Loss of imprinting at the CTCF binding sites in the DLK1-DIO3 domain reactivates microRNA expression in acute promyelocytic leukaemia. Homo sapiens

We report the DNA-methylation profiling of 10 regions selected from the DLK1-DIO3 domain on chromosome 14q32 in BM/PB samples from patients with acute promyelocytic leukaemia (APL), other subclasses of acute myeloid leukaemia and healthy donors, using high-throughput amplicon bisulfite sequencing with Roche 454 technology. We identify monoallelic-hypermethylation in APL only at the differentially methylated region (DMR) located upstream from the MEG3 gene (MEG3-DMR), whereas no changes in the DNA methylation profile were detected at the imprinting control region of the domain (IG-DMR) among the samples analysed. We show that the expression profile of 6 miRNAs clustered downstream from the MEG3-DMR correlates with the methylation profile at both DMRs. We demonstrate that miRNAs expression negatively correlates with DNA-methylation at the IG-DMR and MEG3 gene-body, whereas the correlation was positive for the CpGs located in the promoter of MEG3, including the binding sites for the insulator CTCF. We propose a loss of imprinting at the CTCF binding sites in patients with APL. These results are consistent with the previously reported DLK1-DIO3 miRNAs overexpression in APL, indicating a possible involvement of these ncRNAs in the pathogenesis of the disease. Overall design: Investigation of the epigenetic regulation of the miRNAs clustered in 14q32 by next-generation sequencing

本研究报道了针对急性早幼粒细胞白血病（acute promyelocytic leukaemia, APL）、其他亚型急性髓系白血病患者及健康供者的骨髓（BM）/外周血（PB）样本，采用罗氏454技术开展高通量扩增子亚硫酸氢盐测序，对14号染色体14q32区域DLK1-DIO3结构域中筛选出的10个区域进行DNA甲基化谱分析。我们仅在位于MEG3基因上游的差异甲基化区域（differentially methylated region, DMR）处，于APL样本中检测到单等位基因超甲基化现象；而在所分析的所有样本中，该结构域的印记控制区域（imprinting control region, IG-DMR）均未出现DNA甲基化谱的改变。我们发现，位于MEG3-DMR下游的6个成簇微小RNA（miRNA）的表达谱，与两个差异甲基化区域的甲基化谱均存在相关性。研究表明，微小RNA的表达与IG-DMR及MEG3基因体区域的DNA甲基化水平呈负相关，而与MEG3启动子区域（包含绝缘子蛋白CTCF的结合位点）内的CpG位点呈正相关。我们提出，APL患者存在CTCF结合位点处的印记丢失现象。本研究结果与此前报道的APL中DLK1-DIO3微小RNA过表达结论一致，提示这些非编码RNA（non-coding RNAs, ncRNAs）可能参与了该病的发病机制。整体实验设计：通过下一代测序技术探究14q32区域成簇微小RNA的表观遗传调控机制。