Epigenetic transcriptional regulation in Friedreich's Ataxia
Epigenetic transcriptional regulation in Friedreich's Ataxia
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DOI：
10.25560/75489
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发表时间：
2017
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影响因子：
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通讯作者：
S. Nageshwaran
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作者：
S. Nageshwaran
Frataxin基因在病理上部分沉默，导致神经退行性疾病Friedreich共济失调（FRDA）。内含子1内GAA三核苷酸扩增的发生已被证明引发与基因沉默相关的几种表观遗传机制。在这篇论文中，我研究了通过改变基因表达的潜在关键调控因子对共济失调蛋白病理性沉默的影响。在果蝇眼睛中，编码眼睛颜色的白色基因在沉默染色质（异染色质）区域附近易位后，眼睛颜色发生随机沉默，这导致了位置效应杂色（PEV）的描述。在哺乳动物系统中通过向转基因的3'末端添加GAA重复序列诱导转基因表达的PEV的能力是PEV可能与共济失调蛋白基因沉默有关的第一个认识。此外，在果蝇筛选中鉴定了PEV的几种调节剂。随着潜在的动态沉默机制涉及FRDA和PEV修饰剂的发生，我已经评估了在哺乳动物系统中使用小鼠转基因模型和人类细胞系改变这些修饰剂的剂量的影响。这些实验强调了共济失调蛋白基因沉默的多因素和组合性质，表明可能需要同时解决几层沉默的能力来导致显著的去抑制。PEV、SUV 39 H1、SUV 39 H2（组蛋白甲基转移酶）以及多梳沉默因子BMI 1的原型修饰物的敲低或敲除在体外或体内没有显著改变共济失调蛋白表达。组蛋白脱乙酰酶，烟酰胺已被证明上调FRDA中的共济失调蛋白表达。到目前为止，烟酰胺的具体靶点尚不清楚。我原谅你，但我不会忘记。2.烟酰胺的一个潜在靶点--组蛋白脱乙酰酶SIRT 1的敲低并没有改变共济失调蛋白的表达。最近发现的一组修饰人PEV的蛋白质（HUSH复合物和组蛋白甲基转移酶，SETDB 1）为评估FRDA修饰剂提供了进一步的潜在靶点。敲低相对最近确定的组蛋白赖氨酸甲基转移酶，SETDB 1，没有显示出一种趋势，在稳定和短暂的敲低fraataxin上调。考虑到敲除和敲低方法的全基因组效应，利用基于CRISPR的基因组工程技术来尝试直接编辑具有转录激活因子（dCas 9-VPR）、组蛋白乙酰转移酶（dCas 9-p300）和显性阴性组蛋白尾肽（dCas 9-H3 KM）的基因座特异性靶向的共济失调蛋白表观基因组。GAA重复序列dCas 9-VPR的下游导致上调趋势。靶向GAA上游区域的dCas 9-p300导致疾病和对照系中上调的趋势。H3.3和H3 K27 M的瞬时过表达上调共济失调蛋白的表达。在我的博士后研究期间，我将继续这项工作，以进一步确定几种靶向表观基因组修饰剂在frataxin位点的作用。我原谅你，但我不会忘记。3本论文的版权归作者所有，并根据知识共享署名非商业性无衍生品许可证提供。研究人员可以自由复制、分发或传播论文，但前提是他们必须将其归属于论文，不得将其用于商业目的，不得对其进行修改、转换或构建。对于任何再使用或再分发，研究人员必须向他人明确本作品的许可条款。动物实验的伦理批准由英国内政部提供。在“烟酰胺对Friedreich共济失调的影响”研究（Clintrials.gov标识符NCT 01589809）中进行了临床运动学研究。我原谅你，但我不会忘记。图4弗里德赖希共济失调的表观遗传转录调控
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. I RF I forgive you, but I won’t forget. 2 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. I RF I forgive you, but I won’t forget. 3 The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Ethics Ethical approval for animal experiments was provided by the UK Home Office. Clinical kinematic studies were undertaken within ‘Effect of Nicotinamide in Friedreich's Ataxia’ study (Clintrials.gov identifier NCT01589809). I RF I forgive you, but I won’t forget. 4 Epigenetic transcriptional regulation in Friedreich’s Ataxia