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: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-06 至 2025-01-05
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751859
• 关键词: ATAC-seq; Adult; Automobile Driving; Binding; Binding Sites; Biological; Biological Assay; Cell Differentiation process; Cell Nucleolus; Cell model; Cells; Chemicals; Chromatin; Clinical; Competence; Couples; DNA; DNA Transposable Elements; DNase I hypersensitive sites sequencing; Data; Development; Elements; Embryo; Embryonic Development; Endogenous Retroviruses; Endowment; Enhancers; Epigenetic Process; Event; Failure; Fertility; Gametogenesis; Gene Expression; Genes; Genetic; Genome; Germ Cells; Gonadal Ridge; Gonadal structure; Heterochromatin; Human; In Vitro; Infertility; Knockout Mice; Licensing; Link; Long Terminal Repeats; Maintenance; Maps; Mediating; Modeling; Molecular; Morphology; Mus; NPM1 gene; Phenocopy; Pluripotent Stem Cells; Process; Production; Proliferating; Publishing; RNA Polymerase I; RNA Polymerase Inhibitor; Regulation; Ribosomal RNA; Ribosomes; Role; Scaffolding Protein; Site; Specific qualifier value; Structure; Structure of primordial sex cell; System; Technology; Testing; Time; Transcriptional Silencer Elements; Transposase; Up-Regulation; Work; blastomere structure; cell determination; conditional knockout; embryo cell; epigenome; experimental study; fitness; genome-wide; histone modification; in vivo; insight; interest; migration; mouse model; nucleolin; pluripotency; pluripotency factor; programs; promoter; sex; stem cells; transcription factor; transcriptome sequencing; transcriptomics

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.

项目摘要/摘要： 原始生殖细胞是成体生殖系的胚胎前体，其正常发育 相当于身体健康。在胚胎发育过程中，PGCs经历了两个命运限制步骤：1) 规范，其中原生殖细胞表达多能基因，一种被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)决定，其中多能性计划被终止，原生殖细胞 根据胚胎的性别进行区分。虽然分子研究已经仔细地解剖了PGC 规范中，PGC的测定仍然知之甚少。尽管目前最先进的技术允许体外培养 从多能干细胞(PSC)诱导PGC样细胞(PGCL)，这些PGCL代表特定的 PGC和，到目前为止，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不育提供了可能性 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驱动 PGC测定和许可证配子体形成能力。在这个过程中特别令人感兴趣的是监管 转座元件(TES)，其中一些仍然能够转座，因此威胁到 生殖系基因组的完整性。相反，一些长码头重复(LTR)亚类港口的TES 转录和多能性因子结合位点，并可在测定期间发挥调节作用 多能网络的表达。了解LTR元件的调节如何对PGC起作用 我们采用的是体外小鼠模型。这项提议的中心假设是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：调节LTR类转座元件和适当的核仁 功能。为了测试这一点，我们将使用特定于PGC的条件敲除模型TRIM28，允许我们 在活体内询问测定。在目标1中，我将使用ATAC-seq和CutnTag测序来了解损失 TRIM28的突变改变了基因组的可及性和增强子的动态，假设LTR的错误调节 缺少TRIM28的元件导致无法正确调节基因表达网络中的开关 随着PGCS进入决定期。在目标2中，我使用寡核苷酸，一种DNA-FISH方法来评估TRIM28的丢失 影响核仁异染色质和形态，并用体外化学微扰法观察可能 缺失TRIM28的表型复制。这项工作的完成将对我们理解 PGC表观基因组是如何在决定许可配子发生的过程中重新连接的。从这项工作中获得的见解可以 被用来促进体外PGC模型向功能性配子发生发展。