Kdm4 histone demethylase family is required to maintain trophoblast stem cell fate by modulation of H3K9me3 status

Abstract

Faithful maintenance of embryonic and trophoblast lineage resulting from the first differentiation event is crucial to the proper development of embryo and placenta. Epigenetic regulation involving DNA methylation and H3K9me3 has been shown to be important in stabilizing the embryonic fate through the repression of transcriptional program of the unrelated cell lineages. The histone H3K9 demethylase Kdm4 (Jmjd2) family was previously shown contributing to the murine embryonic stem (ES) cell state. However, their roles in trophoblast lineage determination and maintenance remain unclear. Here we show that Kdm4 activity is crucial to the maintenance of trophoblast stemness gene expression in murine trophoblast stem (TS) cells. Induction of TS cell differentiation leads to loss of Kdm4b binding to trophoblast stemness gene Cdx2, which is associated with increased H3K9me3 enrichment and gene repression. In contrast, loss of H3K9me3 at differentiation gene Ascl2 was independent of Kdm4b occupancy, suggesting the specific epigenetic regulation of trophoblast stemness genes by Kdm4 family. Although forced expression of Kdm4b in ES cells demonstrated no significant induction of trophoblastic transdifferentiation, the trophoblast lineage commitment can be further promoted together with 5-azacytidine treatment. Importantly, inhibition of Kdm4 activity in ES cells cultured in TS medium condition (primed ES cells) showed reduced expression of trophoblast lineage genes. We also observed stronger induction of trophoblast differentiation genes by continuous inhibition of Kdm4 activity in the TS-like cells derived from the DNA hypomethylated Np95-/-ES cells, implying the crucial role of Kdm4 family in the maintenance of stem cell state. Our findings suggest that Kdm4 family sustains the transcriptional program associated with stemness in TS cells through epigenetic regulation of the H3K9me3 status.