Iteratively redefining developmental potential through poised enhancers

项目摘要/摘要： 有一个基本的需要了解转录因子如何与它们的共同调节因子一起发挥作用 通过细胞命运的转变来重新定义细胞的发育潜力。我们目前的理解仅限于 少数例子通常基于其上游有转录因子的简单线性通路 协同调节器激活或抑制增强子，增强子又及时调节其同源基因 太空。然而，如果没有更好地了解转录因子和转录因子之间的相互依赖关系， 它们的共同调节因子包括表观遗传酶和协同DNA结合因子，将不可能 预测其中任何一项的操作将如何影响细胞命运和发育潜力。的长期目标是 该实验室将了解细胞命运转变的所有分子控制水平，以便有效地重新编程 细胞到所需的表型。这里的目标是关注转录因子Foxd3，它是必不可少的 以保持各种干细胞的发育潜力。中心假设是使用Foxd3 通过在干细胞中建立稳定的增强子来重新定义细胞的发展潜力 与其协同调节因子和协作性细胞特异性转录因子相关。这一假说是由 从初步数据确定Foxd3作为同时激活和抑制因子的双重功能角色 通过其与多种表观遗传因子的相互作用和调节。因此，它平衡了基因并重新定义了 通过转移到新的增强子位置来研究不同干细胞群体的发展潜力。以下是 具体目标是：1)确定Foxd3、H3K4甲基化、 核小体耗竭和H3K27乙酰化，2)揭示Foxd3运动的机制基础 胚胎干细胞向外胚层细胞的转化，3)鉴定Foxd3在凝集素募集和增强子中的作用 发育基因激活过程中的启动子环。在目标1中，上位性和结构-功能分析将 使用Foxd3及其辅助调节子的突变体来确定 在结合部位建立双功能复合体的因素。在目标2中，翻译后修改和 将评估与其他转录因子的合作，以剖析Foxd3的机制基础 动静。在目标3中，将评估FOXD3的S在凝集素招募和增强子-启动子循环中的作用 采用时间历程实验和上位性实验。这项提议意义重大，因为它将提供新颖的 基因控制的范例对细胞的发展潜力至关重要。这些范例不太可能是 Foxd3所特有的，但反映了茎系转录因子用来保留或 激发干细胞的全部潜能。这些知识将为细胞操作提供更好的设计策略 并提高了针对表观遗传协同调节因子的治疗效果的预测能力。

PROJECT SUMMARY/ABSTRACT: There is a fundamental need to understand how transcription factors function together with their co-regulators to redefine a cell’s developmental potential through cell fate transitions. Our current understanding is limited to a small number of examples often based on simple linear pathways with a transcription factor upstream of its co-regulators either activating or repressing an enhancer, which in turn regulates its cognate gene in time and space. However, without a better understanding of the mutual dependencies between transcription factors and their co-regulators including epigenetic enzymes and collaborative DNA binding factors, it will be impossible to predict how manipulation of either will influence cell fate and developmental potential. The long-term goal of the lab is to understand all levels of molecular control of cell fate transitions in order to efficiently reprogram cells to desired phenotypes. The objective here is to focus on a transcription factor, Foxd3, which is essential to maintain the developmental potential of various stem cells. The central hypothesis is that Foxd3 is used iteratively in stem cells to redefine the cell’s development potential by establishing poised enhancers in association with its co-regulators and collaborative cell specific transcription factors. This hypothesis derives from preliminary data establishing a dual functional role for Foxd3 as a simultaneous activator and repressor through its interaction with and regulation of multiple epigenetic factors. As such it poises genes and redefines the developmental potential of different stem cell populations by moving to new enhancer sites. The following specific aims are proposed: 1) Determine the epistatic relationship between Foxd3, H3K4 methylation, nucleosome depletion, and H3K27 acetylation, 2) Uncover the mechanistic basis for Foxd3 movements during the embryonic stem to epiblast cell transition, 3) Identify role of Foxd3 in cohesin recruitment and enhancer- promoter looping during developmental gene activation. In aim 1, epistasis and structure-function analyses will be performed using mutants of the Foxd3 and its coregulators to determine the interdependencies between the factors in establishing a dual-functional complex at bound sites. In aim 2, post-translational modifications and collaboration with other transcription factors will be evaluated to dissect the mechanistic basis of Foxd3 movements. In aim 3, Foxd3’s role in cohesin recruitment and enhancer-promoter looping will be evaluated using time-course and epistasis experiments. The proposal is highly significant as it will provide novel paradigms of gene control central to a cell’s developmental potential. These paradigms are unlikely to be specific to Foxd3, but rather reflect general strategies used by stemness transcription factors to retain or induce a stem cell’s full potential. Such knowledge will allow for better-designed strategies for cell manipulation and improved ability to predict effects of therapeutics aimed at the epigenetic co-regulators.