The Role of Homeodomain Proteins in Human Decidualization

同源域蛋白在人类蜕膜化中的作用

基本信息

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
项目类别：
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

项目摘要

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.

为了使人子宫接受植入胚胎的接受，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，称为卵巢激素。尽管子宫接受性在妊娠的植入阶段具有不可或缺的作用，并且由于子宫缺乏，许多昂贵的体外受精程序证明了未能成功的，但决定性决定性化的分子途径仍被严格理解。决定性化研究的一个主要发展是产生永生的人子宫内膜基质细胞（HESC），该细胞对卵巢激素的孕酮和雌激素对卵巢激素和雌激素做出了牢固的反应。作为拟议研究的初步数据，我们从hESC中产生了一个记者细胞系，该细胞系在左右启动子的控制下表达YFP，因此，当用卵巢激素的鸡尾酒处理时，对催乳素启动子的控制表现出一致且可靠的YFP诱导。称为HESC-Prys的细胞代表了一种在各种基因组和环境应力下评估决定性化的强大筛选工具。使用这些细胞，我们进行了完整的基因组siRNA筛选以绘制人类的决定性遗传网络，并发现正常决定性化所需的最具影响力的转录家族中有同源域的转录因子，包括MSX2，EN1，IRX1/3，DLX1/2/2/2/6，LHX1/2/2/2/3/9和SIX3/6。拟议的研究旨在首先使用QPCR和免疫印迹，首先绘制hESC中同源域蛋白的暂时诱导概况，以响应激素。接下来，同源域蛋白在决定性化中的要求将由为每个候选因素生成基因敲除hESC-PRLY。将使用几个参数评估基因敲除细胞进行决策：YFP诱导，QPCR和Immunoboblet的替代决定标记（IGFBP1和FOXO1）的表达，通过3D共培养和子宫内膜上皮细胞的3D共培养测定法，以及对葡萄孢子蛋白瘤的共培养物的接受性。每个基因敲除细胞系还将通过用质粒编码每个因子进行瞬时转染进行救援实验。最后，“通话卡”测定法，其中融合到PiggyBac转座酶标记结合位点的转录因子与敲除细胞系的RNA测序与野生型对照组的RNA测序将用于识别HESC中最具影响力的两个最具影响力的同源蛋白的直接靶标。这些数据将共同定义同源域转录因子在决策中的强大作用，并确定在治疗女性植入水平不孕症时潜在药理调节的途径。访问最新的技术，有见地的讲座，定期研讨会和培养协作氛围将是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的首要领导者提供了无与伦比的建议环境。