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

H3.3介导的发育二价基因的表观遗传调控，用于重编程和分化

• 批准号: 9750724
• 负责人: Duancheng Wen
• 金额: $ 35.6万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750724
• 关键词: Animal Model; Animals; Cell Line; Cell Lineage; Cells; ChIP-seq; Chromatin; Clinical; Data; Development; Ensure; Epigenetic Process; Ethics; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Histones; Human; Knock-out; Knowledge; Mediating; Methods; Modification; Mus; Patients; Play; Pluripotent Stem Cells; Property; Public Health; Regenerative Medicine; Regulation; Reporter; Reporting; Role; Somatic Cell; Stem cells; Testing; Therapeutic; Tissues; Transcriptional Activation; Variant; Work; base; cell type; embryonic stem cell; epigenetic regulation; histone modification; induced pluripotent stem cell; pluripotency; promoter; repaired; stem cell differentiation; stem cell therapy; tool; transcription factor

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.

项目摘要：将体细胞重新编程为多能干细胞 基于干细胞的疗法前景广阔，但主要障碍之一在于其能力 确定哪些干细胞是真正的多能性 我们已经证明诱导多能性。 来自小鼠的干细胞 (iPSC) 具有明显不同的产生“全 iPS”的潜力 动物，即使细胞具有相似的转录谱，这种差异表明存在差异。 表观遗传机制在调节干细胞多能性中发挥重要作用。 发育二价（DB）基因在谱系定型中被激活，它们被沉默，但 准备稍后在多能干细胞中激活，我们目前无法对此进行探测。 必要的平衡状态，因为 iPSC 的转录谱并未揭示出 我们之前报道过 H3.3 是建立多能性所必需的。 在重编程过程中，并且需要维持 ESC 中 DB 基因的二价性。 我们的初步数据表明H3.3在许多DB基因的启动子处富集，并且其 缺乏富集与 iPSC 发育潜力受损密切相关。 研究结果表明H3.3在调节DB基因表达中发挥着关键作用，因此 根据我们的观察，我们追求H3.3是建立的必要条件。 重编程过程中的二价性以及启动子处的表观遗传特征决定了 DB 我们的长期目标是阐明分化后激活的基因。 建立和维持干细胞多能性的机制 其目的。 提议是定义组蛋白变体 H3.3 调节 DB 基因的机制 以及干细胞的发育特性，我们计划使用独特的方法来检验这一假设。 动物模型将使我们能够在任何中间获得足够的遗传均匀细胞 这将使我们能够使用 ChIP 测序来研究 H3.3 和组蛋白修饰。 我们在此应用中提出以下两个目标： 目标 1：识别。 H3.3如何调节DB基因表观遗传特征的建立 目标 2：确定 H3.3 富集标记的功能。 iPSC 分化期间 DB 基因中的启动子将成功完成这些目标。 让我们能够确定 DB 基因启动子处 H3.3 的富集如何影响 这项工作将提供干细胞分化为特定细胞谱系的潜力。 关于多能性表观遗传调控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。