Defining Novel Molecular Pathways to Totipotency

项目摘要 全能性，一个显着的细胞可塑性的一个细胞在产生一个完整的有机体与 胚胎和胚外的贡献，是多细胞和发展必不可少的。在小鼠中，完全 全能细胞短暂存在于受精卵和卵裂期卵裂球中，尽管全能2-细胞（2C）- 类细胞（2CLC）也可以在培养的胚胎干细胞（ESCs）中零星产生或遗传诱导产生。 它们是多能性的，只能形成胚胎谱系。最近，扩大潜在的干细胞和 衍生了具有全能性特征的扩展多能干细胞，统称为EPSC， 使用两组不同的小分子抑制剂在体外稳定培养。然而，2CLC和两个EPSC 细胞系在培养条件、转录组和核心多能性因子的表达方面存在差异，提示 替代全能国家。2CLC和EPSC是容易获得的细胞来源，在体外具有价值 理解全能性的模型，这方面的知识对于捕获和稳定 全能2CLC状态，这是目前不可能的。定义这些不同的分子途径 全能细胞将有助于解开全能性的复杂调节机制， 全能2CLC的捕获/稳定。据报道，全能2CLC重新激活转录， 内源性逆转录病毒（ERV），特别是MERVL，其激活现已被发现是致病性的 在我们的初步研究中获得全能性。ZSCAN 4在2C期胚胎中也有明显表达， 从ESC状态向2CLC退出的那些零星ESC。通过表征MERVL/ZSCAN 4双 报告基因阳性（DR+/+）ESCs，我们已经开始剖析2CLC的分子途径 全能性和发现miR-344及其直接靶ZMYM 2作为新的正和负调节剂， 分别用于MERVL活化和2CLC全能性。本申请的目的是扩展我们的 了解ESC培养中不同的细胞潜能状态，并定义MERVL对 全能性和探索全能性的转录和转录后机制。我们 假设2CLC代表一种由新分子轴调节的替代全能状态 包括转录和转录后机制。我们建议进行以下研究， 验证我们的假设1)定义EPSC和2CLC中替代全能状态的分子路线图。（二） 确定MERVL元件对2CLC全能性的功能贡献。我们将定义和区分 单个MERVL元件在促进2CLC全能性中的功能作用。3)剖析分子 通过将miR-344建立为理解的新范式，2CLC全能性的潜在机制 哺乳动物全能性了解驱动和诱导全能特征的分子机制， 体外对于理解如何实现最大程度的细胞可塑性至关重要， 维持，从而为有效的重编程和潜在的治疗途径提供更多的选择。

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.