Abstract
Friedreich ataxia (FRDA) is a multisystem genetic disorder caused by GAA repeat expansion mutations within the FXN gene, resulting in heterochromatin formation and deficiency of frataxin protein. Elevated levels of the FXN antisense transcript (FAST-1) have previously been detected in FRDA. To investigate the effects of FAST-1 on the FXN gene expression, we first stably overexpressed FAST-1 in non-FRDA cell lines and then we knocked down FAST-1 in FRDA fibroblast cells. We observed decreased FXN expression in each FAST-1 overexpressing cell type compared to control cells. We also found that FAST-1 overexpression is associated with both CCCTC-Binding Factor (CTCF) depletion and heterochromatin formation at the 5′UTR of the FXN gene. We further showed that knocking down FAST-1 in FRDA fibroblast cells significantly increased FXN expression. Our results indicate that FAST-1 can act in trans in a similar manner to the cis-acting FAST-1 overexpression that has previously been identified in FRDA fibroblasts. The effects of stably transfected FAST-1 expression on CTCF occupancy and heterochromatin formation at the FXN locus suggest a direct role for FAST-1 in the FRDA molecular disease mechanism. Our findings also support the hypothesis that inhibition of FAST-1 may be a potential approach for FRDA therapy.
Highlights
(FXN-TSS) and silences the promotor activity[6]
No significant difference was detected in Friedreich ataxia (FRDA) fibroblast cells treated with scrambled shRNA (Fig. 9). It is more than 20 years since the identification of the genetic mutation underlying FRDA, the exact mechanisms by which GAA repeat expansions lead to FXN gene silencing are still not fully understood
It has been suggested that abnormal triplex conformation, such as sticky DNA adopted by GAA repeat expansion and/or RNADNA hybrids, might impede the transcription of the FXN gene[24,25]
Summary
Severe depletion of the chromatin insulator protein CTCF has been identified at the 5′ untranslated region (UTR) of the FXN gene in FRDA patients. An antisense transcript named FAST-1 (FXN Antisense Transcript – 1), whose sequence overlaps with the CTCF binding site, has been discovered. FAST-1 expression is significantly increased in FRDA and is associated with the severe CTCF depletion and heterochromatin formation in the 5′UTR of the FXN gene[14,19]. Literature supporting the notion that antisense transcripts are involved in heterochromatin formation and the regulation of their partner mRNA expression inspired us to further investigate the characteristics of FAST-1. Mapping the 3′ and 5′ ends of the FAST-1 transcript onto the genome showed that FAST-1 transcription overlaps with the FXN-TSS and CTCF binding site in the 5′UTR of the FXN gene. We show that knocking down FAST-1 expression results in increased FXN expression in FRDA fibroblast cells, thereby revealing FAST-1 to be a potential FRDA therapeutic target
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