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: 10408113
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408113
• 关键词: Animal Model; Animals; Cell Line; Cell Lineage; Cell Therapy; Cells; ChIP-seq; Chromatin; Clinical; Data; Development; Ensure; Epigenetic Process; Ethics; Gene Expression; Gene Expression Profile; Generations; Genes; 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 cell pluripotency; Testing; Tissues; Transcriptional Activation; Variant; Work; base; cell type; embryonic stem cell; epigenetic regulation; histone modification; induced pluripotent stem cell; pluripotency; promoter; regeneration potential; repaired; stem cell differentiation; stem cell therapy; stem cells; 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：重新编程的体细胞成为多能干细胞的持有 基于干细胞疗法的巨大希望，但主要障碍之一在于能够 确定哪些干细胞真正多功能。我们已经表明诱导的多能 小鼠的干细胞（IPSC）可能具有产生“全身”的潜力明显不同 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。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 中毒以在多能干细胞中激活。我们目前无法为此进行调查 必要的中毒状态，因为IPSC的转录轮廓并未揭示潜力 转录激活。我们以前报道说，需要H3.3建立多能 在重编程过程中，需要维持DB基因在ESC中的双价性。实际上， 我们的初步数据表明，H3.3在许多DB基因的启动子上富含 缺乏富集与IPSC中的发育潜力受损密切相关。这 发现表明H3.3在调节数据库基因表达中起着至关重要的作用，因此 多能。根据我们的观察，我们假设需要H3.3建立 重编程过程中的双价，并且在启动子上的表观遗传学签名使DB保持 分化后以后激活的基因。我们的长期目标是阐明钥匙 在干细胞中建立和维持多能性的机制。这个目的 建议是定义组蛋白变体H3.3调节数据库基因的机制 以及干细胞的发育特性。我们计划使用独特的 动物模型将使我们能够在任何中间体中获得足够的遗传均匀细胞 阶段。这将使我们能够使用芯片测序研究H3.3和Hisstone修改 在重新编程期间。我们在此应用程序中提出以下两个目标：AIM 1：确定 H3.3如何调节在DB基因中建立表观遗传学特征 重新编程针对多能。目标2：确定H3.3富集标记的功能 IPSC分化过程中DB基因的启动子。这些目标的成功完成将 允许我们确定DB基因启动子对H3.3的富集如何影响 干细胞分化为特定细胞谱系的潜力。这项工作将提供两者 有关多能性表观遗传调节功能相关性的关键信息，也是一个 评估干细胞多能性的可能临床方法。