Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism
Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism
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DOI：
10.1210/en.2017-00478
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发表时间：
2017-10
期刊：
影响因子：
4.8
通讯作者：
I. Tamura;Yuichiro Shirafuta;Kousuke Jozaki;Takuya Kajimura;Masahiro Shinagawa;R. Maekawa;T. Taketani;H. Asada;Shun Sato;H. Tamura;N. Sugino
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作者：
I. Tamura;Yuichiro Shirafuta;Kousuke Jozaki;Takuya Kajimura;Masahiro Shinagawa;R. Maekawa;T. Taketani;H. Asada;Shun Sato;H. Tamura;N. Sugino
&NA;Wilms肿瘤抑制基因（WT 1）编码调节哺乳动物泌尿生殖系统发育的必需转录因子。然而，WT 1在人类子宫内膜中的功能仍不清楚。目前的研究检查了WT 1参与调节IGF-结合蛋白-1（IGFBP-1）和催乳素（PRL），这是蜕膜化的特异性标志物，在人类子宫内膜基质细胞（ESC）中进行蜕膜化。将从增殖期子宫内膜分离的ESC与环磷酸腺苷（cAMP）孵育以诱导蜕膜化。cAMP通过诱导IGFBP-1和PRL增加WT 1表达。通过小干扰RNA敲低WT 1抑制cAMP诱导的IGFBP-1和PRL表达。cAMP还诱导WT 1向IGFBP-1和PRL启动子的募集。为了研究WT 1被cAMP上调的机制，我们将重点放在C/EBP上，这是一个在蜕膜化过程中调节许多基因表达的基因。C/EBP的敲低降低cAMP-增加WT 1表达。cAMP增加了C/EBP向位于转录起始位点下游约14，000 bp的WT 1增强子的募集。为了测试WT 1增强子区域对WT 1表达的内源性功能，在HEK 293细胞中通过CRISPR/Cas9系统缺失内源性WT 1增强子区域。在增强子缺失的克隆中未观察到cAMP引起的WT 1表达增加。染色质免疫沉淀分析显示，该增强子区域具有高水平的H3 K27 ac和H3 K4 me 1，这是活性增强子标记。这些结果显示了WT 1在调节人ESC中的蜕膜化中的作用。C/EBP是一个上游基因，通过与新的增强子区域结合来调节WT 1的表达。
&NA; 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/EBP, a gene that regulates the expression of many genes during decidualization. Knockdown of C/EBP decreased cAMP‐increased WT1 expression. cAMP increased the recruitment of C/EBP 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/EBP is an upstream gene that regulates WT1 expression by binding to the novel enhancer region.