The role and regulation of the IC2 imprinted cluster in extravillous trophoblast differentiation

About This Grant

PROJECT ABSTRACT The development of the human placenta depends on proper differentiation of the epithelial trophoblasts. Defects in trophoblast differentiation, particularly toward the extravillous trophoblast (EVT) lineage, are associated with adverse pregnancy outcomes. However, the mechanisms that regulate the differentiation of the germinative cytotrophoblasts to EVTs remain unknown. The imprinting disorder Beckwith-Wiedemann Syndrome (BWS) is characterized by abnormal EVT morphology and can offer insights into candidate regulators of EVT differentiation. BWS is most often caused by dysregulation of a cluster of imprinted genes whose expression from the maternal allele is controlled by the imprinting control region IC2. In the human placenta, the IC2- regulated genes are CDKN1C, PHLDA2, SLC22A18, and KCNQ1. Both CDKN1C and PHLDA2 are upregulated over the course of EVT differentiation, are associated with placental phenotypes in knockout mice, and have been previously shown to influence the activity of the transcription factor ASCL2, a critical regulator of EVT differentiation. Therefore, CDKN1C and PHLDA2 most likely drive the EVT phenotypes in BWS. However, the mechanisms underlying the role of the IC2 genes in EVT differentiation and the interactions between these genes remain unexplored. In Aim 1, I will establish the role of the IC2 genes in EVT differentiation. I hypothesize that CDKN1C and PHLDA2 act synergistically to promote EVT differentiation by altering the activity of the transcription factor ASCL2. I will investigate this hypothesis by knockdown of the IC2 genes in human trophoblast stem cells, which can be differentiated into EVTs in vitro, and by characterizing EVTs in BWS patient placentas. Aside from their role in EVT differentiation, the factors that regulate the expression of CDKN1C and PHLDA2 in this process are unknown. Both genes are upregulated in EVT differentiation without changes in methylation at their promoters or the imprinting control region IC2. This implies that there are uncharacterized enhancers regulating the expression of CDKN1C and PHLDA2. For many imprinted genes, promoter-enhancers interactions are mediated by CTCF-dependent contacts with their imprinting control region; however, the CTCF landscape of the IC2 region during EVT differentiation has not been explored. In Aim 2, I will establish the mechanism regulating CDKN1C and PHLDA2 expression in EVT differentiation. I hypothesize that CDKN1C and PHLDA2 are regulated in EVT differentiation by uncharacterized enhancers and that contact with these enhancers depends on contact with IC2 through CTCF-mediated chromatin looping. To test this hypothesis, I will investigate the impact of maternal IC2 deletion and CTCF depletion on IC2 gene expression and EVT differentiation efficiency in iPSC-derived human trophoblast stem-like cells. I aim to establish the role and regulation of the IC2 imprinted cluster in EVT differentiation. By identifying novel regulators of EVT differentiation, I will improve our understanding of this fundamental process in placental development.