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

Quasilocal Conservation Laws: Why We Need Them

We argue that conservation laws based on the local matter-only stress-energy-momentum tensor (characterized by energy and momentum per unit volume) cannot adequately explain a wide variety of even very simple physical phenomena because they fail to properly account for gravitational effects. We construct a general quasi}local conservation law based on the Brown and York total (matter plus gravity) stress-energy-momentum tensor (characterized by energy and momentum per unit area), and argue that it does properly account for gravitational effects. As a simple example of the explanatory power of this quasilocal approach, consider that, when we accelerate toward a freely-floating massive object, the kinetic energy of that object increases (relative to our frame). But how, exactly, does the object acquire this increasing kinetic energy? Using the energy form of our quasilocal conservation law, we can see precisely the actual mechanism by which the kinetic energy increases: It is due to a bona fide gravitational energy flux that is exactly analogous to the electromagnetic Poynting flux, and involves the general relativistic effect of frame dragging caused by the object's motion relative to us.Comment: 20 pages, 1 figur

Replication in mammalian cells recapitulates the locus-specific differences in somatic instability of genomic GAA triplet-repeats

Friedreich ataxia is caused by an expanded (GAA·TTC)(n) sequence in intron 1 of the FXN gene. Small pool PCR analysis showed that pure (GAA·TTC)(44+) sequences at the FXN locus are unstable in somatic cells in vivo, displaying both expansions and contractions. On searching the entire human and mouse genomes we identified three other genomic loci with pure (GAA·TTC)(44+) sequences. Alleles at these loci showed mutation loads of <1% compared with 6.3–30% for FXN alleles of similar length, indicating that somatic instability in vivo is regulated by locus-specific factors. Since distance between the origin of replication and the (CTG·CAG)(n) sequence modulates repeat instability in mammalian cells, we tested if this could also recapitulate the locus-specific differences for genomic (GAA·TTC)(n) sequences. Repeat instability was evaluated following replication of a (GAA·TTC)(115) sequence in transfected COS1 cells under the control of the SV40 origin of replication located at one of five different distances from the repeat. Indeed, depending on the location of the SV40 origin relative to the (GAA·TTC)(n) sequence, we noted either no instability, predominant expansion or both expansion and contraction. These data suggest that mammalian DNA replication is a possible mechanism underlying locus-specific differences in instability of GAA triplet-repeat sequences

Deficiency of RecA-dependent RecFOR and RecBCD

pathways causes increased instability of the (GAA TTC) n sequence when GAA is the lagging strand templat

A Quantitative Proteomic Profile of the Nrf2-Mediated Antioxidant Response of Macrophages to Oxidized LDL Determined by Multiplexed Selected Reaction Monitoring

<div><p>The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.</p> </div