Department of Agricultural and Life Sciences, Faculty of Agriculture, Shinshu University, Nagano, 399-4598, Japan.
Department of Interdisciplinary Genome Sciences and Cell Metabolism, Institute for Biomedical Sciences, ICCER, Shinshu University, Nagano, 399-4598, Japan.
Epigenetics Chromatin. 2018 Sep 29;11(1):55. doi: 10.1186/s13072-018-0227-8.
Genomic imprinting leads to maternal expression of IGF2R in both mouse and opossum. In mouse, the antisense long noncoding (lnc) RNA Airn, which is paternally expressed from the differentially methylated region (DMR) in the second intron of Igf2r, is required to silence the paternal Igf2r. In opossum, however, intriguingly, the DMR was reported to be in a different downstream intron (intron 11) and there was no antisense lncRNA detected in previous analyses. Therefore, clarifying the imprinting mechanism of marsupial IGF2R is of great relevance for understanding the origin and evolution of genomic imprinting in the IGF2R locus. Thus, the antisense lncRNA associated with the marsupial DMR can be considered as the 'missing link'. In this study, we identified a novel antisense lncRNA, ALID, after detailed analysis of the IGF2R locus in an Australian marsupial, the tammar wallaby, Macropus eugenii, and compared it to that of the grey short-tailed opossum, Monodelphis domestica.
Tammar IGF2R showed maternal expression and had a maternally methylated CpG island (CGI) in intron 12 as well as a promoter CGI without differential methylation, but none in the second intron. Re-analysis of the IGF2R of opossum detected the CGI in intron 12, not intron 11, as previously reported, confirming that the DMR in intron 12 is conserved between these marsupials and so is the putative imprinting control region of marsupial IGF2R. ALID is paternally expressed from the middle of the DMR and is approximately 650 bp long with a single exon structure that is extremely short compared to Airn. Hence, the lncRNA transcriptional overlap of the IGF2R promoter, which is essential for the Igf2r silencing in the mouse, is likely absent in tammar. This suggests that fundamental differences in the lncRNA-based silencing mechanisms evolved in eutherian and marsupial IGF2R and may reflect the lack of differential methylation in the promoter CGI of marsupial IGF2R.
Our study thus provides the best candidate factor for establishing paternal silencing of marsupial IGF2R without transcriptional overlap, which is distinct from the Igf2r silencing mechanism of Airn, but which may be analogous to the mode of action for the flanking Slc22a2 and Slc22a3 gene silencing in the mouse placenta.
基因组印迹导致 IGF2R 在鼠和袋熊中均表现为母系表达。在鼠中，来自第二内含子中差异甲基化区域（DMR）的父系表达的长非编码（lnc）RNA Airn 是沉默父系 Igf2r 所必需的。然而，在袋熊中，有趣的是，据报道 DMR 位于不同的下游内含子（内含子 11）中，并且在之前的分析中没有检测到反义 lncRNA。因此，阐明有袋类 IGF2R 的印迹机制对于理解 IGF2R 基因座中基因组印迹的起源和进化具有重要意义。因此，与袋熊 DMR 相关的反义 lncRNA 可以被认为是“缺失的环节”。在这项研究中，我们在澳大利亚有袋动物袋鼩中详细分析了 IGF2R 基因座后，鉴定出一种新型反义 lncRNA ALID，并将其与灰色短尾负鼠 Monodelphis domestica 进行了比较。
袋鼩 IGF2R 表现为母系表达，在 12 号内含子中具有母系甲基化的 CpG 岛（CGI）以及没有差异甲基化的启动子 CGI，但在第二内含子中没有。对负鼠 IGF2R 的重新分析检测到 12 号内含子中的 CGI，而不是之前报道的 11 号内含子，这证实了这些有袋动物中内含子 12 中的 DMR 是保守的，因此也是有袋类 IGF2R 的假定印迹控制区。ALID 从 DMR 的中部父系表达，长度约为 650bp，与 Airn 相比，其单外显子结构极短。因此，对于在鼠中沉默 Igf2r 至关重要的 IGF2R 启动子上的 lncRNA 转录重叠很可能在袋鼩中不存在。这表明，在真兽类和有袋类 IGF2R 中，基于 lncRNA 的沉默机制存在根本差异，这可能反映了有袋类 IGF2R 启动子 CGI 中无差异甲基化的缺失。
因此，我们的研究为在没有转录重叠的情况下建立有袋类 IGF2R 的父系沉默提供了最佳候选因子，这与 Airn 沉默 Igf2r 的机制不同，但可能类似于鼠胎盘侧翼 Slc22a2 和 Slc22a3 基因沉默的作用模式。
