Dynamic transcription factor function in control of pluripotent cell sub-states

干细胞有两个决定性的特征；它们可以对称分裂，产生与自身功能相同的细胞，并可以分化成更成熟的细胞类型，执行我们身体的功能，这一过程被称为分化。为了保持干细胞群的功能，自我更新和分化必须平衡。在实验室中生长的最通用的哺乳动物干细胞具有分化成所有成人体细胞的能力，被称为多能干细胞。最近，科学家们发现，不同类型的小鼠多能干细胞可以在实验室中培养；胚胎干细胞（ES）和外胚干细胞（EpiSCs）。此外，我们小组的工作已经确定了胚胎干细胞群体中的分子异质性，这与细胞之间的功能差异有关。具体来说，未分化的胚胎干细胞在具有高或低浓度的特定转录因子（我们将其命名为Nanog）和具有更大或更小的自我更新可能性的状态之间波动。细胞分化倾向的这种波动变化可能对平衡群体中相反的干细胞特性至关重要。因此，了解这些可逆状态是如何被分子控制的，可能会影响到实现可预测的、统一的分化控制的策略，因此具有重要的战略意义。在本研究中，我们将研究多能性基因调控因子在关键调节自我更新效率的靶基因中的功能，以确定多能性群体如何被分离成有效自我更新的细胞和具有更高分化可能性的细胞。我们已经确定了64个基因的一小组，这些基因在响应Nanog活性时改变转录，并且代表了Nanog功能的良好候选介质。我们将询问这64个基因如何促成功能异质性，以及基因调控因子如何控制相应的基因。目的1：检测Nanog敏感基因是否能充分补充Nanog功能。Esrrb是一种重要的Nanog敏感靶点，当它被添加到Nanog被移除的细胞中时，它可以补充Nanog的几种功能，但不能完全补充Nanog的功能。因此，我们将测试额外的候选物在组合分析中完全补偿Nanog损失的能力。Nanog抑制了几个候选基因，因此我们将测试这些候选基因水平的降低是否能够弥补Nanog的损失。目的2：确定多能性基因调控因子的生化功能。我们将确定Nanog波动是如何产生的。我们将在Nanog基因中定位基因调控因子，以确定哪些调控因子控制Nanog，从而潜在地控制功能亚型之间的划分。我们已经发现Nanog蛋白抑制Nanog基因，我们将探究这是如何发生的，以发现是否有简单的规则控制着Nanog如何打开和关闭不同基因。目的3：比较多能性基因调控在体内和在多能性人细胞中的功能。我们将确定在培养中发生的功能补偿是否也发生在小鼠胚胎中。有趣的是，一些候选调节因子可以将EpiSCs重新编程为胚胎干细胞状态。人类胚胎干细胞比小鼠胚胎干细胞更像小鼠EpiSCs，因此我们将测试候选干细胞影响人类胚胎干细胞生长特性的能力。这有利于简化和降低人类胚胎干细胞培养的成本。这项工作将对培养细胞和胚胎中多能性基因调节因子的作用机制提供更深入、更精细的理解。

Stem cells have two defining features; they can divide symmetrically to produce cells functionally identical to themselves and can specialise into the more mature cell types that carry out our bodies' functions, a process called differentiation. To preserve a functional stem cell population, self-renewal and differentiation must be balanced.The most versatile mammalian stem cell that can grow in the lab has the ability to differentiate into all adult body cells and is called a pluripotent stem cell. Recently, scientists have found that different types of mouse pluripotent stem cells can be grown in the lab; embryonic stem (ES) cells and epiblast stem cells (EpiSCs). In addition, work from our group has identified molecular heterogeneity within ES cell populations that is related to functional differences between the cells. Specifically, undifferentiated ES cells fluctuate between states in which they have high or low concentrations of a particular transcription factor, which we have named Nanog, and that have, respectively, a greater or lesser likelihood of self-renewal. This fluctuating alteration in the propensity of cells to differentiate may be crucial to balancing the opposing stem cell properties in a population. Therefore, understanding how these reversible states are controlled molecularly is likely to impinge on strategies for achieving predictable, uniform control of differentiation and is thus strategically important.In this research we will examine the function of pluripotency gene regulators at target genes that critically modulate self-renewal efficiency to determine how the pluripotent population is segregated into cells that self-renew efficiently and cells that have a higher likelihood of differentiation. We have identified a small set of 64 genes that alter transcription in response to Nanog activity and that represent good candidate mediators of Nanog function. We will ask how these 64 genes contribute to functional heterogeneity and how gene regulators control the corresponding genes.Aim 1: Test whether Nanog-sensitive genes can fully complement Nanog function. A prominent Nanog-sensitive target, Esrrb can complement several Nanog functions when added back to cells from which Nanog has been removed but cannot fully complement Nanog function. Therefore, we will test the ability of additional candidates to fully compensate for loss of Nanog in combinatorial assays.Several candidate genes are repressed by Nanog, so we will test the ability of reduction in the level of these candidates to compensate for loss of Nanog. Aim 2: Determine the biochemical function of pluripotency gene regulators.We will determine how Nanog fluctuations arise. We will localise gene regulators across the Nanog gene to determine which of these control Nanog and therefore potentially control partitioning between functional subtypes. We have found that Nanog protein represses the Nanog gene and we will ask how this happens to find out if simple rules govern how Nanog switches different genes on and off. Aim 3: Compare pluripotency gene regulator function in vivo and in pluripotent human cells.We will determine whether functional compensations occuring in culture also occur in the mouse embryo. Interestingly, some candidate regulators can reprogramme EpiSCs to an ES cell state. Human ES cells are more like mouse EpiSCs than mouse ES cells, so we will test the ability of our candidates to influence the growth properties of human ES cells. This could beneficially simplify and reduce the cost of human ES cell culture. This work will deliver a deeper, more refined understanding of the mechanisms of action of pluripotency gene regulators in cells in culture and in the embryo.