Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSb and endonuclease activity of MutLa. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts

Biophysical investigation of M-DNA

M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA. M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)n•d(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed. These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation

Genome-Wide DNA Methylation in Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a heritable renal disease that causes the enlargement of kidneys due to the bilateral development of fluid-filled cysts. This results in end-stage kidney disease in adults and a reduced life expectancy. While it is known that a mutation within a PKD-causing gene is required for the development of ADPKD, the underlying mechanisms causing cystogenesis and allowing the progression of disease are not well understood. As a result of this poor understanding there are few treatment options for patients with ADPKD, therefore a large proportion of patients will progress to end-stage renal disease for which they will need dialysis or renal transplantation. Epigenetic modifications including DNA methylation are known to be altered in neoplasia, for which there are now several FDA-approved therapeutic drugs. As there are many similarities between ADPKD and neoplasia, we postulate that like tumour tissue, ADPKD tissue contains differentially methylated regions that may be exploited for future therapeutic discovery. To investigate this, we have performed reduced representation bisulfite sequencing (RRBS) on four ADPKD kidney tissue samples, and three non-ADPKD kidney tissue samples. In this analysis we confirm that there are 13 regions in the genome with differential methylation, and there is a global trend of hypomethylation in ADPKD. Furthermore, the 3’ end of the PKD associated gene PKD1 shows increased methylation associated with increased mRNA expression. To investigate whether DNA methylation changes are universally changed in ADPKD cysts, we performed RRBS on a further eight ADPKD samples, each from unique cysts from a single ADPKD patient. In this analysis there were differential methylation patterns in each cyst, however these changes were not consistent between cysts. These data show trends in global methylation in ADPKD not previously reported, and methylation changes within the genes NAGLU and GET4 concomitant with gene expression, which require further investigation to identify their role in ADPKD

Analyse fonctionnelle de la polycystine-1 et de son domaine intracellulaire dans le développement de la polykystose rénale autosomique dominante

La polykystose rénale autosomique dominante (PKRAD) est la maladie génétique rénale la plus commune touchant 1/500 personnes. Elle se caractérise principalement par la formation de kystes rénaux dans tous les segments du néphron, entraînant l’insuffisance rénale, et par des manifestations extrarénales kystiques (foie, pancréas, rate) et non-kystiques (anomalies cardiaques, vasculaires et cérébrales). Deux gènes, PKD1 et PKD2, sont responsables de 85 et 15% des cas respectivement. Ces gènes encodent les polycystine-1 (PC-1) et -2 (PC-2) qui forment un complexe à la membrane plasmique et ciliaire des cellules épithéliales rénales. PC-1 est une protéine transmembranaire de 4302 acides aminés possédant un court domaine intracellulaire incluant un motif coiled-coil impliqué dans l’interaction entre PC-1 et PC-2 in-vitro. L’importance du coiled-coil est démontrée par des mutations affectant spécifiquement ce motif chez des patients PKRAD. Le mécanisme pathogénétique responsable de la PKRAD est indéterminé. Chez la souris, la PKRAD se développe suite à l’ablation (Pkd1-/-) ou lors de la surexpression (SBPkd1TAG) de Pkd1, ce qui suggère un effet de dosage. Des anomalies ciliaires sont aussi souvent associées à PKRAD. Mon objectif était de déterminer in-vivo le mécanisme pathogénétique de la polycystine-1 dans le développement des symptômes PKRAD rénaux et extrarénaux et plus spécifiquement, le rôle du motif coiled-coil dans le mécanisme de kystogenèse. Pour ce faire, nous avons généré deux constructions, Pkd1 sauvage (Pkd1TAG) et Pkd1 tronquée de son motif coiled-coil (Pkd1ΔCoiled-coil), par recombinaison homologue à partir du BAC-Pkd1 sauvage comprenant la séquence murine entière de Pkd1. Trois lignées de souris Pkd1TAG générées par microinjection démontrent un niveau d’expression de Pkd1 qui corrèle avec le nombre de copie du transgène (2, 5 et 15 copies). Les souris Pkd1TAG reproduisent la PKRAD en développant des kystes rénaux dans toutes les parties du néphron et des cils primaires plus longs que les contrôles non transgéniques. Les analyses physiologiques supportent que les souris Pkd1TAG développent une insuffisance rénale et démontrent une augmentation du volume urinaire de même qu’une diminution de l’osmolalité, de la créatinine et des protéines urinaires. De plus, les souris Pkd1TAG développent des kystes hépatiques, des anomalies cardiaques associées à des dépôts de calcium et des anévrismes cérébraux. La sévérité du phénotype augmente avec l’expression de Pkd1 appuyant l’hypothèse d’un mécanisme de dosage. Nous avons aussi déterminé que l’expression du transgène Pkd1TAG complémente le phénotype létal-embryonnaire des souris Pkd1-/-. D’autre part, nous avons générés 4 lignées de souris Pkd1ΔCoiled-coil (2 et 15 copies du transgène) dont le nombre de copies corrèle avec le niveau d’expression du transgène. Ces souris Pkd1ΔCoiled-coil, contrairement aux Pkd1TAG de même âge, ne développent pas de kystes et possèdent des cils primaires de longueur normale. Afin d’évaluer le rôle du motif coiled-coil en absence de polycystine-1 endogène, nous avons croisé les souris Pkd1ΔCoiled-coil avec les souris Pkd1-/-. Contrairement aux souris Pkd1-/- qui meurent in-utéro, les souris Pkd1ΔCoiled-coil; Pkd1-/- survivent ~10 à 14 jours après la naissance. Elles démontrent des kystes rénaux et pancréatiques sévères, un retard de croissance et des anomalies pulmonaires. Tous les segments du néphron sont affectés. Mon projet démontre que la surexpression de Pkd1 est un mécanisme pathogénique de la PKRAD tant au niveau rénal qu’extrarénal. De plus, il démontre que le motif coiled-coil est un élément déterminant dans la kystogenèse/PKRAD in-vivo.Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder affecting 1:500 people worldwide, independently from sex and origin. ADPKD is characterized by formation of large bilateral kidney cysts affecting all segments of the nephron and increasing progressively in size and number leading to end stage renal failure by mid-fifty. Moreover, this systemic disease includes several extrarenal symptoms such as intracranial aneurysms, valvular defects and cysts formation in the liver and the pancreas. PKD1 and PKD2 genes mutations are involved in 85 and 15 % of the clinical cases. PKD genes encode polycystin-1 (PC-1) and -2 (PC-2), which both form a complex at the cell and ciliary membrane of renal epithelial cells. PC-1 is a large transmembrane protein with a small intracellular tail including a coiled-coil motif implicated in PC-1/PC-2 interaction in-vitro. Interestingly, specific mutations affecting the coiled-coil motif cause ADPKD in humans. The pathogenetic mechanism of ADPKD is unknown. In mice, both ablation (Pkd1-/-) or overexpression (SBPkd1TAG) of Pkd1 cause ADPKD, suggesting a dosage model. Ciliary anomalies are also linked to polycystic kidney disease. Herein, we evaluated in-vivo the role of Pkd1 in the development of renal and extrarenal manifestations of ADPKD and more specifically, the role of the coiled-coil motif in cystogenesis. We generated two constructions, wildtype Pkd1 (Pkd1TAG) and coiled-coil deleted Pkd1 (Pkd1ΔCoiled-coil), by homologous recombination from the wildtype Pkd1-BAC comprising the whole Pkd1 murine sequence. Three Pkd1TAG mice lines have been generated by microinjection and show expression patterns correlating with the copy number of the transgene (2, 5 and 15 copy). All Pkd1TAG mice develop renal cysts affecting all nephron segments as in ADPKD and longer primary cilia compared to wildtype mice. Physiologic analysis supports renal failure by increased urinary output and decreased of urinary proteins, osmolality and creatinin levels. Pkd1TAG mice also show cysts in the liver, cardiac and valvular anomalies associated with calcium deposition and cerebral aneurysms. The severity of the phenotype increased with Pkd1 expression suggesting a dosage model. Importantly, the Pkd1TAG transgene rescue embryonic lethality of Pkd1-/- mice. Furthermore, we generated 4 lines of Pkd1ΔCoiled-coil mice of 2 and 15 copies of the transgene correlating also to the level of expression. Compared to age-matched Pkd1TAG, Pkd1ΔCoiled-coil mice develop no cysts and show normal cilia length. To gain more insights on the role of coiled-coil motif in absence of endogenous Pc-1, we mated Pkd1ΔCoiled-coil with Pkd1-/- mice. Compared to the lethal embryonic Pkd1-/- mice, Pkd1ΔCoiled-coil; Pkd1-/- live ~ 10 to 14 days. They show severe renal and pancreatic cysts as well as growth retardation and pulmonary defects. My study demonstrates that Pkd1 overexpression is a pathogenic mechanism to induce ADPKD renal and extrarenal phenotype. Moreover, this work shows that the coiled-coil motif of polycystin-1 is a critical determinant in ADPKD cystogenesis

