Wnt/Beta-catenin and Tcf/Lef factor regulation of pluripotency

描述(申请人提供)：以前的突破性发现使体外培养多能胚胎干细胞(ES)成为可能，并将成人细胞重新编程为多能状态。这些进展为再生医学打开了新的机会，为我们提供了细胞起点，从那里我们可以获得具有生理特性的成人细胞类型，用于研究和治疗人类疾病。获得适合于直接培养的细胞类型 治疗应用或作为用于药物开发的人类疾病的细胞模型，都需要与体内细胞对应物类似的实质性水平。大多数情况下，研究人员试图通过胚胎发生复制细胞发育过程的关键方面，从而获得最好的体内模拟。考虑到表观遗传效应通过胚胎发生是累积和渐进的，这一建议的一个中心假设是，谱系指定的初始步骤是关键的，在退出多能性过程中所犯的表观遗传错误可能会产生不良影响，即使在细胞达到靶向分化状态之后也是如此。为了解决这个问题，我们的目标是阐明从谱系特定的细胞类型中消除或“退役”ES细胞特异性基因调控元件(即增强子)的机制。我们发现，一种序列特异的DNA结合转录因子Tcf7l1与ES细胞中的增强子特异结合，这些增强子是退役的目标。鉴于我们之前的研究结果显示Tcf7l1刺激ES细胞分化，我们建议检验Tcf7l1是使ES特异性增强子退役所必需的假设。该提案将进一步确定Tcf7l1的这种效应如何改变细胞对细胞外信号的反应，并使用已建立的体外分化方案来衡量它对真正的谱系指定的重要性。完成这项拟议的研究的直接结果将是更好地理解细胞如何退出多能性状态，并展示早期步骤中的缺陷如何降低后续阶段分化的效率。最后，考虑到可获得的ES特异性增强剂在成人细胞中的致癌潜力，应认识到拟议工作对提高基于细胞的治疗的安全性的重要性。

DESCRIPTION (provided by applicant): Previous groundbreaking discoveries have made it possible to grow pluripotent embryonic stem (ES) cells in vitro and to reprogram adult cells back to a pluripotent state. These advances have opened new opportunities for regenerative medicine by providing the cellular starting point from which we can derive adult cell types with physiological properties to study and treat human disease. Attaining cell types suitable for direct therapeutic application or as cellular models of human disease for drug development both require a substantial level of similarly to the in vivo cellular counterpart. Most often, investigaors attempt to attain the best in vivo mimics by replicating key aspects of a cell's developmental progression through embryogenesis. Given the appreciation that epigenetic effects are cumulative and progressive through embryogenesis, a central hypothesis of this proposal is that the initial steps in lineage specification are critical, and epigenetic mistakes made during exit from pluripotency can have undesirable effects even after cells have reached the targeted differentiated state. To address this problem, we aim to elucidate the mechanisms that eliminate or "decommission" ES cell- specific gene regulatory elements (i.e. enhancers) from lineage specified cell types. We discovered that a sequence-specific DNA-binding transcription factor, Tcf7l1, binds specifically to enhancers in ES cells that are targeted for decommissioning. Given our previous findings that showed Tcf7l1 stimulated differentiation of ES cells, we propose testing the hypothesis that Tcf7l1 is necessary to decommission ES specific enhancers. The proposal will further determine how this effect of Tcf7l1 changes the responses of cells to extracellular signals, and measure how important it is for genuine lineage specification using established in vitro differentiation protocols. The immediate outcome of completing the proposed research will be a better understanding of how cells exit the pluripotent state, and a demonstration of how defects during early steps can diminish the efficiency of subsequent stages of differentiation. Finally, given the oncogenic potential of accessible ES- specific enhancers in adult cells, the importance of the proposed work for increasing the safety of cell-based therapies should be appreciated.