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

描述（由申请人提供）：以前的突破性发现已经使体外培养多能胚胎干（ES）细胞和将成体细胞重编程回到多能状态成为可能。这些进展为再生医学开辟了新的机会，提供了细胞起点，我们可以从中获得具有生理特性的成体细胞类型，以研究和治疗人类疾病。获得适合直接 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相当水平的相似性。大多数情况下，研究者试图通过复制胚胎发生过程中细胞发育进程的关键方面来获得最佳的体内模拟物。考虑到表观遗传效应是通过胚胎发生累积和进行的，该提议的中心假设是谱系特化的初始步骤是关键的，并且在退出多能性期间所犯的表观遗传错误即使在细胞已经达到靶向分化状态之后也可能具有不期望的影响。为了解决这个问题，我们的目标是阐明消除或“退役”ES细胞特异性基因调控元件（即增强子）从谱系指定的细胞类型的机制。我们发现，序列特异性DNA结合转录因子Tcf7l1，特异性结合到ES细胞中的增强子，这些细胞被靶向退役。鉴于我们以前的研究结果表明，Tcf7l1刺激ES细胞的分化，我们建议测试的假设，Tcf7l1是必要的退役ES特异性增强子。该提案将进一步确定Tcf7l1的这种作用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谱系规范的重要性。完成拟议研究的直接结果将是更好地了解细胞如何退出多能状态，并证明早期步骤中的缺陷如何降低后续分化阶段的效率。最后，考虑到可接近的ES特异性增强子在成体细胞中的致癌潜力，应该理解所提出的工作对于增加基于细胞的疗法的安全性的重要性。

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.