Molecular Mechanisms Guiding TRIM28 Contribution to Determination

项目概要/摘要： 原始生殖细胞（PGCs）是成体生殖系的胚胎前体，其正常发育是由原始生殖细胞（PGCs）和成体生殖细胞（PGCs）共同完成的。 等同于生物体的健康状况。在胚胎发育过程中，PGCs经历两个命运限制步骤： 说明，其中PGCs表达多能基因，一种称为潜伏多能性的状态，同时经历 无数的表观遗传重塑和2）决定，其中多能性程序被熄灭，PGCs 根据胚胎的性别来区分。虽然分子研究仔细剖析了PGC 规范，PGC测定仍然知之甚少。尽管目前的最先进技术允许在体外 从多能干细胞（PSC）诱导PGC样细胞（PGCLC），这些PGCLC代表特定的 PGC，迄今为止，不能可靠地诱导进行体外测定。这是一个重大的 体外配子发生的障碍，这为临床缓解夫妇不孕症提供了可能性， 任何一方都不能产生自己的配子。我们假设特定的表观遗传变化 PGC测定和许可配子发生能力。在这一过程中，特别令人感兴趣的是， 转座因子（TEs），其中一些仍然能够转座，因此威胁到 生殖细胞基因组的完整性。相反，长末端重复序列（LTR）亚类的一些TE具有 转录-和多能性-因子结合位点，并可以在确定调节的时间内发挥作用， 多能性网络的表达。了解LTR元件的调节如何有助于PGC 我们采用体外小鼠模型。该建议的核心假设是，PGC 决定是依赖于TRIM 28的表观遗传转变，TRIM 28是高度保守的表观遗传支架 蛋白质，两个独立的过程：调节LTR类转座因子和适当的核仁 功能为了测试这一点，我们将采用TRIM 28的PGC特异性条件性敲除模型，使我们能够 体内询问测定。在目标1中，我将使用ATAC-seq和CutnTag测序来了解损失是如何发生的。 TRIM 28的表达改变了基因组的可接近性和增强子动力学，假设LTR的错误调节 在缺乏TRIM 28的情况下，元件驱动基因表达网络中正确调节开关的失败 当PGCs进入测定时。在目标2中，我使用OligoPaint，一种DNA-FISH方法，来评估TRIM 28损失 影响核仁异染色质和形态，并使用化学微扰离体观察可能的 TRIM 28缺失的表型复制。这项工作的完成将对我们理解 PGC表观基因组如何在决定许可配子发生的过程中重新连接。从这项工作中获得的见解可以 可以利用体外PGC模型促进功能性配子发生。

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.