Friedreich?s ataxia (FRDA) is the most common autosomal recessive ataxia. It is caused by reduced levels of the mitochondrial protein frataxin (FXN) as a result of large expansions of GAA trinucleotide repeats located in the first intron of the FXN gene. Although the FXN coding sequence in FRDA patients is unaltered, transcription of the gene is suppressed as a consequence of the large GAA expansions. Downregulation of FXN expression is associated with a transition of chromatin surrounding the GAAs from an active to a repressed state, however the underlying molecular mechanism of FXN silencing remains largely unknown. At the present time there is no effective treatment for FRDA and transcriptional silencing of FXN is one of the primary targets for therapeutic intervention. Therefore, understanding the mechanism governing GAA-induced silencing is of critical importance for therapy development. Based on our preliminary studies we hypothesize that long, expanded GAA repeats induce replication stress leading to changes of the replication program at the endogenous FXN locus. A resulting collision between transcription and replication suppresses transcription elongation and stimulates expansions of GAA repeats. The transcription elongation defect is further amplified in trans by deficiency of specific transcriptional co-factors. To address this hypothesis, we will focus on three fundamental questions regarding the molecular pathogenesis of Friedreich?s ataxia: 1) How does interplay between transcription and replication at the endogenous FXN locus affect gene silencing and expansions of GAA repeats? 2) Which step of the transcription process is affected by expanded GAA repeats in FRDA cells? 3) What is the contribution of trans-factors to the transcriptional defect in FRDA? First, we will dissect mechanisms of molecular interplay between transcription and replication in the endogenous FXN locus using a set of CRISPR/Cas9 engineered FRDA cells. Furthermore, we will employ the precision nuclear run-on sequencing (PRO-seq) technique to determine the profile of nascent transcription at the FXN locus, while also defining the exact step of transcription affected by expanded GAA repeats. Additionally, we will define the influence of reactivation of FXN transcription on progressive expansions of the GAAs to evaluate potential risks associated with long-term reactivation of FXN expression. Lastly, our preliminary data from transcriptome profiling of a large cohort of FRDA and control cells demonstrated a profound downregulation of a set of transcription elongation co-factors in FRDA cells. We will elucidate the mechanism whereby these trans-factors affect transcriptional processivity of the FXN gene to identify new therapeutic targets for FRDA. To answer these questions, we will use a battery of FRDA cell models generated in our laboratory, including FRDA patient fibroblasts, induced pluripotent stem cells, neuronal and cardiac cells differentiated from the pluripotent cells. Collectively, successful completion of this project will uncover the molecular events occurring at the FXN locus in FRDA cells and define cis- elements as well as trans-factors critical for repeat-induced FXN silencing and GAAs expansion. Combined approaches of genome editing, pharmacological modulation, and high resolution transcriptome analyses performed in a spectrum of thoughtfully chosen FRDA models will fuel development of new therapeutic approaches

Public Health Relevance

Friedreich ataxia (FRDA), the most common inherited ataxia, is a progressive neurodegenerative disease caused by transcriptional silencing of the FXN gene. Proposed studies will identify molecular mechanisms leading to the silencing of this gene and expansion of the GAA repeat tract. This work will uncover new avenues for therapies of Friedreich?s ataxia and other repeat expansion diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
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Gubitz, Amelie
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University of Alabama Birmingham
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Long, Ashlee; Napierala, Jill S; Polak, Urszula et al. (2017) Somatic instability of the expanded GAA repeats in Friedreich's ataxia. PLoS One 12:e0189990
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