Epigenetic upregulation of lncRNAs at 13q14.3 in leukemia is linked to the In Cis downregulation of a gene cluster that targets NF-kB.
Epigenetic upregulation of lncRNAs at 13q14.3 in leukemia is linked to the In Cis downregulation of a gene cluster that targets NF-kB.
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DOI：
10.1371/journal.pgen.1003373
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发表时间：
2013-04
期刊：
影响因子：
4.5
通讯作者：
Mertens D
中科院分区：
文献类型：
--
作者：
Garding A;Bhattacharya N;Claus R;Ruppel M;Tschuch C;Filarsky K;Idler I;Zucknick M;Caudron-Herger M;Oakes C;Fleig V;Keklikoglou I;Allegra D;Serra L;Thakurela S;Tiwari V;Weichenhan D;Benner A;Radlwimmer B;Zentgraf H;Wiemann S;Rippe K;Plass C;Döhner H;Lichter P;Stilgenbauer S;Mertens D
非编码RNA比以前认为的要常见得多。然而，对于绝大多数非编码RNA，细胞功能仍然是谜。两个长的非编码RNA（lncRNA）基因DLEU 1和DLEU 2映射到染色体带13q14.3处的关键区域，其在实体瘤和造血系统恶性肿瘤如慢性淋巴细胞白血病（CLL）中反复缺失。虽然在13q14.3的蛋白编码候选基因中没有发现点突变，但它们在恶性细胞中失调，表明表观遗传肿瘤抑制机制。因此，我们在CLL细胞中表征了13q14.3的表观遗传组成，并使用5种不同的半定量和定量方法（aPRIMES，BioCOBRA，MCIP，MassARRAY和亚硫酸氢盐测序）通过染色质免疫沉淀（ChIP）发现了与DLEU 1和DLEU 2转录起始位点的激活转录和显著DNA去甲基化相关的组蛋白修饰。这些表观遗传畸变与邻近候选肿瘤抑制基因的转录失调相关，表明该基因簇的顺式共调节。我们发现，13q14.3基因除了其先前已知的功能调节NF-κ B的活性，我们可以显示后过表达，siRNA介导的敲低，显性负突变基因，通过使用Western印迹与以前未描述的抗体，通过定制的ELISA以及报告基因测定。此外，我们对810种人类miRNAs进行了无偏筛选，并将13q14.3处的miR-15/16家族基因鉴定为NF-κ B活性的最强诱导剂。总之，13q14.3的肿瘤抑制机制是由两个lncRNA基因控制的一组基因，这两个基因受DNA甲基化和组蛋白修饰的调控，其成员都调控NF-κ B。因此，13q14.3中的肿瘤抑制机制强调了表观遗传畸变和lncRNA基因在人类肿瘤发生中的作用，并且是功能相关基因簇共定位的一个例子。最近的结果表明，不编码蛋白质的基因组区域被读取并转录成RNA。虽然大多数产生的非编码RNA分子的功能仍不清楚，但其中一些根据其长度（通常为200- 2，000个核苷酸）被称为长非编码RNA（lncRNA）基因，其在调节靶基因的活性中起作用。在大多数情况下，这种失调涉及与DNA相关的所谓“表观遗传”标记的变化，这些标记遗传给细胞后代而不改变DNA序列。在这里，我们描述了一个例子，其中两个lncRNA基因（DLEU 1和DLEU 2）与一组相邻的蛋白质编码的肿瘤抑制基因在几乎所有的慢性淋巴细胞白血病患者的表观遗传失调。这种共同的调节表明受影响的基因参与了相同的细胞途径。根据这一观点，13q14.3基因调节NF-κ B信号通路，诱导或抑制其活性。先前已经显示NF-kB的活化促进白血病细胞的存活，强调了13q14.3肿瘤抑制基因座对于该疾病的病理机制的重要性。
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster. Recent results suggest that genome regions not coding for proteins are read and transcribed into RNA. While the function for the majority of the resulting non-coding RNA molecules remains unclear, some of them are termed according to their length (typically 200–2,000 nucleotides) as long non-coding RNA (lncRNA) genes that play a role in regulating the activity of target genes. In most instances, this deregulation involves changes of so-called “epigenetic” marks associated with the DNA that are inherited to the cellular progeny without changes in the DNA sequence. Here we describe an example where two lncRNA genes (DLEU1 and DLEU2) are epigenetically deregulated together with a cluster of neighboring protein-coding tumor suppressor genes in almost all patients suffering from chronic lymphocytic leukemia. Such a common regulation suggests that the affected genes are involved in the same cellular pathway. In line with this notion, the 13q14.3 genes modulate the NF-kB signalling pathway, either inducing or repressing its activity. An activation of NF-kB has previously been shown to promote survival of the leukemic cells, underlining the importance of the 13q14.3 tumor suppressor locus for the pathomechanism of the disease.
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