Transcriptional and Epigenetic Control of Pluripotency and Self-Renewal by Honey Bee Royalactin and its human structural analog

目前人类许多先天性和获得性疾病的治疗选择存在局限性，包括 出生缺陷，癌症，退行性疾病和糖尿病，突出了开发新的 再生医学的方法，以显着改善组织修复。哺乳动物的多能性 胚胎干细胞（ESC）使其成为研究早期发育方面的方便模型， 这是为再生医学获得许多治疗相关细胞的宝贵起点。 尽管在破译驱动ESC多能性的机制方面取得了显着进展， 人类胚胎干细胞（hESC）是如何调控多能状态的。如果我们要 使用hESC作为胚胎发育的高保真模型，如果我们希望改善hESC的结果， 分化和细胞重编程对多能性的保真度，那么我们必须了解 hESC如何适应哺乳动物胚胎发育的模式。在这个项目中，我们试图回答一些 通过表征和验证一种潜在的替代性多能性状态来解决这些基本问题。我们 范式转换假说源于我们的意外发现，即蜜蜂蜂王制造蛋白， Royalactin及其在哺乳动物中的结构类似物Regina具有意想不到的强大多能性维持能力 对哺乳动物干细胞的影响。我们假设Regina/Royalactin稳定并捕获了一个关键的 多能状态不同于现有的植入前（幼稚）和植入后（致敏）相关的干细胞状态。 我在这里概述了一个计划，从分子上表征这种新的细胞亚稳态，有3个具体目标。在Aim中 1，我们将分离和表征ESCs中受体复合物的组成和活性。我们 假设Regina/Royalactin作为分泌分子，可能直接与受体伴侣相互作用， 反应细胞的膜，以影响基因表达和随后的细胞行为。我们将确定 通过多个高通量正向遗传学和蛋白质组学策略来研究受体。在目标2中，我们将导出 并维持鼠和人ESC以在功能上证明Regina/Royalactin介导的 多能性可以追溯到参与谱系特化和维持的信号通路， 胚胎本身。建立一个新的哺乳动物多能性的独特阶段将是一个重要的进展 我们对早期血统承诺的理解最后，在目标3中，我们将阐明和描述关键的 里贾纳/Royalactin和下游表观遗传和转录组事件之间的接口机制。 全基因组分析将与当前确定的条件进行比较，以确定是否存在遗传和 存在与发育潜能受损相关的ESC的表观遗传不稳定性。总的来说，我们的 数据将直接确定一种新的内源性哺乳动物多能性因子如何在哺乳动物中引发命运决定， ESCs，并提供了一个新的平台，研究的原则，管理细胞的潜力，表观遗传调控， 调节幼稚多能干细胞发育过程的机制。

Limitations in current treatment options for many congenital and acquired diseases in humans, including birth defects, cancer, degenerative disorders and diabetes, highlight the need for the development of novel approaches for regenerative medicine to dramatically improve tissue repair. The pluripotent nature of mammalian embryonic stem cells (ESCs) makes them a convenient model for studying aspects of early development and an invaluable starting point for deriving numerous therapeutically relevant cells for regenerative medicine. Despite the remarkable progress made in deciphering mechanisms driving ESC pluripotency, fundamental gaps remain in understanding how human embryonic stem cells (hESCs) regulate the pluripotent state. If we are to use hESCs as a high-fidelity model for embryonic development, and if we wish to improve outcomes of hESC differentiation and the fidelity of cellular reprogramming to pluripotency, then it is imperative that we understand how hESCs fit into the paradigm of mammalian embryonic development. In this project we seek to answer some of these fundamental questions, by characterizing and validating a potential alternative pluripotency state. Our paradigm-shifting hypothesis stems from our unexpected discovery that the honey bee queen-maker protein, Royalactin, and its structural analog in mammals, Regina, have unexpected robust pluripotency maintenance effects in mammalian stem cells. We hypothesize that Regina/Royalactin stabilize and capture a pivotal pluripotent state distinct from the existing pre- (naïve) and post- (primed) implantation associated stem cell states. I outline here a plan to molecularly characterize this novel cellular metastable state with 3 specific aims. In Aim 1, we will Isolate and characterize the composition and activity of the receptor complex(es) in ESCs. We hypothesize that Regina/Royalactin, as secreted molecules, likely directly interact with a receptor partner on the membrane of responsive cells to affect gene expression and subsequent cellular behavior. We will identify the receptor(s) through multiple high throughput forward genetics and proteomic strategies. In Aim 2, we will derive and maintain murine and human ESCs to functionally demonstrate that the Regina/Royalactin-mediated state of pluripotency can be related back to the signaling pathways involved in lineage specification and maintenance in the embryo itself. Establishment of a new distinct stage of mammalian pluripotency will be an important advance in our understanding of early lineage commitment. Lastly, in Aim 3 we will elucidate and characterize the critical mechanisms that interface between Regina/Royalactin and downstream epigenetic and transcriptomic events. The genome-wide analyses will be compared to current established conditions to determine whether genetic and epigenetic instability of the ESCs, associated with impaired developmental potential, exists. Taken together, our data will directly establish how a novel endogenous mammalian pluripotency factor instigates fate decisions in ESCs, and provide a new platform to study the principles governing cell potency, epigenetic regulation, and the mechanisms that regulate developmental processes in naïve pluripotent stem cells.