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

项目摘要：重编程体细胞成为多能干细胞 干细胞疗法前景广阔，但主要障碍之一在于 确定哪些干细胞是真正的多能性。我们已经证明，诱导多能 来自小鼠的干细胞（iPSC）可以具有显著不同的产生“全iPS”的潜力。 动物，即使细胞具有相似的转录谱。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。当 发育二价基因在谱系定型中被激活，它们被沉默， 为以后在多能干细胞中激活做好准备。我们目前还没有办法去探测这个 这是必要的平衡状态，因为iPSC的转录谱并没有揭示出 转录激活我们以前报道过，H3.3是建立多能性所必需的 在重编程过程中，并需要保持胚胎干细胞中DB基因的二价性。事实上， 我们的初步数据显示H3.3在许多DB基因的启动子处富集，并且其 缺乏富集与iPSC中受损的发育潜力密切相关。这 这一发现表明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H3.3是建立 在重编程过程中的二价，并且启动子处的表观遗传标记使DB 分化后激活的基因。我们的长期目标是阐明 建立和维持干细胞多能性的机制。的目的 一个建议是确定组蛋白变体H3.3调节DB基因的机制 以及干细胞的发育特性。我们计划用独特的 动物模型，这将使我们能够获得足够的遗传均匀的细胞在任何中间 阶段这将使我们能够使用ChIP测序研究H3.3和组蛋白修饰 在重新编程过程中。在本申请中，我们提出以下两个目标： H3.3如何调节DB基因中表观遗传标记的建立， 重编程向多能性发展。目的2：确定H3.3富集标记在 在iPSC分化期间DB基因中的启动子。成功实现这些目标将 使我们能够确定DB基因启动子处H3.3的富集如何影响DB基因的表达。 干细胞分化为特定细胞谱系的潜力。这项工作将提供两个 关于多能性的表观遗传调节的功能相关性的关键信息，以及 可能的临床方法来评估干细胞的多能性。

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.