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Molecular Mechanisms Guiding TRIM28 Contribution to Determination

Molecular Mechanisms Guiding TRIM28 Contribution to Determination
指导 TRIM28 对测定的分子机制
批准号:
10751859
负责人:
Jonathan Adam DiRusso
金额:
$3.81万
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-07-06 至 2025-01-05

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中文摘要
翻译
项目摘要/摘要: 原始生殖细胞 (PGC) 是成体生殖系的胚胎前体,其正常发育 相当于有机体健康。在胚胎发育过程中,PGC 经历两个命运限制步骤:1) 规范,其中 PGC 表达多能基因,一种称为潜在多能性的状态,同时经历 无数的表观遗传重塑和 2) 测定,其中多能性程序被消灭并且 PGC 根据胚胎的性别来区分。虽然分子研究已经仔细剖析了 PGC 规范中,PGC 测定仍然知之甚少。尽管目前最先进的技术允许在体外 从多能干细胞 (PSC) 诱导 PGC 样细胞 (PGCLC),这些 PGCLC 代表特定的 迄今为止,PGC 还不能可靠地诱导进行体外测定。这构成了一个重要的 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 任何一方都无法产生自己的配子。我们假设特定的表观遗传变化驱动 PGC 测定和许可配子发生能力。在此过程中特别令人感兴趣的是监管 转座因子(TE),其中一些仍然能够转座,因此威胁到 种系基因组的完整性。相反,长终端重复 (LTR) 子类港口的一些 TE 转录和多能因子结合位点,并且可以在测定期间发挥作用以调节 多能性网络的表达。了解 LTR 元件的调控如何促进 PGC 我们采用体外小鼠模型来确定。该提案的中心假设是 PGC 决定是依赖于TRIM28的表观遗传转变,TRIM28是一种高度保守的表观遗传支架 蛋白质,用于两个独立的过程:LTR 类转座元件和适当核仁的调节 功能。为了测试这一点,我们将采用 TRIM28 的 PGC 特异性条件敲除模型,使我们能够 询问体内测定。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 TRIM28 改变基因组可及性和增强子动力学,假设 LTR 的错误调节 缺乏 TRIM28 的元件导致无法正确调节基因表达网络中的开关 当 PGC 进入测定阶段时。在目标 2 中,我使用 OligoPaint(一种 DNA-FISH 方法)来评估 TRIM28 损失如何 影响核仁异染色质和形态,并使用离体化学扰动来观察可能的 TRIM28 缺失的表型。这项工作的完成将对我们的理解产生广泛的影响 PGC 表观基因组在决定配子发生许可过程中如何重新连接。这项工作的见解可以 可用于推进体外 PGC 模型向功能性配子发生方向发展。
英文摘要
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.
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