Quantitative investigations of FXN transcription and epigenetic modifications, including histone acetylation and methylation, in FRDA human and mouse tissues

Abstract

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder characterized by progressive gait and, limb ataxia, cardiomayopathy, diabetes mellitus, optic atrophy and hearing loss. It is most often caused by homozygous expanded (GAA)∙(TTC)n repeats within intron 1 of the FXN gene, resulting in severely reduced levels of frataxin protein. The exact mechanisms of how the expanded (GAA)∙(TTC)n repeats reduce FXN transcription are not fully understood. However, many studies have suggested that the expanded repeat may induce epigenetic modifications that cause the FXN transcription inhibition to occur. In the past few years, epigenetic modifications have been given considerable attention as an important mechanism that is contributing to the aetiology of FRDA. This thesis investigated histone acetylation and methylation in three different regions of the FXN gene: FXN promoter, upstream GAA and downstream GAA, using chromatin immunoprecipitation (ChIP) and quantitative reverse transcriptase PCR (qRT-PCR) of the human and transgenic mouse brain tissues. Furthermore, the frataxin mRNA levels were investigated in autopsied brain tissues from an FRDA patient and FXN transgenic mouse brain, heart and liver tissues. In addition, a preliminary study that investigated the effect of a histone deacetylase inhibitor (HDACi) on FXN transcription and histone modifications (acetylation) of transgenic mouse brain, heart and liver tissues was conducted. Results showed an overall significant decrease in the acetylation pattern of H3K9ac and H4K16ac residues in all three regions within FXN gene. Moreover, a significant increase in the di- and trimethylation pattern of the H3K9me2 and the H3K9me3 residues was identified in all three regions of the FXN gene. The results were comparable between the FRDA patient and transgenic mouse (YG8, YG22) brain tissues. The FXN mRNA levels showed a significant decrease in all transgenic mouse brain, heart and liver tissues, which is comparable with the FXN mRNA level of the FRDA patient brain and heart tissues.Results for the preliminary HDACi study showed an approximate 20-30 % increase in the FXN mRNA level in different transgenic mouse tissues after 3 days intake at 150mg/kg dose. In addition, there was an increase in the acetylation pattern of the H3K9ac and the H4K12ac in the HDACi treated transgenic mouse brain tissues. These studies will aid the understanding of FXN epigenetic modifications and their contribution to FRDA disease; this is an exciting challenge leading to a new effective FRDA therapeutic pathway.