Friedreich's ataxia (GAA)n•(TTC)n repeats strongly stimulate mitotic crossovers in Saccharomyces cerevisae.
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ABSTRACT:
SUBMITTER: Tang W
PROVIDER: S-EPMC3020933 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich's ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.
| S-EPMC7515735 | biostudies-literature
Project description:The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAA?TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA?TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA?TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells and occupies FXN intron 1, and shRNA silencing of MSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.
| S-EPMC2987635 | biostudies-literature
Project description:Friedreich's ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the frataxin gene (FXN) due to expansions of GAA repeats in intron 1. FRDA manifests with multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7-9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient's lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.
| S-EPMC5736210 | biostudies-other
Project description:Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats responsible for the human disease Friedreich's ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeat's orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.
| S-EPMC2722067 | biostudies-literature
Project description:Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual's lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich's ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well-particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich's ataxia pathogenesis.
| S-EPMC7865128 | biostudies-literature
Project description:Friedreich's ataxia (FRDA) is a common hereditary degenerative neuro-muscular disorder caused by expansions of the (GAA)n repeat in the first intron of the frataxin gene. The expanded repeats from parents frequently undergo further significant length changes as they are passed on to progeny. Expanded repeats also show an age-dependent instability in somatic cells, albeit on a smaller scale than during intergenerational transmissions. Here we studied the effects of (GAA)n repeats of varying lengths and orientations on the episomal DNA replication in mammalian cells. We have recently shown that the very first round of the transfected DNA replication occurs in the lack of the mature chromatin, does not depend on the episomal replication origin and initiates at multiple single-stranded regions of plasmid DNA. We now found that expanded GAA repeats severely block this first replication round post plasmid transfection, while the subsequent replication cycles are only mildly affected. The fact that GAA repeats affect various replication modes in a different way might shed light on their differential expansions characteristic for FRDA.
| S-EPMC3351192 | biostudies-literature
Project description:Friedreich's ataxia (FRDA) is an autosomal recessive neurological disease caused by expansions of guanine-adenine-adenine (GAA) repeats in intron 1 of the frataxin (FXN) gene. The expansion results in significantly decreased frataxin expression. We report that human FRDA cells can be corrected by zinc finger nuclease-mediated excision of the expanded GAA repeats. Editing of a single expanded GAA allele created heterozygous, FRDA carrier-like cells and significantly increased frataxin expression. This correction persisted during reprogramming of zinc finger nuclease-edited fibroblasts to induced pluripotent stem cells and subsequent differentiation into neurons. The expression of FRDA biomarkers was normalized in corrected patient cells and disease-associated phenotypes, such as decreases in aconitase activity and intracellular ATP levels, were reversed in zinc finger nuclease corrected neuronal cells. Genetically and phenotypically corrected patient cells represent not only a preferred disease-relevant model system to study pathogenic mechanisms, but also a critical step towards development of cell replacement therapy.
| S-EPMC4817761 | biostudies-literature
Project description:Friedreich's ataxia (FRDA) is caused by biallelic expansion of GAA repeats leading to the transcriptional silencing of the frataxin (FXN) gene. The exact molecular mechanism of inhibition of FXN expression is unclear. Herein, we analyze the effects of hyperexpanded GAA repeats on transcription status and chromatin modifications proximal and distal to the GAA repeats. Using chromatin immunoprecipitation and quantitative PCR we detected significant changes in the chromatin landscape in FRDA cells relative to control cells downstream of the promoter, especially in the vicinity of the GAA tract. In this region, hyperexpanded GAAs induced a particular constellation of histone modifications typically associated with heterochromatin-like structures. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Furthermore, we observed similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation. We also demonstrated that histone modifications associated with hyperexpanded GAA repeats are independent of initiation and progression of transcription. Our data provide strong evidence that FXN deficiency in FRDA patients results from a block of transition from initiation to a productive elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.
| S-EPMC3201871 | biostudies-literature
Project description:Lymphoblast cells from a patient with Freidriech's Ataxia were incubated with pyrrole-imidazole polyamides targeted to the GAA triplet repeat in the intron 1. The polyamides were shown in cell culture to increase levels of endogenous frataxin mRNA. A normal sibling derived lymphoblast cell line was used as a control. Keywords: human lymphoblast cells
2006-06-14 | GSE5040 | GEO
Project description:Friedreich's ataxia is caused by large homozygous, intronic expansions of GAA repeats in the frataxin (FXN) gene, resulting in severe downregulation of its expression. Pathogenic repeats are located in intron one, hence patients express unaffected FXN protein, albeit in low quantities. Although FRDA symptoms typically afflict the nervous system, hypertrophic cardiomyopathy is the predominant cause of death. Our studies were conducted using cardiomyocytes differentiated from induced pluripotent stem cells derived from control individuals, FRDA patients, and isogenic cells corrected by zinc finger nucleases-mediated excision of pathogenic expanded GAA repeats. This correction of the FXN gene removed the primary trigger of the transcription defect, upregulated frataxin expression, reduced pathological lipid accumulation observed in patient cardiomyocytes, and reversed gene expression signatures of FRDA cardiomyocytes. Transcriptome analyses revealed hypertrophy-specific expression signatures unique to FRDA cardiomyocytes, and emphasized similarities between unaffected and ZFN-corrected FRDA cardiomyocytes. Thus, the iPSC-derived FRDA cardiomyocytes exhibit various molecular defects characteristic for cellular models of cardiomyopathy that can be corrected by genome editing of the expanded GAA repeats. These results underscore the utility of genome editing in generating isogenic cellular models of FRDA and the potential of this approach as a future therapy for this disease.
| S-EPMC6853280 | biostudies-literature