Nodal signaling plays critical roles during embryonic development. The Nodal gene is not expressed in adult tissues but is frequently activated in cancer cells, contributing to progression toward malignancy. Although several regulatory elements of the Nodal gene have been identified, the epigenetic mechanisms by which Nodal expression is regulated over the long term remain unclear. We found a region exhibiting dynamic changes in DNA methylation at approximately -3.0 kb to -0.4 kb upstream from the transcriptional start site (TSS) that we termed the epigenetic regulatory element (ERE). The ERE was unmethylated in mouse embryonic stem cells (mESCs) but became increasingly methylated in differentiated cells and tissues, concomitant with the downregulation of Nodal mRNA expression. In vitro reporter assays identified an Oct3/4 binding motif within the ERE, indicating that the ERE is responsible for the activation of Nodal in mESCs. Furthermore, the ERE was a target of differentiation-associated Polycomb silencing, and the chromatin condensed when mESCs differentiated to embryoid bodies (EBs). Pharmacological inhibition of PRC2 led to the reactivation of Nodal expression in EBs and mouse embryonic fibroblasts (MEFs). The ERE was also targeted by PRC2 in normal human cells. In NODAL-expressing human cancer cells, accumulation of EZH2 and trimethylation of H3K27 at the ERE were diminished. In conclusion, Nodal is epigenetically controlled through the ERE in the mouse embryo and human cells.
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