Chromatin Dynamics During Epithelial Commitment

上皮定型期间的染色质动力学

• 批准号: 9981936
• 负责人: Anthony E Oro
• 金额: $ 8.12万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-12 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981936
• 关键词: ATAC-seq; Address; Binding; Binding Sites; Biological Markers; Cell Therapy; Cells; Chromatin; Clinical; Data; Development; Disease; Enhancers; Epidermolysis Bullosa Dystrophica; Epigenetic Process; Epithelial; Epithelium; Exhibits; Family member; Feedback; Genes; Genetic; Genetic Transcription; Genomics; Goals; Homeostasis; Human; Knowledge; Link; Mediating; Modeling; Molecular Conformation; Patients; Pattern; Process; Production; Protocols documentation; Regenerative Medicine; Regulation; Repression; Role; Simple Epithelium; Skin; Specificity; Surface; Surface Ectoderm; TFAP2A gene; TFAP2C gene; TP53 gene; Technology; Tissue Differentiation; Tissue Engineering; Tissues; Transcription Initiation Site; Tretinoin; United States National Institutes of Health; Validation; base; bone morphogenic protein; engineered stem cells; epigenetic regulation; genome editing; induced pluripotent stem cell; innovation; insight; keratinocyte; keratinocyte differentiation; morphogens; novel; palliation; promoter; skin disorder; stem; transcription factor; transcriptome sequencing

Project Summary Recent advances with induced pluripotent stem (iPS) cells and tissue engineering have opened the door to keratinocyte-based tissue replacement for patients with Recessive Dystrophic Epidermolysis bullosa (RDEB). While our lab has shown that bone morphogenic protein (BMP) and retinoic acid (RA) morphogens can induce ES/iPS-derived graftable human keratinocytes, detailed mechanistic insights into keratinocyte differentiation remain a major roadblock to efficient tissue manufacturing and a goal of the NIH Regenerative Medicine Innovation Project. Without RA/BMP, master regulator p63 binds but exhibits few transcriptional changes, highlighting the importance of morphogen-lineage selector interactions. HiChIP analysis shows that RA/BMP induces chromatin conformational changes that connect chromatin-bound p63 to linked enhancers/promoters and determine transcriptional specificity at each gene. Through our novel network transcription factor (TF) inference model we discovered that forced expression of a single transcription factor, TFAP2C, can induce functional keratinocytes in the absence of RA/BMP. We subsequently identified a two-step mechanism where the TFAP2C initiates the simple epithelial landscape, and induces expression of and opens additional binding sites for the p63. In turn, p63 matures the TFAP2C-patterned epigenetic landscape resulting in p63 positive autoregulation and the closing of a subset of TFAP2C binding sites, shifting the landscape to a p63-centric keratinocyte TF network. These data support the intriguing hypothesis that RA/BMP initiates epigenetic changes through TFAP2 family members that ultimately result in p63-driven epigenetic maturation of the landscape to keratinocytes. In this proposal we aim to address key gaps in our knowledge through: Functional validation of the TFAP2- centric network in epigenetic landscape initiation by confirming TFAP2 necessity for RA/BMP- mediated epigenetic landscape change, determining TFAP2C functional requirement for the early TF network, and defining the morphogen-inducible TFAP2C interactome; Elucidation how p63 matures the chromatin landscape during keratinocyte production by determining how TFAP2 and p63 cooperate to allow p63 positive autoregulation, determining if keratinocyte maturation requires p63- dependent TFAP2 repression, and completing and validating an inference TF model of keratinocyte differentiation. Successful completion of this proposal will provide deep mechanistic insights into the chromatin dynamics of tissue differentiation and establish a detailed epigenetic characterization that enables development of our novel cell therapy for a previously untreatable genetic skin disorder.

项目概要 诱导多能干 (iPS) 细胞和组织工程的最新进展开启了 隐性营养不良性表皮松解症患者基于角质形成细胞的组织替代疗法的大门 大疱（RDEB）。虽然我们的实验室已经证明骨形态发生蛋白（BMP）和视黄酸 (RA) 形态发生素可诱导 ES/iPS 衍生的可移植人角质形成细胞，详细机制 对角质形成细胞分化的深入了解仍然是高效组织制造的主要障碍 以及 NIH 再生医学创新项目的目标。不带 RA/BMP，主调节器 p63 结合但表现出很少的转录变化，凸显了形态发生素谱系的重要性 选择器交互。 HiChIP 分析表明 RA/BMP 诱导染色质构象 将染色质结合的 p63 连接到相关增强子/启动子的变化并确定 每个基因的转录特异性。通过我们新颖的网络转录因子（TF）推断 在模型中，我们发现强制表达单个转录因子 TFAP2C 可以诱导 在缺乏 RA/BMP 的情况下，功能性角质形成细胞。我们随后确定了两步走 TFAP2C 启动简单上皮景观并诱导表达的机制 并为 p63 打开额外的结合位点。反过来，p63 使 TFAP2C 模式成熟 表观遗传景观导致 p63 正向自动调节和 TFAP2C 子集的关闭 结合位点，将景观转移到以 p63 为中心的角质形成细胞 TF 网络。这些数据支持 RA/BMP 通过 TFAP2 家族成员启动表观遗传变化这一有趣的假设 最终导致 p63 驱动的表观遗传景观成熟为角质形成细胞。在这个 我们的提案旨在通过以下方式解决我们知识中的关键差距： TFAP2-的功能验证 通过确认 RA/BMP- 的 TFAP2 必要性，表观遗传景观启动中的中心网络 介导的表观遗传景观变化，确定早期 TF 的 TFAP2C 功能需求 网络，并定义形态发生素诱导的 TFAP2C 相互作用组；阐明 p63 如何成熟 通过确定 TFAP2 和 p63 如何影响角质形成细胞生成过程中的染色质景观 合作以允许 p63 积极的自动调节，确定角质形成细胞成熟是否需要 p63- 依赖性 TFAP2 抑制，并完成和验证角质形成细胞的推理 TF 模型 差异化。该提案的成功完成将为我们提供深入的机制见解 组织分化的染色质动力学，并建立详细的表观遗传特征 使我们能够开发出针对以前无法治疗的遗传性皮肤病的新型细胞疗法。