10 research outputs found
Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA·TTC)n sequence when GAA is the lagging strand template
The most common mutation in Friedreich ataxia is an expanded (GAA·TTC)n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA·TTC)n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA·TTC)n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA·TTC)n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA·TTC)n sequence
Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription
Background: Over 15 inherited diseases are caused by expansion of triplet-repeats. Friedreich ataxia (FRDA) patients are homozygous for an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality. Methodology/Principal Findings: We found a severe depletion of the chromatin insulator protein CTCF (CCCTC-binding factor) in the 59UTR of the FXN gene in FRDA, and coincident heterochromatin formation involving the +1 nucleosome via enrichment of H3K9me3 and recruitment of heterochromatin protein 1. We identified FAST-1 (FXN Antisense Transcript – 1), a novel antisense transcript that overlaps the CTCF binding site in the 59UTR, which was expressed at higher levels in FRDA. The reciprocal relationship of deficient FXN transcript and higher levels of FAST-1 seen in FRDA was reproduced in normal cells via knockdown of CTCF. Conclusions/Significance: CTCF depletion constitutes an epigenetic switch that results in increased antisense transcription, heterochromatin formation and transcriptional deficiency in FRDA. These findings provide a mechanistic basis for the transcriptional silencing of the FXN gene in FRDA, and broaden our understanding of disease pathogenesis in triplet-repea
Deficiency of RecA-dependent RecFOR and RecBCD
pathways causes increased instability of the (GAA TTC) n sequence when GAA is the lagging strand templat
Reversal of epigenetic promoter silencing in Friedreich ataxia by a class I histone deacetylase inhibitor
Increased DNA methylation at the <i>FXN</i> locus in the 1-month-old YG8sR mouse.
<p><b>(A)</b> Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. <b>(B)</b> Normalized melting curves in a methylation sensitive—high resolution melting (MS-HRM) assay to detect CpG methylation in lymphoblastoid cell lines from three FRDA (red curve) and three non-FRDA control subjects (blue curve) at the three CpG sites upstream of the GAA-TR mutation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) showing a clear separation of the curves indicating that the MS-HRM assay is able to detect a relative increase in methylation at the three CpG sites. <b>(C-H)</b> Normalized melting curves in a MS-HRM assay to detect CpG methylation in fibroblast cell lines and multiple tissues from 1-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues and fibroblasts. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. LBCLs = lymphoblastoid cell lines; CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.</p
<i>FXN</i> transcriptional deficiency in the YG8sR mouse extends both upstream and downstream of the expanded GAA-TR mutation.
<p><b>(A)</b> Relevant portions of the <i>FXN</i> gene are depicted schematically, with the GAA-TR mutation in intron 1, the <i>FXN</i> transcriptional start site (arrow) at position -59 relative to the initiation codon (“A” in ATG as +1), the three CpG sites in intron 1 used for DNA methylation analysis (relative to the first “G” in the GAA-TR sequence). Quantitative RT-PCR was performed to measure <i>FXN</i> transcript both upstream (Ex1; immediately downstream of the transcriptional start site) and downstream (Ex3-Ex4) of the GAA-TR mutation. Amplicons used for measuring the length of the GAA-TR sequence (GAA-PCR) and for methylation sensitive—high resolution melting (MS-HRM) are also depicted. Solid lines above the gene depict the shorter predicted <i>FXN</i> transcripts caused by defects in transcriptional elongation through the expanded GAA-TR mutation and by deficient transcriptional initiation due to <i>FXN</i> promoter silencing. Deficiency of transcript at both upstream and downstream locations would suggest a defect in transcriptional initiation, and deficiency of only Ex3-Ex4 would suggest a defect in transcriptional elongation. <b>(B)</b> PCR analysis to measure the length of the GAA-TR sequence in intron 1 of the <i>FXN</i> gene in various tissues from Y47R and YG8sR mice (for each tissue, the paired samples depict Y47R and YG8sR in the left and right lanes, respectively). The precise length of the GAA-9 product from Y47R fibroblasts and the GAA-133 product from YG8sR fibroblasts were determined by direct sequencing, which also showed that the repeat tract was pure (i.e., absence of non-GAA repeat sequence). <b>(C, D)</b> Quantitative RT-PCR showing deficiency of <i>FXN</i> transcript in 1-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. <b>(E)</b> Quantitative RT-PCR showing deficiency of <i>FXN</i> transcript in fibroblasts from YG8sR compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. <b>(F, G)</b> Quantitative RT-PCR showing deficiency of <i>FXN</i> transcript in 12-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle. Data shown in panels C through G represent three complete experiments using tissues isolated from two YG8sR and two Y47R individuals. Error bars represent +/-SEM. ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p
Increased DNA methylation at the <i>FXN</i> locus in the 12-month-old YG8sR mouse.
<p><b>(A)</b> Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. <b>(B-F)</b> Normalized melting curves in a MS-HRM assay to detect CpG methylation in multiple tissues from 12-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.</p
Metabolic labeling of nascent <i>FXN</i> transcript in primary fibroblasts showing deficiency of transcriptional initiation in the YG8sR mouse.
<p><b>(A, B)</b> Quantitative RT-PCR of metabolically labeled nascent transcript for the indicated incubation times (1, 2 and 4 hours) is shown for <i>FXN</i> mRNA upstream (“Ex1” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) and downstream (“Ex3-Ex4” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138437#pone.0138437.g001" target="_blank">Fig 1A</a>) of the GAA-TR sequence in intron 1. YG8sR cells showed 2.0–3.4 fold less nascent <i>FXN</i> transcript (exact fold changes are indicated) compared with Y47R cells at all the time points assayed. Graphs represent cumulative data from four independent metabolic labeling experiments. Error bars represent +/-SEM. * = <i>p</i><0.05; ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p