Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus.
Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus.
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DOI：
10.1371/journal.pone.0074956
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Pook MA
中科院分区：
文献类型：
--
作者：
Al-Mahdawi S;Sandi C;Mouro Pinto R;Pook MA
弗里德赖希共济失调（FRDA）是由FXN基因内含子1内的纯合GAA重复扩增突变引起的，其诱导表观遗传变化和FXN基因沉默。亚硫酸氢盐测序研究已经确定5-甲基胞嘧啶（5 mC）DNA甲基化是可能参与这一过程的表观遗传变化之一。然而，通过亚硫酸氢盐测序分析样品是一个耗时的过程。此外，最近有研究表明5-羟甲基胞嘧啶（5 hmC）也存在于哺乳动物DNA中，亚硫酸氢盐测序无法区分5 hmC和5 mC。我们开发了特异性MethylScreen限制性酶切和基于qPCR的方案，以更快速地定量FXN上游GAA区域中四个CpG位点的DNA甲基化。在FRDA小脑和心脏组织中的所有四个CpG位点都证实了DNA甲基化的增加。我们还使用能够区分5 hmC和5 mC的方法分析了FRDA小脑和心脏组织中的DNA甲基化状态。我们的分析表明，FRDA和未受影响的组织中的大多数DNA甲基化实际上包含5 hmC而不是5 mC。我们还发现在相同的FRDA小脑组织中，FXN 5' UTR区域的染色质绝缘子蛋白CTCF（CCCTC结合因子）的占用率降低。FXN上游GAA区域（主要是5 hmC而不是5 mC）的DNA甲基化增加以及FXN 5' UTR处CTCF占据率降低与FRDA疾病相关的人体组织相关。这些分子机制在FRDA发病机制中的作用目前尚待确定。
Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5hmC and 5mC. We have developed specific MethylScreen restriction enzyme digestion and qPCR-based protocols to more rapidly quantify DNA methylation at four CpG sites in the FXN upstream GAA region. Increased DNA methylation was confirmed at all four CpG sites in both FRDA cerebellum and heart tissues. We have also analysed the DNA methylation status in FRDA cerebellum and heart tissues using an approach that enables distinction between 5hmC and 5mC. Our analysis reveals that the majority of DNA methylation in both FRDA and unaffected tissues actually comprises 5hmC rather than 5mC. We have also identified decreased occupancy of the chromatin insulator protein CTCF (CCCTC-binding factor) at the FXN 5’ UTR region in the same FRDA cerebellum tissues. Increased DNA methylation at the FXN upstream GAA region, primarily 5hmC rather than 5mC, and decreased CTCF occupancy at the FXN 5’ UTR are associated with FRDA disease-relevant human tissues. The role of such molecular mechanisms in FRDA pathogenesis has now to be determined.
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