Iteratively redefining developmental potential through poised enhancers

项目总结/摘要： 有一个基本的需要了解如何转录因子的功能与他们的共同调节 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 少数例子通常基于简单的线性途径，其上游有转录因子， 辅助调节因子激活或抑制增强子，增强子又及时调节其同源基因， 空间然而，如果没有更好地理解转录因子之间的相互依赖性， 它们的协同调节因子包括表观遗传酶和协同DNA结合因子， 预测操纵任何一个将如何影响细胞命运和发育潜力。的长期目标 该实验室旨在了解细胞命运转换的所有分子控制水平，以便有效地重新编程 将细胞转化为所需的表型。这里的目标是关注转录因子Foxd 3，这是必不可少的。 来维持各种干细胞的发育潜能。核心假设是使用Foxd 3 迭代地在干细胞中，通过建立平衡的增强子来重新定义细胞的发育潜力， 与其共调节因子和协同细胞特异性转录因子的关联。这一假设源于 根据初步数据，确定Foxd 3作为同时激活剂和抑制剂的双重功能作用， 通过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，重新定义了 通过移动到新的增强子位点来增强不同干细胞群体的发育潜力。以下 提出了具体的目的：1）确定Foxd 3，H3 K4甲基化， 核小体耗竭和H3 K27乙酰化，2）揭示Foxd 3在细胞内运动的机制基础。 胚胎干细胞向上胚层细胞的转变，3）确定Foxd 3在粘附素募集和增强子中的作用， 在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Foxd 3及其共调节子的突变体进行，以确定 在结合位点建立双功能复合物的因素。在目标2中，翻译后修饰和 与其他转录因子的合作将被评估，以剖析Foxd 3的机制基础 动作在目标3中，将评估Foxd 3在粘附素募集和增强子-启动子成环中的作用 采用时程和上位性实验。该提案非常重要，因为它将提供新颖的 基因控制的范例对细胞的发育潜力至关重要。这些范例不太可能是 特异于Foxd 3，而是反映了茎型转录因子用于保留或 激发干细胞的全部潜能这些知识将允许更好地设计细胞操作策略 和提高的预测针对表观遗传辅助调节因子的治疗效果的能力。

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.