Transcriptional Reprogramming and Chromatin Remodeling Accompanies Oct4 and Nanog Silencing in Mouse Trophoblast Lineage

Abstract

In mouse blastocysts, CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. However, the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression are poorly understood. To address this, a Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system. Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 h. Consistent with the down-regulation of Oct4 and Nanog transcripts, a time-dependent increase in CDX2 binding and a decrease in RNA polymerase II (RNAPII) and OCT4 binding was observed within 48 h (P<0.05). To test whether transcriptionally active epigenetic marks were erased during differentiation, histone H3K9/14 acetylation and two of its epigenetic modifiers were evaluated. Accordingly, a significant decrease in histone H3K9/14 acetylation and loss of p300 and HDAC1 binding at the Oct4 and Nanog regulatory elements was observed by 48 h. Accompanying these changes, there was a significant increase in total histone H3 and a loss of chromatin accessibility at both the Oct4 and Nanog regulatory elements (P<0.05), indicative of chromatin remodeling. Lastly, DNA methylation analysis revealed that methylation did not occur at Oct4 and Nanog until 96 to 120 h after induction of CDX2. In conclusion, our results show that silencing of Oct4 and Nanog is facilitated by sequential changes in transcription factor binding, histone acetylation, chromatin remodeling, and DNA methylation at core regulatory elements.