Epigenetic Control of Trophoblast Differentiation

About This Grant

PROJECT SUMMARY/ABSTRACT The major cell types of the placenta are derived from trophoblast stem cells undergoing a choreographed allocation of cell fates in response to signaling cues from the developing fetus and the uterine environment. Early defects in the differentiation of trophoblast stem cells can lead to abnormal placentation which is thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. The long-term goal of this project is to uncover the critical regulatory mechanisms of human trophoblast cell fate specification to improve health outcomes of these pregnancies. In this proposal, we will generate insights into the epigenetic events that control the formation of the syncytiotrophoblast cells of the mouse labyrinth and human chorionic villi. These structures separate the maternal and fetal blood and orchestrate the exchange of oxygen, nutrients, and fetal waste. The first aim focuses Polycomb Repressive Complex 1.1 (PRC1.1), an epigenetic silencing complex which is thought to facilitate differentiation by repressing stem cell genes and maintain lineage fidelity by repressing genes that promote alternate lineages. The PRC1.1 component BCOR (BCL6 CORepressor), interacts with DNA-binding factors BCL6 and KDM2B to recruit PRC1.1 to its genomic targets. BCOR is an X-linked gene with essential roles in placental development in mice, is mutated in human syndromes involving low birth weight and intrauterine growth restriction, and women with a heterozygous null mutation in BCOR can experience recurrent miscarriages. Extraembryonic deletion of Bcor mice leads to a reduced labyrinth and an expanded junctional zone. We will use PRC1.1 as an entry point for understanding how epigenetic changes in the genome control differentiation of trophoblast stem cells by profiling cell types with single nuclei RNA-seq and ATAC-seq and changes in H3K27ac, H3K27me3 and DNA methylation with or without BCOR loss in all trophoblast cells or specific placental cell subtypes. The second aim will elucidate the roles of eight candidate DNA-binding factors that may control gene regulatory networks in differentiating trophoblast cells. Using inducible CRISPR/Cas9 genome editing of human trophoblast stem cells we have found that loss-of-function mutations in four of these candidates (ELF5, FIRRE, IKZF4, and ZBTB7C) alter trophoblast differentiation. We will use single nucleus RNA-seq and genome-wide profiling of H3K27ac, H3K27me3, and single nucleus ATAC-seq in autonomously differentiating 3D cultures of wild-type and knockout cell lines. We will also determine the immunolocalization and genome-wide occupancy of these factors in 3D cultures and in vivo placenta samples using authenticated antibodies. Our functional genomic approaches will identify new genes involved in trophoblast differentiation that provide diagnostic and therapeutic targets for preclinical studies. Together these aims will reveal how cell fate specification in the developing placenta is controlled by the coordinated action of epigenetic modifiers and DNA-binding factors through modifications to chromatin structure.