Dissecting mechanisms of pioneer transcription factor-mediated lineage reprogramming

项目摘要/摘要 将丰富和可获得的细胞重新编程为治疗有用的细胞类型是很有希望的 用于再生医学。细胞重新编程可以通过异位表达转录来实现 直接将一种分化的细胞类型转化为另一种细胞类型的因子(TF)，绕过胚胎状态 尝试提高目标细胞生产的速度和效率。已经有许多不同类型的细胞 由这种“直接谱系重新编程”方法产生，但它们的实际用途受到限制， 在大多数协议中，只有一小部分信元被成功转换为目标信元类型。即便如此， 由此产生的种群通常是部分分化的或不完全指定的。大多数细胞工程 方法需要使用至少一个先锋因素，即能够访问其 沉默染色质中的结合部位。先锋TF有能力在特定背景下传授血统能力- 具体方式，并在发展中发挥核心作用，这体现在它们在不同的 发展计划。我们的长期目标是了解先锋因子介导的直接作用机制 世系重编。在这个提案中，我们雇佣了典型的先驱TF，FoxA家族，来驱动 成纤维细胞向内胚祖细胞样状态的转化，代表了一种直接血统的范例 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 谱系重新编程，先驱TF重新接合发育中的GRN，这取决于 它们被引入的细胞。我们的三个独立但又相互关联的目标是为了理解： (目标1)重新编程过程中转录变化的性质及其与来源的关系 发展计划；(目标2)FoxA先锋因子及其辅因子HNF4a和它们的直接靶点 推动命运改变的活动；(目标3)染色质环境对先锋TF靶标可及性的影响 以及这如何影响重新编程的效率和产生的电池的潜力。在这里，我们应用一个 记录细胞内转铁蛋白结合历史的创新单细胞谱系追踪方法和基因组技术 正在进行重新编程。总而言之，这将产生前所未有的数字量化 重新编程过程，并将揭示成功转换细胞身份的障碍。一种改进的 了解先驱介导的重新编程机制将有助于提高转换效率 和保真度，跨越一系列重新编程战略，并提高对这一重要行动的认识 转录调节器的一类。

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.