Molecular Mechanisms Guiding TRIM28 Contribution to Determination

项目摘要/摘要：

Project Summary/ Abstract: Primordial Germ Cells (PGCs) are embryonic precursors to the adult germline, the proper development of which is tantamount to organismal fitness. During embryonic development PGCs undergo two fate-restriction steps: 1) specification, in which PGCs express pluripotent genes, a state called latent pluripotency, while undergoing myriad epigenetic remodeling and 2) determination, in which the pluripotency program is extinguished and PGCs differentiate according to the sex of the embryo. While molecular studies have carefully dissected PGC specification, PGC determination remains poorly understood. Although the current state-of-the-art allows in vitro induction of PGC-Like-Cells (PGCLCs) from pluripotent stem cells (PSCs), these PGCLCs represent specified PGCs and, thus far, cannot be reliably induced to undergo determination in vitro. This constitutes a significant roadblock for in vitro gametogenesis, which offers a possibility for clinical relief of infertility in couples where either partner is unable to produce their own gametes. We hypothesize that specific epigenetic changes drive PGC determination and license gametogenic capacity. Of particular interest during this process is the regulation of Transposable Elements (TEs), some of which remain capable of transposition and therefore threaten the integrity of the germline genome. Conversely, some TEs of the Long Terminal Repeat (LTR) subclass harbor transcription- and pluripotency- factor binding sites and could function during the time of determination to regulate expression of the pluripotency network. To understand how regulation of LTR elements contributes to PGC determination we employ an in vitro mouse model. The central hypothesis of this proposal is that PGC determination is an epigenetic transition that is reliant on TRIM28, a highly conserved epigenetic scaffolding protein, for two independent processes: regulation of LTR-class transposable elements and proper nucleolar function. To test this, we will employ a PGC-specific conditional knockout model of TRIM28, allowing us to interrogate determination in vivo. In Aim 1, I will use ATAC-seq and CutnTag sequencing to understand how loss of TRIM28 alters genome accessibility and enhancer dynamics, hypothesizing that misregulation of LTR elements in the absence of TRIM28 drives a failure to correctly regulate the switch in gene expression networks as PGCs enter determination. In Aim 2, I use OligoPaint, a DNA-FISH approach, to assess how TRIM28 loss effects nucleolar heterochromatin and morphology, and use chemical perturbation ex vivo to observe possible phenocopy with loss of TRIM28. Completion of this work will have broad implications in our understanding of how the PGC epigenome is rewired during determination to license gametogenesis. Insights from this work can be leveraged to advance in vitro PGC models towards functional gametogenesis.