Chromatin Dynamics During Epithelial Commitment

上皮定型期间的染色质动力学

• 批准号: 10612007
• 负责人: Anthony E Oro
• 金额: $ 44.35万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612007
• 关键词: ATAC-seq; Address; Binding; Binding Sites; Biological Markers; Bone Morphogenetic Proteins; Cell Therapy; Cells; Chromatin; Clinical; Data; Development; Dimensions; Disease; Enhancers; Epidermolysis Bullosa Dystrophica; Epigenetic Process; Epithelium; Exhibits; Family member; Feedback; Genes; Genetic; Genetic Transcription; Genomics; Goals; Homeostasis; Human; Knowledge; Link; Mediating; Modeling; Molecular Conformation; Patients; Pattern; Process; Production; Regenerative Medicine; Regulation; Repression; Role; Simple Epithelium; Skin; Specificity; Surface; Surface Ectoderm; TFAP2A gene; TFAP2C gene; TP53 gene; Tissue Differentiation; Tissue Engineering; Tissues; Transcription Initiation Site; Tretinoin; United States National Institutes of Health; Validation; differentiation protocol; engineered stem cells; epigenetic regulation; genome editing; induced pluripotent stem cell; induced pluripotent stem cell technology; innovation; insight; keratinocyte; keratinocyte differentiation; manufacture; morphogens; novel; palliation; promoter; replacement tissue; 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.

项目摘要 诱导多能茎（IPS）细胞和组织工程的最新进展已经打开 为凹性营养不良表皮分解患者的角质形成细胞替代的门置换 Bullosa（rdeb）。虽然我们的实验室表明骨形态发生蛋白（BMP）和视黄酸 （RA）形态剂可以诱导ES/IPS衍生的可嫁接人角质形成细胞，详细的机理 对角质形成细胞分化的见解仍然是有效组织制造的主要障碍 NIH再生医学创新项目的目标。没有RA/BMP，主管主管 p63结合，但几乎没有转录变化，强调了形态学的重要性 选择器交互。 HICHIP分析表明，RA/BMP诱导染色质构象 将结合染色质p63连接到链接的增强剂/启动子的更改并确定 每个基因的转录特异性。通过我们的新型网络转录因子（TF）推断 模型我们发现，单个转录因子TFAP2C的强迫表达可以诱导 在没有RA/BMP的情况下，功能性角质形成细胞。随后我们确定了两步 TFAP2C启动简单上皮景观的机制，并诱导表达 并为p63打开其他结合位点。反过来，p63成熟了TFAP2C模式 表观遗传景观导致p63阳性自动调节和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家族成员引发表观遗传变化的有趣假设 最终导致p63驱动的表观遗传学成熟到角质形成细胞。在这个 提案我们旨在通过以下方面解决知识中的关键差距：TFAP2-的功能验证 通过确认TFAP2的必要性，表观遗传景观启动的中心网络 介导的表观遗传景观变化，确定早期TF的TFAP2C功能需求 网络，并定义形态诱导的TFAP2C相互作用组；阐明p63如何成熟 通过确定TFAP2和p63的生产过程中染色质景观 合作以允许p63阳性自动调节，确定角质形成细胞是否成熟是否需要p63- 依赖的TFAP2抑制，并完成和验证角质形成细胞的推理TF模型 分化。该提案的成功完成将为您提供深刻的机械见解 组织分化的染色质动力学，并建立一个详细的表观遗传表征 使我们的新型细胞疗法发展为以前不可治疗的遗传性皮肤疾病。