Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states

Abstract

Background: Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). Results: As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. Conclusions: Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.

Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies

“…The generation of embryoids are very new and no studies have yet examined their epigenome. However, as ESCs are prone to loose imprinting upon prolonged culture, careful examination of the imprint status in the embryoids will be of major importance [ 170 , 171 ]. On the other hand, embryo culture has been recognised as a major factor contributing to adverse effects of ART onto placental function [ 172 ].…”

Lineage-specific dynamics of erasure of X-upregulation during inactive-X reactivation

“…chromosome, the evolution of X-chromosome inactivation happened in female cells 3 . While X-inactivation has been extensively studied, the existence of X-chromosome upregulation process remains controversial and has been challenged [4][5][6] . Recent studies by us and others have provided substantial shreds of evidence for the existence of upregulated active-X chromosome in both males and females [7][8][9][10][11][12][13][14][15] .…”

A multi-omics integrative analysis based on CRISPR screens re-defines the pluripotency regulatory network in ESCs

“…The RNA-seq data of three germ layer directional differentiation samples (E-MTAB-4904) 71 was downloaded from the BioStudies database ( https://www.ebi.ac.uk/biostudies/ ). The RPKM processed expression data of mESCs (GSE53387) were from the supplemental data of reference 72 , 73 .…”