Dicer is associated with ribosomal DNA chromatin in mammalian cells.
Dicer is associated with ribosomal DNA chromatin in mammalian cells.
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DOI：
10.1371/journal.pone.0012175
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发表时间：
2010-08-13
期刊：
影响因子：
3.7
通讯作者：
Svoboda P
中科院分区：
文献类型：
--
作者：
Sinkkonen L;Hugenschmidt T;Filipowicz W;Svoboda P
RNA沉默是利用小RNA作为序列特异性向导来抑制基因表达的途径的常用术语。RNA沉默机制的组分涉及许多生物体中染色质功能的不同方面。然而，在哺乳动物细胞中RNA沉默与染色质的关联仍然不清楚。免疫染色的有丝分裂染色体与抗体可视化无论是内源性或异位表达的Dicer在哺乳动物细胞揭示协会的蛋白质与核糖体DNA（rDNA）重复。染色质免疫沉淀和亚硫酸氢盐测序实验表明，Dicer与间期染色体rDNA阵列中的活性和沉默基因的转录区域相关。缺乏Dicer的小鼠胚胎干（ES）细胞的代谢标记没有显示出rRNA生物合成的明显缺陷，尽管这些细胞中的前rRNA合成减少，可能是由于miRNA的丢失导致其生长较慢。我们详细分析了rDNA的染色质结构，但在Dicer−/− ES细胞中没有发现rDNA位点的表观遗传变化。相反，我们发现rDNA甲基化在原代组织中相当低，与转化细胞系中的rDNA甲基化模式形成对比。我们发现，Dicer，RNA沉默途径的关键组成部分，可以检测到与哺乳动物细胞中的rDNA染色质。Dicer的这种特定定位的作用并不明显，因为该酶与rDNA基因相关，而不管它们的转录活性如何。然而，Dicer定位于转录区域表明转录可能有助于Dicer沉积在rDNA染色质上。我们假设Dicer在维持rDNA阵列的完整性中起作用。
RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer−/− ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.
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