially responsible for A3A-mediated L1 inhibition. My thesis work represents the first mechanistic explanation for inhibition of an autonomous retroelement by an A3 cytidine deaminase, providing new insight into the dynamic interplay between endogenous retrotransposons and host genomes.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102491/1/risandra_1.pd

    LINE-1 Integration Preferences in Human Somatic Cells

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    Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active transposable element in the human genome. The vast majority of L1s are inactive, but a small number (~80-100 per human genome) retain the ability to mobilize by a ‘copy and paste’ mechanism called retrotransposition. L1 encodes two proteins (ORF1p and ORF2p) required for retrotransposition. ORF2p is a 150kDa protein that has endonuclease (EN) and reverse transcriptase (RT) activities that are responsible for initiating L1 integration by a mechanism termed target-site primed reverse transcription (TPRT). During canonical TPRT, the L1 EN makes a single-strand endonucleolytic nick at a double-stranded genomic DNA target sequence (typically 5’-TTTT/A-3’ and variants of that sequence), to expose a 3’-hydroxyl group that is used as a primer by the L1 RT to reverse transcribe L1 messenger RNA. Different types of transposable elements (TEs) have evolved convergent strategies to target genomic ‘safe havens,’ where TE insertions are predicted to have relatively minimal effects on host fitness and gene expression. Whether L1 integrates into specific genomic regions requires elucidation. In this thesis, I have examined L1 integration preferences in four human cell lines that are proxies for in vivo cell types known to accommodate endogenous de novo L1 retrotransposition events. By combining cultured cell, molecular biological, the Pacific Bioscience sequencing platform, and computational approaches, I characterized 65,079 de novo engineered human L1 integration sites. I compared our L1 insertion dataset to a weighted random model, which assumes that L1 integration preferences are mediated solely by the presence of a degenerate L1 EN consensus cleavage site in the human genome. The data suggest that gene content, transcriptional activity, strand bias, epigenetic environment, and DNA replication status have minimal effects on L1 integration. Thus, L1 EN is the principal determinant of L1 integration. In contrast to canonical EN-dependent L1 retrotransposition, previous studies indicated that L1s could also integrate at sites of DNA damage, including dysfunctional telomeres, by an endonuclease-independent (ENi) mechanism in certain cultured cell lines that contain mutations in genes that render the non-homologous end-joining (NHEJ) pathway of DNA repair and p53 inactive. Here, we explored whether the disruption of other DNA repair pathways influence ENi L1 integration. We observed ENi retrotransposition in certain tissue culture cell lines containing defects in the Fanconi anemia (FA) DNA repair pathway. Since defects in the FA pathway can lead to the accumulation of inter-strand DNA crosslinks that, if left unrepaired, can interfere with DNA replication, we hypothesized that lesions arising at stalled DNA replication forks may provide substrates for enhanced ENi retrotransposition. Indeed, the examination of L1 EN mutant integration sites in FANCD2-deficient cells, suggests that a 3’-hydroxyl group present at Okazaki fragments and/or double-strand DNA breaks generated at collapsed DNA replication forks might be used as a primer to initiate ENi L1 retrotransposition. In sum, our results suggest that ENi L1 retrotransposition may represent an ancestral mobilization mechanism used by LINE-like retrotransposons prior to the acquisition of an endonuclease domain. Under this scenario, LINE-like elements were reliant upon genomic features (e.g., sites of genomic DNA damage, replication forks, and, less frequently, dysfunctional telomeres) to initiate TPRT in the absence of an endonuclease. Indeed, we posit that the acquisition of an endonuclease domain allowed L1 to autonomously insert throughout the genome and, as originally implied by its name, become an interspersed retrotransposon.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140966/1/daflasch_1.pd