Defining Novel Molecular Pathways to Totipotency

定义全能性的新分子途径

• 批准号: 9982114
• 负责人: Jianlong Wang
• 金额: $ 33.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982114
• 关键词: ATAC-seq; Animal Experimentation; Binding; CCL3 gene; Cell Culture Techniques; Cells; Chromatin; Code; Complex; Conceptus; Data; Development; Elements; Embryo; Embryonic Development; Endogenous Retroviruses; Generations; Genes; Genetic Transcription; In Vitro; Individual; Knowledge; Length; Messenger RNA; MicroRNAs; Molecular; Mus; Organ; Organism; Pathway interactions; Pluripotent Stem Cells; Production; Proteins; Proteome; Proteomics; RNA; Regulator Genes; Regulatory Element; Reporter; Reporting; Role; Source; Suggestion; Testing; Therapeutic; Time; Totipotency; Totipotent; Totipotent cell; Transplantation; Untranslated RNA; base; embryo cell; embryo stage 2; embryonic stem cell; gag-pol Fusion Proteins; in vitro Model; in vivo; novel; pluripotency; self-renewal; small molecule inhibitor; stem; stem cells; transcriptome; zygote

PROJECT SUMMARY Totipotency, a remarkable cellular plasticity of a single cell in generating a complete organism with both embryonic and extraembryonic contribution, is essential for multicellularity and development. In mice, fully totipotent cells exist transiently in the zygote and cleavage-stage blastomeres, although totipotent 2-cell (2C)- like cells (2CLCs) can also arise sporadically or induced genetically in cultured embryonic stem cell (ESCs) that are pluripotent and can only contribute to embryonic lineages. Recently, expanded potential stem cells and extended pluripotent stem cells, collectively known as EPSCs, with totipotency features were derived and stably cultured in vitro using two distinct sets of small molecule inhibitors. However, 2CLCs and the two EPSC lines differ in their culture conditions, transcriptomes, and expression of core pluripotency factors, suggestive of alternative totipotent states. 2CLCs and EPSCs represent easily accessible cell source and valuable in vitro models for understanding totipotency, the knowledge of which will be critical to capture and stabilize the totipotent 2CLC state, which is not currently possible. Defining molecular pathways underlying these various totipotent cells will facilitate unraveling the complex regulatory mechanisms of totipotency and achieving the capture/stabilization of totipotent 2CLCs. The totipotent 2CLCs were reported to reactivate transcription of endogenous retroviruses (ERVs), in particular MERVL, whose activation has now been found to be causative for totipotency in our preliminary studies. ZSCAN4 is also sharply expressed in 2C-stage embryo and marks those sporadic ESCs exiting from the ESC state towards 2CLCs. By characterizing MERVL/ZSCAN4 double reporter positive (DR+/+) ESCs, we have begun to dissect the molecular pathways underlying 2CLC totipotency and discovered miR-344 and its direct target ZMYM2 as novel positive and negative regulators, respectively, for MERVL activation and 2CLC totipotency. The objective of this application is to extend our knowledge of the divergent cellular potency states in ESC culture, and define the MERVL contribution to totipotency and explore transcriptional and post-transcriptional mechanisms underlying totipotency. We hypothesize that 2CLCs represent an alternative totipotent state that is regulated by a novel molecular axis encompassing the transcriptional and post-transcriptional mechanisms. We propose the following studies to test our hypothesis. 1) Define a molecular roadmap for alternative totipotent states in EPSCs and 2CLCs. 2) Establish the functional contribution of MERVL elements to 2CLC totipotency. We will define and distinguish functional roles of individual MERVL elements in contributing to 2CLC totipotency. 3) Dissect the molecular mechanism underlying 2CLC totipotency by establishing miR-344 as a new paradigm for understanding mammalian totipotency. Understanding the molecular mechanisms that drive and induce totipotent features in vitro is essential to understanding of how a maximum degree of cellular plasticity can be achieved and maintained, thereby providing more options for efficient reprogramming and potential therapeutic avenues.

项目摘要 全能量，单个细胞的显着细胞可塑性在产生一个完整的生物体中 胚胎和外囊外贡献对于多细胞性和发展至关重要。在老鼠中，完全 整数细胞在合子和裂解阶段胚胎中瞬时存在，尽管全能2细胞（2C） - 像细胞（2CLC）一样，也可以在培养的胚胎干细胞（ESC）中偶发地或遗传诱导。 具有多能，只能导致胚胎谱系。最近，扩展了潜在的干细胞和 扩展的多能干细胞（统称为EPSC）具有能力特征，并得出 使用两组不同的小分子抑制剂在体外稳定培养。但是，2CLC和两个EPSC 线的培养条件，转录组和核心多能因素的表达有所不同，暗示了 替代宗旨状态。 2CLC和EPSC代表易于访问的细胞源，体外有价值 理解能力的模型，其知识对于捕获和稳定至关重要 全能2CLC状态，目前是不可能的。定义分子途径，这些途径 全能细胞将有助于阐明整数的复杂调节机制并实现 捕获/稳定2CLC。据报道，全能2CLC重新激活 内源性逆转录病毒（ERV），尤其是Mervl，现在发现其激活是病因 在我们的初步研究中进行全能。 ZSCAN4在2C阶段的胚胎和标记中也迅速表达 那些零星的ESC从ESC州向2CLC退出。通过表征mervl/zscan4 double 记者正（DR+/+）ESC，我们开始剖析2Clc的分子途径 全能量并发现miR-344及其直接靶标ZMym2是新型正和负调节剂， 分别用于MERVL激活和2CLC的能力。此应用的目的是扩展我们 对ESC培养中分歧的细胞效能状态的了解，并定义了MERVL的贡献 全能和转录后的转录和转录后机制。我们 假设2Clcs代表了由新分子轴调节的替代性整合状态 涵盖转录和转录后机制。我们建议以下研究 检验我们的假设。 1）定义EPSC和2CLC中替代替代状态的分子路线图。 2） 建立MERVL元素对2CLC计数的功能贡献。我们将定义和区分 单个MERVL元素在有助于2CLC能力中的功能作用。 3）剖析分子 通过建立miR-344作为一种新的理解范式，通过建立miR-344来理解的机制 哺乳动物的能力。了解在 体外对于了解如何达到最大程度的细胞可塑性至关重要 维护，从而提供了更多的选择，以有效地重编程和潜在的治疗途径。