The Role of Homeodomain Proteins in Human Decidualization

同源域蛋白在人类蜕膜化中的作用

• 批准号: 10086328
• 负责人: Meade Elspeth Haller
• 金额: $ 6.74万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10086328
• 关键词: 3-Dimensional; Basic Science; Binding Sites; Biological Assay; Biological Models; Categories; Cell Line; Cells; Choriocarcinoma; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Contraceptive Agents; DNA-Binding Proteins; Data; Data Set; Decidual Cell Reactions; Defect; Development; Developmental Process; Differentiation and Growth; Embryo; Endometrial; Endometrial Stromal Cell; Endometrium; Enhancers; Environment; Epithelial Cells; Estrogens; Exhibits; Expression Profiling; FOXO1A gene; Family; Female; Female infertility; Fertilization; Fertilization in Vitro; Fostering; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomics; Homeodomain Proteins; Hormone use; Hormones; Human; Human Cloning; IGFBP1 gene; Implant; Infertility; Influentials; Knock-out; LHX1 gene; Maps; Modernization; Molecular; Organoids; Ovarian hormone; Pathway interactions; Pattern; Pharmacology; Phase; Placentation; Plasmids; Pregnancy; Procedures; Process; Progesterone; Prolactin; Proteins; Reaction; Reporter; Role; Screening procedure; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Stress; Technology; Testing; Training; Transcript; Transfection; Translational Research; Transposase; Universities; Uterus; Washington; Western Blotting; base; collaborative environment; design; differential expression; early pregnancy loss; endometrial stroma; experimental study; genome wide screen; healthy pregnancy; homeodomain; hormone sensitivity; implantation; knock-down; lectures; natural Blastocyst Implantation; novel; promoter; response; screening; symposium; three dimensional cell culture; transcription factor; transcriptome sequencing; uterine receptivity; whole genome

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.

为了使人类子宫能够接受植入的胚胎，子宫内膜基质细胞必须对卵巢激素进行复杂的增殖和分化反应，称为蜕膜化。尽管子宫容受性在妊娠着床阶段发挥着不可或缺的作用，并且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，但令人遗憾的是，蜕膜化的分子途径仍然没有得到充分的研究。蜕膜化研究的一项重大进展是永生化人类子宫内膜基质细胞（hESC）的产生，其对卵巢激素孕激素和雌激素的转录反应强烈。作为拟议研究的初步数据，我们生成了从 hESC 亚克隆的报告细胞系，该细胞系在催乳素启动子的控制下表达 YFP，因此在用卵巢激素混合物处理时表现出一致且强大的 YFP 诱导。这些细胞被称为 hESC-PRLY，是评估各种基因组和环境应激下蜕膜化的强大筛选工具。使用这些细胞，我们进行了全基因组 siRNA 筛选，以绘制人类蜕膜化遗传网络图谱，发现正常蜕膜化所需的最有影响力的转录家族是同源域转录因子，包括 MSX2、EN1、IRX1/3、DLX1/2/6、LHX1/2/3/9 和 SIX3/6。拟议的研究旨在首先使用 qPCR 和免疫印迹绘制 hESC 中同源域蛋白响应激素的时间诱导图谱。接下来，蜕膜化中同源域蛋白的需求将由以下确定： 为每个候选因子生成敲除 hESC-PRLY。将使用多个参数评估敲除细胞的蜕膜化：YFP 诱导、通过 qPCR 和免疫印迹表达替代蜕膜标记（IGFBP1 和 FOXO1）、通过与子宫内膜上皮细胞 3D 共培养进行类器官形成测定，以及共培养物对绒毛膜癌球体的接受性。每个敲除细胞系还将通过用编码每个因子的质粒瞬时转染来进行拯救实验。最后，“名片”测定（其中融合到 PiggyBac 转座酶的转录因子标记结合位点），结合敲除细胞系与野生型对照的 RNA 测序，将用于识别 hESC 中最有影响力的两种同源域蛋白的直接靶点。这些数据将共同定义同源域转录因子在蜕膜化中的强大作用，并确定治疗女性着床水平不孕症的潜在药理学调节途径。获取最新技术、富有洞察力的讲座、定期研讨会和营造协作氛围将成为培训过程中不可或缺的一部分。华盛顿大学为转化应用基础研究最重要的领导者提供了无与伦比的建议环境。