Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming

Abstract

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.

Somatic instability of the expanded GAA repeats in Friedreich’s ataxia

“…Amplification of GAA repeat expansions in the FXN gene was performed by PCR using primers GAA_F: and GAA_R: as previously described [ 26 , 28 , 29 ]. Amplifications were conducted in 40 or 50μL reactions containing 50-200ng of DNA template.…”

“…Similar to the results obtained from patient tissues, the mouse tissues showed an expansion bias, particularly in the cerebellum and DRG, as well as an age-dependent increase in mutation load [ 17 , 23 ]. Lastly, in vitro studies on cultured immortalized lymphoblast cell lines and induced pluripotent stem cells (iPSCs), obtained by reprogramming of FRDA fibroblasts, demonstrated somatic instability when serially passaged [ 24 – 26 ].…”

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

“…The vast majority of FRDA patients are homozygous for expanded GAA trinucleotide repeats in the first intron of the frataxin (FXN) gene. The expanded GAA repeats impede transcription of the FXN gene leading to severe downregulation of its mRNA and protein levels [5,6]. Frataxin (FXN) is a small mitochondrial protein involved in the regulation of iron homeostasis and the biosynthesis of iron-sulfur clusters (Fe-S).…”

Experimental DNA- or RNA-Directed Therapies for Trinucleotide Repeat Disease

“…Pharmacological Modulation during Somatic Cell Reprogramming. Another recently evaluated strategy was based on the hypothesis that the epigenetic modifications close to GAA repeats present in Friedreich's ataxia can be reversed by pharmacological modulation during somatic cell reprogramming [55]. In this study, researchers induced the generation of pluripotent stem cells by reprogramming Friedreich's ataxia fibroblasts in the presence of various small molecules that target DNA methylation as well as histone acetylation and methylation.…”

“…In this study, researchers induced the generation of pluripotent stem cells by reprogramming Friedreich's ataxia fibroblasts in the presence of various small molecules that target DNA methylation as well as histone acetylation and methylation. Treatment of these stem cells with two compounds (sodium butyrate and Parnate [tranylcypromine]), led to increased expression of the frataxin gene, which persisted for several passages [55]. However, they observed that prolonged culture of those epigenetically modified cells resulted in progressive expansion of triplet repeats and a consequent decrease in gene expression.…”