Systemic Lupus Erythematosus (SLE) is a chronic multi-organ autoimmune disease considered a prototype for autoantibody and immune complex-mediated tissue injury. Although autoantibodies against a wide diversity of self-antigens are characteristically found in this disease, an important hallmark is the presence of autoantibodies to nuclear antigens. Despite this common clinical feature, individual patients vary widely in the organ systems afflicted, disease severity, disease course, and response to treatment. These characteristics make clinical management of SLE challenging and highlight the need for effective and less toxic therapeutic interventions. Susceptibility to lupus has been shown in both human studies and mouse models to be dependent on genetic predisposition. Therefore, it is likely that knowledge of the genetic basis of SLE will be required before full understanding of SLE pathogenesis can be achieved. In this thesis, studies to define the genetic basis of lupus in an induced and two spontaneous models of the disease are presented. These studies encompass mapping, characterization of interval congenic mice, and cloning of the Lmb3 locus gene. In the first study, a genomewide mapping study was performed to define the genetic basis for resistance of the DBA/2 mice to mercury-induced autoimmunity. On chromosome 1, a single quantitative trait was linked with resistance to HgIA. These results linked the locus Hmr1 to a late stage of lupus with GN. Interval congenic mice are important tools to define and characterize the roles of different loci in lupus-like diseases. The second paper identifies the effect of NZB and NZW Lbw2 alleles on lupus susceptibility by using BWF1 mice with none, one or two copies of the lupus-predisposing NZB.Lbw2 locus. The lack of the NZB locus significantly reduced mortality, GN and B cell activation. IgM anti-chromatin levels in genome-wide mapping was linked only to Lmb2 and none of the known B cell hyperactivity-promoting genes were present in this location, which might indicate a novel B cell activation gene. The third study used reciprocal single locus interval-specific congenic mice to characterize the contribution of Lmb1-4 on the MRL-Faslpr and B6-Faslpr backgrounds. The Lmb3 locus on chromosome 7 was found to have the most prominent phenotype with clear effects on lymphoproliferation, GN and mortality. In the fourth paper the Lmb3 was cloned and shown to be a spontaneous nonsense mutation in the Coro1a gene that encodes an actin-binding and -regulatory protein. Upon further characterization, this genetic alteration was discovered to be a new lupus suppressing mutation that reduced T cell migration, activation, and survival. Our findings highlight the complexity of the genetics of lupus, and further suggest that genes involved in controlling the actin cytoskeleton might be potential targets for autoimmune therapeutics.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations. / Abstract : Germline mutations may arise from several endogenous and exogenous mechanisms in both male and female. However, recent next-generation sequencing (NGS) data confirmed that de novo mutations arise primarily in males. This observation suggests that specific spermatogenesis events are involved in the male mutation bias. One potential origin for male-driven mutations is the differentiation of spermatids into spermatozoa, which involves one of the most striking and global chromatin remodeling processes, where histone-bound chromatin is converted into highly condensed protaminated DNA toroid. Using pulse-field gel electrophoresis and comet assay on flow cytometry sorted cells, it was established that chromatin remodeling process is characterized by a transient surge in DNA double strand breaks (DSBs) in the whole population of murine spermatids, which get repaired by the end of spermiogenesis. Using a highly active nuclear extract and immunofluorescences, topoisomerases and markers of DNA repair systems were shown at these steps. Since haploid cells cannot rely on homologous recombination for templated DNA repair, it was hypothesized that this process may be genetically unstable and largely responsible for the observed male de novo mutations bias. Although very challenging, a method allowing the specific genome-wide mapping of DSBs using NGS was developed to establish the genomic distribution of DSBs during chromatin remodeling. It was shown that intergenic regions were enriched in DSBs, particularly LINE1, satellite DNA and simple repeats. Motif finding on potential hotspots showed that proteins from FOX and PRDM families may be implicated. Although homologous recombination cannot take place during spermiogenesis, an enrichment in BRCA1 motif was found, which is also known to be implicated in NHEJ and removal of topoisomerase adducts. Topoisomerase-like SPO11 motif was also enriched suggesting that the meiotic machinery may also be implicated during chromatin remodeling. Moreover, although DSBs tend to accumulate in intergenic regions, gene ontology analysis of hotspot-containing genes showed a marked enrichment in genes related to neurons and brain development. This result hence supports the fact that neurological disease associated mutations are also male biased and associated with advanced paternal age. Since DSB formation during spermiogenesis is conserved through evolution, these results suggest that chromatin remodeling in spermatids represents a significant component in the reported male de novo mutation bias.