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Use of genomic dna-reporter tools to dissect pathological mechanisms caused by GAA expansions in Friedreich’s Ataxia
Publication . Silva, Ana Maria Ferreira da, 1983-; Wade-Martins, Richard; Outeiro, Tiago Fleming, 1976-
In Friedreich’s ataxia (FRDA), abnormal GAA repeat expansions in intron 1 of the frataxin gene (FXN) cause epigenetic changes and reduce FXN mRNA levels in averaged cell samples though a poorly understood mechanism. Dissecting the silencing mechanism in FRDA in situ is crucial to improve our understanding of the disease. Here, I use novel FRDA human cell models suitable for screening compounds able to upregulate FXN expression and to analyse the link between FXN nuclear localisation and expression in single cells. FXN-Luc, FXN-GAA-Luc, FXN-MS2-Luc and FXN-GAA-MS2-Luc stable human clones carry a site-specific integration of a single copy of the whole FXN locus with either 6 (FXN-Luc and FXN-MS2-Luc) or ~310 (FXN-GAA-Luc and FXN-GAA-MS2-Luc) GAA repeats in intron 1. To fluorescently label the transgenic FXN mRNA, I inserted MS2 binding sites into exon 2 of FXN-MS2-Luc and FXN-GAA-MS2-Luc transgenes by homologous recombination. The ~310 GAA repeats recapitulate the characteristic FXN gene repression and epigenetic changes seen in FRDA. I report a single-cell analysis of FXN repression in which I identify the nuclear lamina (NL) as a novel and key player in FXN transcriptional impairment and silencing. Using a multidisciplinary approach, including analysis in both fixed and living single cells, I show that expanded GAA repeats increase FXN positioning at the NL, leading to decreased numbers of FXN mRNA molecules and slower transcription kinetics in the FXN-GAA-MS2 cell model. Restoring histone acetylation reverses NL positioning. I observe the same abnormal repositioning to the NL in carrier and FRDA patient cells and show that this tightly correlates with a marked decrease in the number of actively expressing FXN alleles. Furthermore, I show that those few active expanded FXN alleles located at the NL express at a significantly lower level than the alleles located in the interior of the nucleus. Finally, I demonstrate that expanded GAA repeats predominantly disrupt FXN transcription initiation. Collectively, these results suggest repressive epigenetic modifications at the expanded GAA-FXN locus may lead to NL relocation, where further repression may occur. The mechanisms described may extend to other genetic diseases mediated by repeat expansions within regions of non-coding DNA.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/61048/2009