title: Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.
abstract: 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.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Skirkanich Hall, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Paul
authors.initials: P
authors.lastname: Ginart
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Kalish
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Connie L
authors.initials: CL
authors.lastname: Jiang
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Alice C
authors.initials: AC
authors.lastname: Yu
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Marisa S
authors.initials: MS
authors.lastname: Bartolomei
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Skirkanich Hall, Philadelphia, Pennsylvania 19104, USA;
authors.forename: Arjun
authors.initials: A
authors.lastname: Raj
mesh_terms: D000483:Alleles; D000818:Animals; D002478:Cells, Cultured; D019175:DNA Methylation; D057890:Epigenomics; D005786:Gene Expression Regulation; D018392:Genomic Imprinting / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009030:Mosaicism; D009154:Mutation; D062085:RNA, Long Noncoding / Q000235:genetics*; D059010:Single-Cell Analysis
chemical_list: C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D007335:Insulin-Like Growth Factor II
keywords: gene expression; imprinting; single cell