Transcription factor control of dynamic transitions within and beyond pluripotency

细胞身份取决于转录因子和环境信号的作用，它们共同读取基因组。细胞身份在发育过程中改变的机制是基础发育和干细胞生物学的特别兴趣。特别是多能细胞，它既能自我更新，又能分化，在体外和体内产生所有谱系，是研究细胞身份维持和变化的理想选择。这些知识的应用对于设计强大的体外分化方案至关重要，该方案用于细胞移植和药物发现，而非生殖系肿瘤起始细胞。多能干细胞的自我更新是由以转录因子OCT4、SOX2和NANOG为中心的多能基因调控网络控制的。虽然在鉴定额外的多能性基因调控网络成分和分析转录因子的整体染色质结合方面取得了相当大的进展，但这些强大的方法并没有告诉我们细胞是如何做出决定的。为了更好地理解自我更新和分化之间的区别，我们将使用精确的机制分析来确定单个转录因子如何影响多能性基因调控网络的运作。多能细胞在分化过程中经历连续的状态。生殖系和体细胞系的分离发生于过渡性的“形成性”多能状态。一个关键的未解决的问题是为什么许多多能转录因子（包括OCT4， SOX2和NANOG）也在种系基因调控网络中起作用，并且对原始生殖细胞（PGC）功能至关重要。由于这些细胞类型可以说具有最根本的不同的潜能，这是自相矛盾的。通过研究OCT4、SOX2和NANOG，以及在从naïve多能胚胎干细胞（ESCs）到形成性上皮样细胞（EpiLCs）和从EpiLCs到PGCs转变的不同阶段的细胞中进行研究，我们旨在解决这一悖论，并揭示表型细胞身份的基础原理。我们的研究将把小鼠的知识扩展到人类的种系进入。这一建议有三个目的，重点关注多能细胞功能的不同方面：多能性在体内是如何被指定和丧失的？转录因子在体外naïve多能性的维持和丧失中是如何起作用的？哪些调控相互作用决定了进入生殖系的效率？通过评估转录因子如何作为一个整体来控制ESC身份，以及揭示转录因子如何在不同的发育阶段被重新利用，实现上述目标将推动该领域的发展。这将通过确定细胞退出特定多能状态的关键机制，特别是当它们进入种系或开始进入原始条纹时，开辟新的领域。总之，这些知识将为严格控制多能细胞的统一分化提供所需的见解，这是未来再生医学策略接受者所要求的。

Cell identity depends on the action of transcription factors and environmental signals that together read the genome. The mechanisms by which cell identity changes during development are of particular interest to fundamental developmental and stem cell biology. In particular, pluripotent cells, which can both self-renew and differentiate to give rise to all lineages in vitro and in vivo, are ideally suited to studying maintenance and changes in cell identity. Application of such knowledge is crucial to the design of robust protocols for in vitro differentiation of cells free from germline tumour-initiating cells for use in cell transplantation and drug discovery. Pluripotent stem cell self-renewal is governed by a pluripotency gene regulatory network centred on the transcription factors OCT4, SOX2 and NANOG. While considerable advances have been made in identifying additional pluripotency gene regulatory network components and in analysing global chromatin binding by transcription factors, these powerful approaches do not tell us how cell decisions are actually made. To better understand the distinction between self-renewal and differentiation we will use precise mechanistic analysis to determine how individual transcription factors affect the operation of the pluripotency gene regulatory network.Pluripotent cells pass through successive states during differentiation. Segregation of germline and somatic lineages occurs from a transitional 'formative' pluripotent state. A key unanswered question is why many pluripotency transcription factors (including OCT4, SOX2 and NANOG) also operate in the germline gene regulatory network and are critical for primordial germ cell (PGC) function. As these cell types have arguably the most radically divergent potencies, this is paradoxical. By studying OCT4, SOX2 and NANOG and by doing so in cells at distinct stages during the transition from naïve pluripotent embryonic stem cells (ESCs) to formative epiblast-like cells (EpiLCs) and from EpiLCs to PGCs, we aim to resolve this paradox and to reveal principles at the very foundation of phenotypic cell identity. Our studies will extend knowledge from the mouse to address germline entry in humans. This proposal has three aims, focused on distinct aspects of pluripotent cell function:1. How is pluripotency specified and lost in vivo?2. How do transcription factors act in the maintenance and loss of naïve pluripotency in vitro?3. What regulatory interactions determine the efficiency of entry to the germline?Delivering the above aims will advance the field by assessing how transcription factors operate as an ensemble to control ESC identity, and by revealing how transcription factors are repurposed at distinct developmental stages. This will break new ground by identifying key mechanisms by which cells exit specific pluripotent states, particularly as they enter the germline, or initiate entry into the primitive streak. Together, this knowledge will provide the insights needed to rigorously command the uniform differentiation of pluripotent cells demanded by future recipients of regenerative medicine strategies.