Dissecting mechanisms of pioneer transcription factor-mediated lineage reprogramming

项目总结/摘要 将丰富且可获得的细胞重新编程为治疗上有用的细胞类型具有很大的希望 用于再生医学细胞重编程可以通过异位表达转录来实现 这些因子（TF）直接将一种分化的细胞类型转化为另一种，绕过胚胎状态， 试图提高靶细胞生产的速度和效率。许多不同的细胞类型已经被 通过这种“直接谱系重编程”方法产生，但它们的实际效用受到限制， 在大多数方案中，只有一小部分细胞被成功转化为目标细胞类型。即便如此， 由此产生的种群通常是部分分化的或不完全特定的。大多数细胞工程 方法需要使用至少一个先锋因子，这是一类独特的TF，能够访问它们的 沉默染色质中的结合位点。先驱TF有能力在一个环境中传授血统能力- 他们在发展中发挥核心作用，这反映在他们在不同的 发展方案。我们的长期目标是了解先锋因子介导的直接 谱系重编程在这个建议中，我们采用原型先驱TF，FoxA家族， 成纤维细胞转化为内胚层祖细胞样（iEP）状态，代表直接谱系的范例 重新编程根据我们的初步结果和目前的证据，我们假设，在直接 在谱系重编程中，先驱TF重新参与发育GRNs，这取决于细胞的染色质状态。 它们被引入的细胞。我们的三个独立但相关的目标是为了理解： (Aim 1）转录变化的性质在重编程过程中从它们的起源和它们与 （目的2）FoxA先驱TF及其辅因子Hnf 4a及其 活性，以驱动命运的变化;（目的3）染色质背景的影响，对目标的可及性先锋TF 以及这如何影响重编程的效率和所产生的细胞的潜力。在这里，我们应用一个 创新的单细胞谱系追踪方法和基因组技术记录TF在细胞中的结合历史 正在重新编程总之，这将产生一个前所未有的数字量化的 重编程过程，并将揭示细胞身份成功转换的障碍。一种改进 了解先驱介导的重编程机制将有助于提高转化效率 和保真度在一系列的重编程策略，并提高知识的行动，这一重要的 一类转录调节因子。

PROJECT SUMMARY/ABSTRACT The reprogramming of abundant and accessible cells into therapeutically useful cell types holds great promise for regenerative medicine. Cellular reprogramming can be achieved by ectopically expressing transcription factors (TFs) that directly convert one differentiated cell type into another, bypassing embryonic states in an attempt to boost the speed and efficiency of target cell production. A number of different cell types have been generated by such “direct lineage reprogramming” methods, but their practical utility has been limited because, in most protocols, only a small percentage of cells are successfully converted to the target cell type. Even then, the resulting populations are often partially differentiated or incompletely specified. Most cell engineering methods require the use of at least one pioneer factor, a unique class of TFs that are able to access their binding sites in silent chromatin. Pioneer TFs have the capacity to impart lineage competence in a context- specific manner, and play central roles in development, as reflected by their redeployment across disparate developmental programs. Our long-term goal is to understand the mechanism of pioneer factor-mediated direct lineage reprogramming. In this proposal we employ prototypical pioneer TFs, the FoxA family, to drive conversion of fibroblasts to an endoderm progenitor-like (iEP) state, representing a paradigm for direct lineage reprogramming. Based on our preliminary results and current evidence, we hypothesize that during direct lineage reprogramming, pioneer TFs re-engage developmental GRNs, depending on the chromatin state of the cells into which they are introduced. Our three independent yet related aims are directed at understanding: (Aim 1) the nature of transcriptional changes during reprogramming from their origin and their relation to developmental programs; (Aim 2) the direct targets of FoxA pioneer TFs and their cofactor, Hnf4a and their activity to drive fate change; (Aim 3) the influence of chromatin context on target accessibility of pioneer TFs and how this impacts efficiency of reprogramming and the potential of cells generated. Here we apply an innovative single-cell lineage tracing methodology and genomic technology to record TF binding history in cells undergoing reprogramming. Together, this will generate an unprecedented digital quantification of the reprogramming process, and will reveal barriers to the successful conversion of cell identity. An improved understanding of pioneer-mediated reprogramming mechanisms will facilitate enhanced conversion efficiency and fidelity across an array of reprogramming strategies, and improve knowledge of the action of this important class of transcriptional regulators.