The Wilms tumor suppressor gene (WT1) encodes an essential transcription factor regulating mammalian urogenital development. However, the function of WT1 in human endometrium is still unclear. The current study examined the involvement of WT1 in the regulation of IGF-binding protein-1 (IGFBP-1) and prolactin (PRL), which are specific markers of decidualization, in human endometrial stromal cells (ESCs) undergoing decidualization. ESCs isolated from proliferative-phase endometrium were incubated with cyclic adenosine monophosphate (cAMP) to induce decidualization. cAMP increased WT1 expression with the induction of IGFBP-1 and PRL. Knockdown of WT1 by small interfering RNA inhibited cAMP-induced expression of IGFBP-1 and PRL. cAMP also induced the recruitment of WT1 to the IGFBP-1 and PRL promoters. To investigate the mechanism by which WT1 is upregulated by cAMP, we focused on C/EBPβ, a gene that regulates the expression of many genes during decidualization. Knockdown of C/EBPβ decreased cAMP-increased WT1 expression. cAMP increased the recruitment of C/EBPβ to the WT1 enhancer that is located approximately 14,000 bp downstream from the transcription start site. To test the endogenous function of the WT1 enhancer region on WT1 expression, the endogenous WT1 enhancer region was deleted by CRISPR/Cas9 system in HEK293 cells. The increase of WT1 expression by cAMP was not observed in the enhancer-deleted clones. Chromatin immunoprecipitation assay revealed that this enhancer region has high levels of H3K27ac and H3K4me1, which are active enhancer marks. These results show the role of WT1 in regulating decidualization in human ESCs. C/EBPβ is an upstream gene that regulates WT1 expression by binding to the novel enhancer region.
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