Epigenetic Barrier in Differentiation of Mouse Embryonic Stem Cells Towards Trophoblast Lineage

Abstract

The first cell differentiation event in mammalian embryo development is the formation of trophoectoderm (TE) and inner cell mass (ICM). It is now clear that the diversity of cell differentiation and the corresponding gene expression pattern is mediated by means of epigenetic mechanisms, such as DNA methylation and covalent histone modifications. With a global comparison of promoter DNA methylation patterns between mouse embryonic stem (ES) cell and mouse trophoblast stem (TS) cell, we have identified a transcription factor, Elf5, which regulates the determination of trophoblast cell fate through its DNA methylation status. However, the mechanism which directs the setup of differential Elf5 methylation in embryonic precursor cells remains to be elucidated. Recent studies demonstrated that both H3K9 methylation and DNA methylation can be established at the same loci by a repressor protein complex. We therefore hypothesize that H3K9 methylation is involved in the regulation of trophoblast lineage differentiation, potentially associated with the setup of DNA methylation at Elf5 promoter. To manipulate the cellular H3K9 methylation level, we have constitutively overexpressed Kdm4 (Jmjd2) family histone demethylases in mouse ES cells. While overexpression of Kdm4 family members in ES cells demonstrated no significant induction of trophoblast differentiation under normal TS culture condition, the addition of 5-aza-deoxycytidine triggered significant increase of Elf5 expression, with the exception of Kdm4b overexpressing ES cells. Further analysis of the Kdm4b overexpressing ES cells indicated persistent expression of Nanog under 5-azadeoxycytidine treatment. Our results thus suggest a role of Kdm4b in the maintenance of pluripotent state of ES cells, and prevent the differentiation to trophoblast lineage. Investigation of the functions of Kdm4 members using the ES cell differentiation system enables us to unveil the details of epigenetic mechanism as well as identify novel lineage determination genes in trophoblast development. Studies on the epigenetic regulation in trophoblast differentiation can provide valuable information on the formation of the placenta, which is crucial for the survival and normal development of an embryo.