Imprinted expression in cystic embryoid bodies shows an embryonic and not an extra-embryonic pattern

Abstract

A large subset of mammalian imprinted genes show extra-embryonic lineage (EXEL) specific imprinted expression that is restricted to placental trophectoderm lineages and to visceral yolk sac endoderm (ysE). Isolated ysE provides a homogenous in vivo model of a mid-gestation extra-embryonic tissue to examine the mechanism of EXEL-specific imprinted gene silencing, but an in vitro model of ysE to facilitate more rapid and cost-effective experiments is not available. Reports indicate that ES cells differentiated into cystic embryoid bodies (EBs) contain ysE, so here we investigate if cystic EBs model ysE imprinted expression. The imprinted expression pattern of cystic EBs is shown to resemble fetal liver and not ysE. To investigate the reason for this we characterized the methylome and transcriptome of cystic EBs in comparison to fetal liver and ysE, by whole genome bisulphite sequencing and RNA-seq. Cystic EBs show a fetal liver pattern of global hypermethylation and low expression of repeats, while ysE shows global hypomethylation and high expression of IAPEz retroviral repeats, as reported for placenta. Transcriptome analysis confirmed that cystic EBs are more similar to fetal liver than ysE and express markers of early embryonic endoderm. Genome-wide analysis shows that ysE shares epigenetic and repeat expression features with placenta. Contrary to previous reports, we show that cystic EBs do not contain ysE, but are more similar to the embryonic endoderm of fetal liver. This explains why cystic EBs reproduce the imprinted expression seen in the embryo but not that seen in the ysE.

Mapping the mouse Allelome reveals tissue-specific regulation of allelic expression

“…In our analysis of RNA-seq data, we found 71 of these known genes including five disputed genes ( Pon3, Peg3os, Cd81, Osbpl5, and Hymai ) ( Figure 4A ). Three of these genes disputed in placenta ( Pon3 , Cd81 and Osbpl5 ) ( Okae et al, 2012 ; Proudhon and Bourc'his, 2010 ), we have previously confirmed to show imprinted expression in VE ( Hudson et al, 2011 ; Kulinski et al, 2015 ). Peg3os and Hymai may be false positives due to overlap with known imprinted genes that show the same imprinted expression status, with Peg3os overlapped in anti-sense by Peg3 (due to incomplete strand-specificity of RNA-seq) and Hymai overlapped in sense by Plagl1 .…”

“…Primary mouse embryonic fibroblasts (MEFs) were derived from reciprocal FVB x CAST crosses from E12.5 embryos ( Andergassen et al, 2015 ). Mouse ES cells were derived from reciprocal crosses from FVB x CAST crosses from mouse blastocysts ( Kulinski et al, 2015 ). The passage number is given for each cell line used, and for each experiment in Supplementary file 1 , sheet A.…”

“…Visceral yolk sac and mammary glands were processed as previously described to isolate VE and mammary epithelial cells ( Hudson et al, 2011 ; Joshi et al, 2010 ). ESCs were derived following an established protocol ( Bryja et al, 2006 ; Kulinski et al, 2015 ), and adapted to 2i media without feeders (ESGRO-2i Medium, Millipore) ( Ying and Smith, 2003 ; Ying et al, 2008 ). Note that the RNA-seq data from MEFs was described in an earlier study ( Andergassen et al, 2015 ).…”

The Airn lncRNA does not require any DNA elements within its locus to silence distant imprinted genes

“…Interestingly, Kcnq1 itself has been reported to be subject to lncRNA independent imprinted silencing despite Kcnq1ot1 lying within one of its introns, and to lose imprinted expression during heart development [43]. Therefore, it remains to be tested if the model can explain lncRNA mediated imprinted silencing of non-overlapped genes in all tissues, or if it is restricted to the specific epigenetic environment present in extra-embryonic tissues, known to have unique features such as low levels of DNA methylation [44,45].…”

JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

“…Furthermore, the increase in the size of EBs formed from cells expressing JAZF1-SUZ12 was associated with the formation of multiple cystic cavities (Figure 5D). Production of cystic cavities is associated with expression of endoderm markers such as Gata4 (Fujikura et al, 2002;Kulinski et al, 2015), consistent with enhanced expression of this gene in EBs containing JAZF1-SUZ12. We conclude that JAZF1-SUZ12 dysregulates gene expression and that this disrupts normal patterns of cell differentiation.…”