PROJECT SUMMARY New mechanistic insights into placentation and the signaling pathways that maintain proper trophoblast differentiation and invasion of the endometrium will provide the foundation for new therapeutics against pre- eclampsia. Senescent cells are characteristic of the developing and mature placenta, and changes in the senescence-associated secretory phenotype (SASP) have been observed in placental dysfunction. In patients with pre-eclampsia, increased SASP factors are associated with their disease status. Senolytics and senomorphics have recently been proposed as therapeutic interventions for patients with pre-eclampsia, reducing the intensity of SASP. Histone variants organize the senescent genome, and a newly characterized histone variant, H2AJ, is enriched in the placenta and is known to control SASP. This proposal aims to perturb H2AJ in human embryonic stem cells and establish foundational knowledge about H2AJ and its role in trophoblast development and genome architecture. Our human trophoblast cells derived from human embryonic stem cells recapitulate many aspects of fetal placental development and upregulate H2AJ (Figure 5,6,7). This study will serve the long-term goal of identifying new regulators of trophoblast development by identifying protein partners of H2AJ that may serve as therapeutic drug targets. Differentiating human embryonic stem cells with and without H2AJ, we will examine transcriptional, chromatin, and secretory changes in the generated trophoblast lineages. Using protein immunoprecipitation, we will identify unknown interactors of H2AJ, providing new insight into the role of H2AJ loading in the genome. It has been shown that during the transition to senescence, cells undergo dramatic chromatin changes, segregating their heterochromatin into large aggregates in the nuclear interior while maintaining SASP genes in highly expressed euchromatin (Figure 8). The mobilization of heterochromatin away from the nuclear periphery and the maintenance of euchromatic boundaries are not well understood in trophoblast cells. While it is known that cells become senescent in the placenta, there are few studies describing their chromatin architecture. As H2AJ appears critical for the upregulation of transcription at specific SASP loci, loss of H2AJ may be associated with demarcation errors between the strict domains of euchromatin and heterochromatin. In this proposal, we will identify H2AJ's deposition in the genome and its role in transcription, characterizing the three-dimensional chromatin architecture of H2AJ depleted and control trophoblast cells. Furthermore, we will perform this system's first Hi-C and chromatin tracing studies. To date, this will be the first study to characterize H2AJ in human development and its role in trophoblast cells. This knowledge will be crucial for discovering new treatment modalities for patients experiencing pre-eclampsia.