Elucidation of non-B DNA-induced mutagenesis mechanisms : DNA repair proteins are required for the processing of H-DNA and Z-DNA in eukaryotes

The vast majority of all cancers result from some form of genetic instability, thus it is important to study the mechanisms involved. The integrity of DNA can be influenced by secondary structure, and DNA can adopt alternative structures that do not conform to the Watson-Crick B-DNA helix (i.e. non-B DNA). To date, >12 different types of non-B DNA structures have been described including H-DNA and Z-DNA, and these structure-forming sequences are abundant in the human genome, occurring 1/3,000 and 1/50,000 base-pairs for Z-DNA and H-DNA, respectively. Non-B DNA can alter DNA metabolism and contribute to the development of many human diseases. Specific to this project, translocations occurring in the c-MYC and BCL-2 genes, which are shown to contain non-B DNA-forming sequences at translocation breakpoint “hotspots”, are characteristic of certain leukemias and lymphomas. However, the mechanism(s) involved in this process remains undefined. Previously, we found these structures to be mutagenic in bacteria, human cells, and mice, largely by stimulating the formation of DNA double strand breaks (DSBs). We speculated that the helical distortions produced by non-B DNA may be recognized as “damage” by the cell, eliciting an error-prone repair response, resulting in genomic instability. Using genetic-based mutation-reporter assays, we have shown for the first time that these structures are mutagenic in yeast. Furthermore, we have identified DNA repair proteins from both the nucleotide excision repair (NER) and mismatch repair (MMR) pathways to be involved in H-DNA and Z-DNA-induced mutagenesis via distinct mechanisms in both yeast and human cells. We further characterized the functions of these proteins using biochemical and molecular biology assays and found that they are enriched at sites of H-DNA and/or Z-DNA, and have cleavage activity at or near the structure. Taken together, these results suggest that non-B structures are processed in an error-prone fashion via various novel structure-specific repair pathways in which repair proteins from multiple pathways cooperate. The results obtained have enhanced our knowledge of DNA structure-induced genetic instability in disease etiology, and will guide future studies in the development of novel strategies to treat and/or prevent genetic diseases.Pharmac