The Role of Homeodomain Proteins in Human Decidualization

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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.