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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.

为了使人类子宫能够接受胚胎的植入，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，称为脱胞化。尽管子宫接受性在妊娠着床阶段起着不可或缺的作用，尽管许多昂贵的体外受精程序由于子宫缺陷而失败，但脱个体化背后的分子途径仍未得到充分研究。脱胞化研究的一个主要进展是产生永生化的人子宫内膜基质细胞（hESCs），这些细胞对卵巢激素黄体酮和雌激素有强烈的转录反应。作为该研究的初步数据，我们从hESCs中克隆了一个亚克隆细胞系，该细胞系在催乳素启动子的控制下表达YFP，因此在卵巢激素混合物处理下表现出一致和强大的YFP诱导。这些细胞被称为hESC-PRLYs，是评估各种基因组和环境压力下去个性化的强大筛选工具。利用这些细胞，我们进行了全基因组siRNA筛选，绘制了人类脱个体化遗传网络，发现正常脱个体化所需的最具影响力的转录家族是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。本研究旨在首先利用qPCR和免疫印迹技术绘制hESCs中同源结构域蛋白在激素作用下的时间诱导谱。接下来，同位结构域蛋白在去个体化过程中的需求将由