H3.3-mediated epigenetic regulation of developmental bivalent genes for reprogramming and differentiation

项目摘要：将体细胞重新编程为多能干细胞 干细胞疗法大有可为，但主要障碍之一在于 确定哪些干细胞是真正的多能干细胞。我们已经证明了诱导多能性 来自小鼠的干细胞(IPSCs)可能具有明显不同的生成“ALL-iPS”的潜力 动物，即使这些细胞有相似的转录图谱。这种差异表明 表观遗传机制在调节干细胞多能性中的重要作用。什么时候 发育二价(DB)基因在谱系承诺中被激活，它们被沉默，但 为以后激活多能干细胞做好准备。我们目前无法对此进行调查 必要的平衡状态，因为ipscs的转录图谱不能揭示 转录激活。我们之前报道过H3.3是建立多能性所必需的 在重新编程过程中，并需要维持胚胎干细胞中DB基因的二价性。事实上, 我们的初步数据显示，H3.3富含在许多DB基因的启动子上，其 缺乏富集物与IPSCs的发展潜力受损密切相关。这 研究结果表明，H3.3在调控DB基因表达中起着关键作用，因此 多能性。根据我们的观察，我们假设需要H3.3来建立 在重编程过程中的二价性，以及启动子上的这种表观遗传特征平衡Db 分化后激活的基因。我们的长期目标是阐明 干细胞多能性的建立和维持机制。这样做的目的是 建议定义组蛋白变异体H3.3调节DB基因的机制 以及干细胞的发育特性。我们计划使用UNIQUE来检验这个假设 动物模型将允许我们在任何中间阶段获得足够的基因一致的细胞 舞台。这将使我们能够使用芯片测序来研究H3.3和组蛋白修饰 在重新编程期间。我们在本申请中提出了以下两个目标：目标1：识别 H3.3如何调控Db基因表观遗传特征的建立 重新编程以实现多能性。目的2：测定H3.3富集素的功能 Db基因在IPSC分化过程中的启动子。这些目标的成功实现将 使我们能够确定DB基因启动子上H3.3的浓缩如何影响 干细胞分化为特定细胞系的可能性。这项工作将提供 关于多能性的表观遗传调控的功能相关性的关键信息，以及 评估干细胞多能性的可能临床方法。

PROJECT SUMMARY: Reprogramming somatic cells to become pluripotent stem cells holds great promise for stem-cell-based therapeutics, but one of the major barriers lies in the ability to identify which of the stem cells are truly pluripotent. We have shown that induced pluripotent stem cells (iPSCs) from mice can have markedly different potential to generate “all-iPS” animals, even when the cells have similar transcription profiles. This difference suggests an essential role for epigenetic mechanisms in regulating stem cell pluripotency. When developmental bivalent (DB) genes are activated in lineage commitment, they are silenced but poised for later activation in pluripotent stem cells. We currently have no way to probe for this necessary poised state, as transcriptional profiles of iPSCs do not reveal potential for transcriptional activation. We previously reported that H3.3 is required to establish pluripotency during reprogramming and is required to maintain the bivalency of DB genes in ESCs. In fact, our preliminary data showed that H3.3 is enriched at the promoter of many DB genes, and its lack of enrichment correlates tightly with compromised developmental potential in iPSCs. This finding indicates that H3.3 plays a critical role in regulating DB gene expression, and thus pluripotency. Based on our observations, we hypothesize that H3.3 is required to establish bivalency during reprogramming and that this epigenetic signature at the promoter poises DB genes for later activation upon differentiation. Our long-term goal is to elucidate the key mechanisms of establishing and maintaining pluripotency in stem cells. The objective of this proposal is to define the mechanisms by which the histone variant H3.3 regulates the DB genes and the developmental properties of stem cells. We plan to test the hypothesis using unique animal models that will permit us to obtain enough genetically uniform cells at any intermediate stage. This will allow us to study both H3.3 and histone modifications using ChIP sequencing during reprogramming. We propose the following two aims in this application: Aim 1: Identify how H3.3 regulates the establishment of epigenetic signatures in DB genes during reprogramming toward pluripotency. Aim 2: Determine the function of H3.3 enrichment mark at the promoter in DB genes during iPSC differentiation. Successful completion of these aims will allow us to identify how the enrichment of H3.3 at the promoter for DB genes impacts the potential for differentiation of stem cells into specific cell lineages. This work will provide both key information on the functional relevance of epigenetic regulation of pluripotency, and also a possible clinical approach to evaluate the pluripotency of stem cells.