Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.
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.
Expansion of tandem repeat sequences is responsible for more than 20 human diseases. Several cis elements and trans factors involved in repeat instability (expansion and contraction) have been identified. However no comprehensive model explaining large intergenerational or somatic changes of the length of the repeating sequences exists. Several lines of evidence, accumulated from different model studies, indicate that transcription through repeat sequences is an important factor promoting their instability. The persistent interaction between transcription template DNA and nascent RNA (RNA•DNA hybrids, R loops) was shown to stimulate genomic instability. Recently, we demonstrated that cotranscriptional RNA•DNA hybrids are preferentially formed at GC-rich trinucleotide and tetranucleotide repeat sequences in vitro as well as in human genomic DNA. Additionally, we showed that cotranscriptional formation of RNA•DNA hybrids at CTG•CAG and GAA•TTC repeats stimulate instability of these sequences in both E. coli and human cells. Our results suggest that persistent RNA•DNA hybrids may also be responsible for other downstream effects of expanded trinucleotide repeats, including gene silencing. Considering the extent of transcription through the human genome as well as the abundance of GC-rich and/or non-canonical DNA structure forming tandem repeats, RNA•DNA hybrids may represent a common mutagenic conformation. Hence, R loops are potentially attractive therapeutic target in diseases associated with genomic instability.
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