R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome.
R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome.
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DOI：
10.1371/journal.pgen.1004318
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发表时间：
2014-05
期刊：
影响因子：
4.5
通讯作者：
Gromak N
中科院分区：
文献类型：
--
作者：
Groh M;Lufino MM;Wade-Martins R;Gromak N
弗里德赖希共济失调（FRDA）和脆性X综合征（FXS）是40种与重复序列扩展（TRED）相关的疾病。虽然他们的分子病理学还没有得到很好的理解，抑制性染色质和不寻常的DNA结构的重复区域的形成被认为发挥了作用。我们的研究现在表明，在患者细胞中，与FRDA和FXS相关的内源性FXN和FMR 1基因的扩增重复序列上形成RNA/DNA杂合体（R环）。这些转录依赖性R环是稳定的，与抑制性H3 K9 me 2染色质标记共定位，并阻碍患者细胞中的RNA聚合酶II转录。我们研究了FXN基因上抑制性染色质标记和R环之间的相互作用。我们发现，抑制性H3 K9 me 2染色质标记的减少对R环水平没有影响。重要的是，通过用DNA拓扑异构酶抑制剂喜树碱处理增加R环水平导致抑制性染色质标记的上调，导致FXN转录沉默。这提供了R环和TRED病理之间的直接分子联系，表明R环作为促进FXN和FMR 1沉默的初始触发物。因此，R环代表了核苷酸扩增障碍的共同特征，并为治疗干预提供了新的靶点。弗里德赖希共济失调和脆性X综合征是与重复序列扩展相关的40种人类疾病之一。在这两种疾病中，重复扩增导致基因沉默，其分子机制尚不清楚，阻碍了治疗这些疾病的特定疗法的发展。有人提出，在重复区域上形成不寻常的DNA结构（RNA/DNA杂交体或R环）可能起作用，但它们的分子功能尚未在体内研究。我们表明，R环形成的FXN和FMR 1基因在FRDA和FXS患者的细胞中的扩展重复序列。这些R环是稳定的，与抑制性染色质标记相关，并阻碍患者细胞中的FXN转录。我们研究了抑制性染色质和R环之间的关系。抑制性染色质数量的减少对R环水平没有影响。相反，R环的增加导致FXN基因的转录沉默和抑制性染色质的形成，提供了R环和扩张疾病的病理之间的直接分子联系。这一发现对于理解扩张性疾病病理学的基本分子机制非常重要。R环触发转录沉默的能力使其成为未来治疗这些疾病的治疗方法的有吸引力的靶标。
Friedreich ataxia (FRDA) and Fragile X syndrome (FXS) are among 40 diseases associated with expansion of repeated sequences (TREDs). Although their molecular pathology is not well understood, formation of repressive chromatin and unusual DNA structures over repeat regions were proposed to play a role. Our study now shows that RNA/DNA hybrids (R-loops) form in patient cells on expanded repeats of endogenous FXN and FMR1 genes, associated with FRDA and FXS. These transcription-dependent R-loops are stable, co-localise with repressive H3K9me2 chromatin mark and impede RNA Polymerase II transcription in patient cells. We investigated the interplay between repressive chromatin marks and R-loops on the FXN gene. We show that decrease in repressive H3K9me2 chromatin mark has no effect on R-loop levels. Importantly, increasing R-loop levels by treatment with DNA topoisomerase inhibitor camptothecin leads to up-regulation of repressive chromatin marks, resulting in FXN transcriptional silencing. This provides a direct molecular link between R-loops and the pathology of TREDs, suggesting that R-loops act as an initial trigger to promote FXN and FMR1 silencing. Thus R-loops represent a common feature of nucleotide expansion disorders and provide a new target for therapeutic interventions. Friedreich ataxia and Fragile X syndrome are among 40 human diseases associated with expansion of repeated sequences. In both disorders repeat expansion leads to gene silencing, the molecular mechanism of which is not well understood, impeding the development of specific therapies to treat these disorders. It is proposed that formation of unusual DNA structures (RNA/DNA hybrids, or R-loops) over repeat regions may play a role, but their molecular function has not been investigated in vivo. We show that R-loops form on expanded repeats of FXN and FMR1 genes in cells from FRDA and FXS patients. These R-loops are stable, correlate with repressive chromatin marks and hinder FXN transcription in patient cells. We studied the relationship between repressive chromatin and R-loops. Decrease in the amount of repressive chromatin has no effect on R-loop levels. In contrast, increase in the R-loops leads to transcriptional silencing of FXN gene and formation of repressive chromatin, providing a direct molecular link between R-loops and pathology of expansion diseases. This discovery is important for understanding the basic molecular mechanism underlying the pathology of expansion diseases. The ability of R-loops to trigger transcriptional silencing makes them an attractive target for future therapeutic approaches to treat these diseases.
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