The Role of Homeodomain Proteins in Human Decidualization

为了使人类子宫能够接受植入胚胎，子宫内膜间质细胞必须经历对卵巢激素的复杂的增殖和分化反应，称为蜕膜形成。尽管子宫容受性在妊娠的植入阶段发挥着不可或缺的作用，尽管许多昂贵的体外受精手术因子宫缺陷而被证明不成功，但遗憾的是，蜕膜形成的分子途径仍未得到充分的研究。蜕膜化研究的一个主要进展是产生了永生化的人子宫内膜基质细胞(HESCs)，它对卵巢激素孕酮和雌激素有很强的转录反应。作为拟议研究的初步数据，我们从hESCs亚克隆了一个报告细胞系，该细胞系在催乳素启动子的控制下表达YFP，因此在用卵巢激素的鸡尾酒处理时显示出一致和强劲的YFP诱导。这些细胞被称为hESC-PRLY，是一种强大的筛选工具，可以评估在各种基因组和环境压力下的蜕膜形成。我们利用这些细胞进行了全基因组siRNA筛选，以绘制人类蜕膜化遗传网络的图谱，并发现在正常蜕膜化所需的最具影响力的转录家族中，有同源结构域转录因子，包括MSX2、EN1、IRX1/3、DLX1/2/6、LHX1/2/3/9和Six3/6。接下来，蜕膜化过程中对同源结构域蛋白的需求将通过以下方式确定 为每个候选因子生成敲除hESC-PRLY。将使用以下几个参数来评估基因敲除细胞的蜕膜化：YFP诱导，qPCR和免疫印迹检测可替代的蜕膜标志物(IGFBP1和FOXO1)的表达，与子宫内膜上皮细胞共培养的有机类物质形成分析，以及共培养的绒毛膜癌球的容受性。每一种基因敲除的细胞系也将通过瞬时转染法与编码每种因子的质粒一起进行挽救实验。最后，结合基因敲除细胞系与野生型对照细胞株的RNA测序，利用转录因子融合到iggyBac转座酶标记结合位点的“名片”分析，将被用来确定hESCs中最有影响力的两个同源结构域蛋白的直接靶点。这些数据将确定同源结构域转录因子在蜕膜化中的强大作用，并确定在女性着床水平不孕症的治疗中潜在的药物调节途径。获得最新技术、有见地的讲座、定期座谈会和培养合作气氛将是培训过程的一个组成部分。华盛顿大学为翻译应用程序的基础研究提供了无与伦比的咨询环境。

In order for the human uterus to become receptive to an implanting embryo, the endometrial stromal cells must undergo a complex proliferation and differentiation response to ovarian hormones, termed decidualization. Despite the integral role of uterine receptivity in the implantation phase of pregnancy, and despite many expensive in vitro fertilization procedures proving unsuccessful due to uterine deficiency, the molecular pathways underlying decidualization remain woefully understudied. One major development in the study of decidualization was the generation of immortalized human endometrial stromal cells (hESCs), which respond transcriptionally robustly to the ovarian hormones progesterone and estrogen. As preliminary data to the proposed study, we generated a reporter cell line sub-cloned from hESCs that expresses YFP under the control of the prolactin promoter, and therefore exhibits a consistent and robust induction of YFP when treated with a cocktail of ovarian hormones. These cells, termed hESC-PRLYs, represent a powerful screening tool to assess decidualization under various genomic and environmental stresses. Using these cells we performed a full genome siRNA screen to map the human decidualization genetic network, and found that among the most influential transcript families required for normal decidualization are homeodomain transcription factors, including MSX2, EN1, IRX1/3, DLX1/2/6, LHX1/2/3/9, and SIX3/6. The proposed study aims to first map the temporal induction profile of homeodomain proteins in hESCs in response to hormone using qPCR and immunoblot. Next, the requirement of homeodomain proteins in decidualization will be determined by generating knockout hESC-PRLYs for each candidate factor. Knockout cells will be assessed for decidualization using several parameters: YFP induction, expression of alternative decidual markers (IGFBP1 and FOXO1) by qPCR and immunoblot, organoid formation assay via 3D coculture with endometrial epithelial cells, and receptivity of cocultures to choriocarcinoma spheroids. Each knockout cell line will also undergo rescue experiments via transient transfection with plasmid encoding each factor. Lastly, “calling card” assay, in which transcription factors fused to PiggyBac transposase mark binding sites, in conjunction with RNA sequencing of knockout cell lines versus wildtype controls, will be used to identify direct targets of the top two most influential homeodomain proteins in hESCs. Together these data will define the robust role of homeodomain transcription factors in decidualization, and identify pathways for potential pharmacological modulation in the treatment of female implantation-level infertility. Access to the newest technologies, insightful lectures, regular symposia and the fostering of a collaborative atmosphere will be an integral part of the training process. Washington University provides an unparalleled environment of advisement by the foremost leaders of basic research for translational applications.