Demonstration of all-or-none loss of imprinting in mRNA expression in single cells

Abstract

Loss of imprinting (LOI) is the reactivation of the silenced allele of an imprinted gene, leading to perturbation of monoallelic expression. We tested the hypothesis that LOI of PLAGL1, a representative maternally imprinted gene, occurs through an all-or-none process leading to a mixture of fully imprinted and nonimprinted cells. Herein using a quantitative RT-PCR-based experimental approach, we measured LOI at the single cell level in human trophoblasts and demonstrated a broad distribution of LOI among cells exhibiting LOI, with the mean centered at ∼100% LOI. There was a significant (P < 0.01) increase in expression after 2 days of 5-aza-2′-deoxycytidine (AZA) treatment and a significant (P < 0.01) increase in LOI after both 1 and 2 days of AZA treatment, while the distribution remained broad and centered at ∼100% LOI. We propose a transcriptional pulsing model to show that the broadness of the distribution reflects the stochastic nature of expression between the two alleles in each cell. The mean of the distribution of LOI in the cells is consistent with our hypothesis that LOI occurs by an all-or-none process. All-or-none LOI could lead to a second distinct cell population that may have a selective advantage, leading to variation of LOI in normal tissues, such as the placenta, or in neoplastic cells.

Genomic Imprinting in Human Placenta

“…It has been in fact shown in cell lines that the administration of 5-aza-2'-deoxycytidine (AZA) induces complete bi-allelic expression in individual clones (Diplas et al, 2009b). This finding is thus consistent with a total erasure of the DNA methylation readout code therefore leading to a complete binding inhibition of the protein complexes that would otherwise drive the allele silencing.…”

Placental pseudo-malignancy from a DNA methylation perspective: unanswered questions and future directions

“…The authors argued that LOI in the first trimester might be a regulatory mechanism promoting trophoblast invasion and the establishment of placentation. Furthermore, the lack of correlation between LOI and gene expression level, and the all-or-nothing pattern of LOI at the single cell level in cytotrophoblasts, suggest that LOI plays a role in placental development by introducing variation in cell phenotype ( Diplas et al, 2009a ; Pozharny et al, 2010 ).…”

Quantitative Allele-Specific Expression and DNA Methylation Analysis of H19, IGF2 and IGF2R in the Human Placenta across Gestation Reveals H19 Imprinting Plasticity

“…This variation could be due to the heterogeneous nature of the placental villous tissue sampled and H19 lineage-specific imprinting at the single cell level. Cell-specific imprinted gene expression has been proposed as an all or none phenomenon in placental cell lines [59], and H19 biallelic expression has been shown to be specific to extravillous cytotrophoblast cells [47], suggesting there is no intermediate imprinting effect at the single cell level. Therefore, observing variations in relative expression from the imprinted allele in placental tissue may simply reflect the fraction of cells with complete biallelic expression [59].…”

“…Cell-specific imprinted gene expression has been proposed as an all or none phenomenon in placental cell lines [59], and H19 biallelic expression has been shown to be specific to extravillous cytotrophoblast cells [47], suggesting there is no intermediate imprinting effect at the single cell level. Therefore, observing variations in relative expression from the imprinted allele in placental tissue may simply reflect the fraction of cells with complete biallelic expression [59]. As first trimester placental tissue sampling is expected to yield a higher proportion of extravillous cytotrophoblast cells than those collected at term, changes in the level of imprinting across gestation may reflect proportional changes in cell lineage populations as the placenta differentiates.…”