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Title: Epigenetic transcriptional regulation in Friedreich's Ataxia
Author: Kathiresu Nageshwaran, Sathiji
ISNI:       0000 0004 8504 4060
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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The Frataxin gene is pathologically partially silenced causing the neurodegenerative disorder, Friedreich's Ataxia (FRDA). The occurrence of the GAA trinucleotide expansion within intron 1 has been shown to invoke several epigenetic mechanisms associated with gene silencing. In this thesis I have investigated the effect on the pathological silencing of frataxin through alteration of potential key regulators of gene expression. The occurrence of stochastic silencing of eye colour within the Drosophila eye following translocation of the white gene, which encodes eye colour, near a region of silent chromatin (heterochromatin) led to the description of position effect variegation (PEV). The ability to induce PEV of transgene expression in a mammalian system through the addition of GAA repeats to the 3' end of the transgene was the first insight that PEV may be implicated in frataxin gene silencing. Furthermore, several regulators of PEV were identified in Drosophila screens. With the potential dynamic silencing mechanisms implicated in FRDA and the occurrence of PEV modifiers, I have assessed the effect in mammalian systems of altering the dosage of these modifiers using mouse transgenic models and human cell lines. These experiments have underlined the multifactorial and combinatorial nature of frataxin gene silencing, suggesting that the ability to concomitantly address several layers of silencing may be required to result in significant de-repression. Knockdown or knockout of the archetypal modifiers of PEV, SUV39H1, SUV39H2 (histone methyltransferases) as well as the polycomb silencing factor BMI1 did not significantly alter frataxin expression in vitro or in vivo. The histone deacetylase, nicotinamide has been shown to upregulate frataxin expression in FRDA. As yet the specific target of nicotinamide is not known. IRF I forgive you, but I won't forget. Knockdown of one potential target of nicotinamide, the histone deacetylase SIRT1, did not alter frataxin expression. Recent discovery of a group of proteins that modify human PEV (the HUSH complex and histone methyltransferase, SETDB1) provided further potential targets for assessment as FRDA modifiers. Knockdown of the relatively recently identified histone lysine methyltransferase, SETDB1, did show a trend towards frataxin upregulation in both stable and transient knockdowns. Given the genome-wide effects of the knockout and knockdown methodologies, CRISPR based genome engineering technology was utilised to attempt to directly edit the frataxin epigenome with locus-specific targeting of transcriptional activators (dCas9-VPR), the histone acetyltransferase (dCas9-p300) and dominant-negative histone tail peptides (dCas9-H3KM). Downstream of the GAA repeat dCas9-VPR resulted in a trend towards upregulation. dCas9-p300 targeting the upstream region of the GAA resulted in a trend towards upregulation in both disease and control lines. Transient overexpression of H3.3 and H3K27M upregulated frataxin expression. I will carry this work forward to further establish the effect of several targeted epigenome modifiers at the frataxin locus during my postdoctoral fellowship.
Supervisor: Festenstein, Richard Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral