Altered Nucleosome Positioning at the Transcription Start Site and Deficient Transcriptional Initiation in Friedreich Ataxia

Chutake, Yogesh; Costello, Whitney N.; Lam, Christina; Bidichandani, Sanjay I.

doi:10.1074/jbc.m114.566414

Cited by 36 publications

(70 citation statements)

References 32 publications

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“…1A). In agreement with previous findings 11, 12 we noted a severe deficiency of FXN transcript in FRDA cells at both the upstream and downstream locations, supporting the existence of a major defect in transcriptional initiation (Fig. 1B).…”

Section: Resultssupporting

confidence: 93%

“…12 Briefly, reverse transcription was performed either with a mixture of random hexamers and oligo dT primers (for the exon 3-4 amplicon) or with a strand-specific RT primer (for the exon 1 amplicon) using the QuantiTect ® reverse transcription kit (Qiagen). mRNA levels were quantified by real-time PCR relative to expression of the control TBP gene using the ΔΔCt method on the Eppendorf RealPlex-4 Mastercycler with SsoAdvanced ™ SYBR ® green supermix (BioRad).…”

Section: Methodsmentioning

confidence: 99%

“…12 Briefly, lymphoblastoid cell lines were incubated with 5-ethynyl uridine. Following 2h and 4h of incubation, RNA was extracted and used for biotinylation by Click reaction.…”

Section: Methodsmentioning

confidence: 99%

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Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

Chutake

Lam

Costello

et al. 2014

Annals of Neurology

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Objective Friedreich ataxia (FRDA) is caused by an expanded GAA triplet-repeat (GAA-TR) mutation in the FXN gene. Patients are typically homozygous for expanded alleles containing 100–1300 triplets, and phenotypic severity is significantly correlated with the length of the shorter of the two expanded alleles. Patients have a severe deficiency of FXN transcript, which is predominantly caused by epigenetic silencing of the FXN promoter. We sought to determine if the severity of FXN promoter silencing is related to the length of the expanded GAA-TR mutation in FRDA. Methods Patient-derived lymphoblastoid cell lines bearing a range of expanded alleles (200–1122 triplets) were evaluated for FXN transcript levels by quantitative RT-PCR. FXN promoter function was directly measured by quantitative analysis of transcriptional initiation via metabolic labeling of newly synthesized transcripts in living cells. Results FXN transcriptional deficiency was significantly correlated with the length of the shorter of the two expanded alleles, which was noted both upstream (R2=0.84; p=0.014) and downstream (R2=0.89; p=0.002) of the expanded GAA-TR mutation, suggesting that FXN promoter silencing in FRDA is related to repeat length. A bilinear regression model revealed that length-dependence was strongest when the shorter of the two expanded alleles contained <400 triplets. Direct measurement of FXN promoter activity in patients with expanded alleles containing <400 versus >400 triplets in the shorter of the two expanded alleles revealed a significantly greater deficiency in individuals with longer GAA-TR alleles (p<0.05). Interpretation FXN promoter silencing in FRDA is dependent on the length of the expanded GAA-TR mutation.

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Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

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Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

Chutake

Lam

Costello

et al. 2014

Annals of Neurology

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“…These results could answer questions regarding the chromatin structure associated with shorter-than-average expanded GAA triplet-repeats in FRDA, which is one of the definitive questions for the understanding of the pathogenesis of FRDA at the chromatin level [21]. However, several questions remain unclear.…”

Section: Accepted Manuscriptmentioning

confidence: 92%

“…The GAA triplet-repeats can also recruit heterochromatin-binding proteins. Further, the formation of heterochromatin on intron 1 of the FXN gene may trigger FXN gene silencing through an intrinsic blocking of transcription initiation or elongation [11,18,19,20,21,22,23,24,25,26,27]. Ruan et al found that the efficiency of nucleosome assembly on pure GAA 22 repeat duplex was only half that of the pUC control DNA.…”

Section: Accepted Manuscriptmentioning

confidence: 96%

GAA triplet-repeats cause nucleosome depletion in the human genome

et al. 2015

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Although there have been many investigations into how trinucleotide repeats affect nucleosome formation and local chromatin structure, the nucleosome positioning of GAA triplet-repeats in the human genome has remained elusive. In this work, the nucleosome occupancy around GAA triplet-repeats across the human genome was computed statistically. The results showed a nucleosome-depleted region in the vicinity of GAA triplet-repeats in activated and resting CD4(+) T cells. Furthermore, the A-tract was frequently adjacent to the upstream region of GAA triplet-repeats and could enhance the depletion surrounding GAA triplet-repeats. In vitro chromatin reconstitution assays with GAA-containing plasmids also demonstrated that the inserted GAA triplet-repeats destabilized the ability of recombinant plasmids to assemble nucleosomes. Our results suggested that GAA triplet-repeats have lower affinity to histones and can change local nucleosome positioning. These findings may be helpful for understanding the mechanism of Friedreich's ataxia, which is associated with GAA triplet-repeats at the chromatin level.

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Neuropathology and Cellular Pathogenesis of Spinocerebellar Ataxia Type 12

et al. 2015

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Objective SCA12 is a progressive autosomal-dominant disorder, caused by a CAG/CTG repeat expansion in PPP2R2B on chromosome 5q32, and characterized by tremor, gait ataxia, hyperreflexia, dysmetria, abnormal eye movements, anxiety, depression, and sometimes cognitive impairment. Neuroimaging has demonstrated cerebellar and cortical atrophy. We now present the neuropathology of the first autopsied SCA12 brain and utilize cell models to characterize potential mechanisms of SCA12 neurodegeneration. Methods A fixed SCA12 brain was examined using gross, microscopic, and immunohistochemical methods. The effect of the repeat expansion on PPP2R2B Bβ1 expression was examined in multiple cell types by transient transfection of constructs containing the PPP2R2B Bβ1 promoter region attached to a luciferase reporter. The neurotoxic effect of PPP2R2B overexpression was examined in transfected rat primary neurons. Results Neuropathological investigation revealed enlarged ventricles, marked cerebral cortical atrophy and Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions, consistent with Marinesco bodies, which did not stain for long polyglutamine tracts, alpha-synuclein, tau, or transactive response DNA-binding protein 43. Reporter assays demonstrated that the region of PPP2R2B containing the repeat functions as a promoter, and that promoter activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequence flanking the repeat. Overexpression of PPP2R2B in primary cortical neurons disrupted normal morphology. Conclusions SCA12 involves extensive, but selective, neurodegeneration distinct from Alzheimer’s disease, synucleinopathies, tauopathies, and glutamine expansion diseases. SCA12 neuropathology may arise from the neurotoxic effect of repeat-expansion–induced overexpression of PPP2R2B.

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Altered Nucleosome Positioning at the Transcription Start Site and Deficient Transcriptional Initiation in Friedreich Ataxia

Cited by 36 publications

References 32 publications

Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

GAA triplet-repeats cause nucleosome depletion in the human genome

Neuropathology and Cellular Pathogenesis of Spinocerebellar Ataxia Type 12

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