Department of Medical Genetics, Life Sciences Institute, Molecular Epigenetics Group, 5503-2350 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia, Canada.
Mol Cell Biol. 2011 Jul;31(14):2827-37. doi: 10.1128/MCB.01435-10. Epub 2011 May 16.
The distal region of mouse chromosome 7 contains two imprinted domains separated by a relatively gene-poor interval. We have previously described a transgenic mouse line called Tel7KI, which contains a green fluorescent protein (GFP) reporter inserted 2.6 kb upstream of the Ins2 gene at the proximal end of this interval. The GFP reporter from Tel7KI is imprinted and maternally expressed in postimplantation embryos. Here, we present evidence that the distal imprinting center, KvDMR1 (IC2), is responsible for the paternal silencing of Tel7KI. First, we show that Tel7KI is silenced when the noncoding RNA Kcnq1ot1 is biallelically expressed due to absence of maternal DNA methylation at IC2. Second, we use an embryonic stem (ES) cell differentiation assay to examine the effect of an IC2 deletion in cis to Tel7KI and show that it impairs the ability of the paternal transmission Tel7KI ES cells to silence GFP. These results suggested that Kcnq1ot1 silencing extends nearly 300 kb further than previously reported and led us to examine other transcripts between IC1 and IC2. We found that splice variants of Th and Ins2 are imprinted, maternally expressed, and regulated by IC2, showing that the silencing domain uncovered by our transgenic line also affects endogenous transcripts.
鼠标 7 号染色体的远端区域包含两个印迹域，它们由一个相对基因贫乏的间隔分开。我们之前描述了一种名为 Tel7KI 的转基因小鼠品系，该品系在这个间隔的近端，Ins2 基因的上游 2.6kb 处插入了一个绿色荧光蛋白（GFP）报告基因。Tel7KI 的 GFP 报告基因在植入后胚胎中表现出印迹和母系表达。在这里，我们提供的证据表明，远端印迹中心 KvDMR1（IC2）负责 Tel7KI 的父系沉默。首先，我们表明，由于 IC2 处没有母体 DNA 甲基化，当非编码 RNA Kcnq1ot1 双等位表达时，Tel7KI 被沉默。其次，我们使用胚胎干细胞（ES）细胞分化测定来检查 IC2 缺失对 Tel7KI 的顺式影响，并表明它损害了父系传递 Tel7KI ES 细胞沉默 GFP 的能力。这些结果表明，Kcnq1ot1 的沉默比之前报道的延伸了近 300kb，并促使我们检查 IC1 和 IC2 之间的其他转录本。我们发现 Th 和 Ins2 的剪接变体是印迹的，母系表达，并受 IC2 调控，表明我们的转基因系揭示的沉默域也影响内源性转录本。
