Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.
Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.
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DOI：
10.1101/gad.275958.115
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发表时间：
2016-03-01
影响因子：
10.5
通讯作者：
Raj A
中科院分区：
文献类型：
--
作者：
Ginart P;Kalish JM;Jiang CL;Yu AC;Bartolomei MS;Raj A
印记缺陷可能会导致正常沉默的等位基因的不适当表达，但目前尚不清楚突变有机体中的每个细胞是否都遵循群体平均水平。吉纳特等人。应用一种新的荧光原位杂交方法，测量单个细胞中的等位基因特异性表达，以解决显示H19/Igf2印记异常的突变体中的这个问题。印记是一种经典的哺乳动物表观遗传现象，导致单个亲本等位基因的表达。印记缺陷可以导致正常沉默的等位基因的不适当表达，但目前尚不清楚突变有机体中的每个细胞是否都遵循群体平均水平，这将对人类印记疾病产生深远的影响。在这里，我们应用一种新的荧光原位杂交方法，测量单个细胞中的等位基因特异性表达，以解决显示H19/IGF2(胰岛素样生长因子2)印记异常的突变株的这个问题。我们发现突变的原代胚胎小鼠成纤维细胞由两个亚群组成：一个亚群同时表达H19等位基因，另一个亚群仅表达母系拷贝。仅在后一组细胞中检测到Igf2的表达。此外，这两个亚群是稳定的，因为细胞不会在两种表达模式之间相互转换。结合小输入甲基化分析和转录成像显示，这两个突变亚群在其印迹控制区表现出不同的甲基化模式。一贯地，药物抑制DNA甲基化减少了单等位基因细胞的比例。重要的是，我们观察到同样的两个亚群也存在于体内的小鼠心脏组织中。我们的结果表明，印迹障碍的分子表型可以表现出显著的单细胞异质性，并提示这种异质性可能是人类印迹障碍表观遗传嵌合体的基础。
Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average. Ginart et al. applied a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 imprinting. Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.