The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model

Copyright @ 2012 Elsevier. The article can be accessed from the link below.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions.This study is funded under European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS. This article is made available through the Brunel Open Access Publishing Fund

Pms2 suppresses large expansions of the (GAA·TTC)n sequence in neuronal tissues

Copyright @ 2012 Bourn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.IDB was supported by a postdoctoral fellowship from the National Ataxia Foundation. RMP was supported by Ataxia UK. SA was supported by The Wellcome Trust. This research was made possible by grants from the National Institutes of Health (NIH/NINDS) and the Muscular Dystrophy Association to S.I.B

Role of Mismatch Repair Enzymes in GAA•TTC Triplet-repeat Expansion in Friedreich Ataxia Induced Pluripotent Stem Cells

The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA•TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA•TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA•TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA•TTC triplet repeats expanded at a rate of about two GAA•TTC triplet repeats/replication. However, GAA•TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA•TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA•TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA•TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system

Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase

CAG/CTG repeat expansions cause over 13 neurological diseases that remain without a cure. Because longer tracts cause more severe phenotypes, contracting them may provide a therapeutic avenue. No currently known agent can specifically generate contractions. Using a GFP-based chromosomal reporter that monitors expansions and contractions in the same cell population, here we find that inducing double-strand breaks within the repeat tract causes instability in both directions. In contrast, the CRISPR-Cas9 D10A nickase induces mainly contractions independently of single-strand break repair. Nickase-induced contractions depend on the DNA damage response kinase ATM, whereas ATR inhibition increases both expansions and contractions in a MSH2- and XPA-dependent manner. We propose that DNA gaps lead to contractions and that the type of DNA damage present within the repeat tract dictates the levels and the direction of CAG repeat instability. Our study paves the way towards deliberate induction of CAG/CTG repeat contractions in vivo

MutLα heterodimers modify the molecular phenotype of Friedreich ataxia

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA), the most common autosomal recessive ataxia disorder, is caused by a dynamic GAA repeat expansion mutation within intron 1 of FXN gene, resulting in down-regulation of frataxin expression. Studies of cell and mouse models have revealed a role for the mismatch repair (MMR) MutS-heterodimer complexes and the PMS2 component of the MutLα complex in the dynamics of intergenerational and somatic GAA repeat expansions: MSH2, MSH3 and MSH6 promote GAA repeat expansions, while PMS2 inhibits GAA repeat expansions. Methodology/Principal Findings: To determine the potential role of the other component of the MutLα complex, MLH1, in GAA repeat instability in FRDA, we have analyzed intergenerational and somatic GAA repeat expansions from FXN transgenic mice that have been crossed with Mlh1 deficient mice. We find that loss of Mlh1 activity reduces both intergenerational and somatic GAA repeat expansions. However, we also find that loss of either Mlh1 or Pms2 reduces FXN transcription, suggesting different mechanisms of action for Mlh1 and Pms2 on GAA repeat expansion dynamics and regulation of FXN transcription. Conclusions/Significance: Both MutLα components, PMS2 and MLH1, have now been shown to modify the molecular phenotype of FRDA. We propose that upregulation of MLH1 or PMS2 could be potential FRDA therapeutic approaches to increase FXN transcription. © 2014 Ezzatizadeh et al.This article has been made available through the Brunel Open Access Publishing Fund

Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich Ataxia

Copyright © 2014 Anjomani Virmouni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This article has been made available through the Brunel Open Access Publishing Fund.Background - Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats). Methodology/Principal Findings - We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R. Conclusions/Significance - Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.European Union, Ataxia UK and FARA

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA) n Repeats Associated with Friedreich’s Ataxia

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA) n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach. Keywords: genome instability; repeat expansion; RNA polyadenylation; RNA processing; transcription-replication conflicts; Friedreich’s ataxia; DNA double-strand breaks; trans-modifiers of repeat expansions; genetic screen; whole-genome sequencin

Transcription as a Threat to Genome Integrity

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant