Control of lung alveolar regeneration by Dot1L/H3K79 methylation

通过 Dot1L/H3K79 甲基化控制肺泡再生

• 批准号: 10594734
• 负责人: EDWARD E MORRISEY
• 金额: $ 56.95万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594734
• 关键词: Acceleration; Acute; Acute Lung Injury; Address; Aging; Alleles; Alveolar; Alveolus; Biological Assay; Cell Differentiation process; Cell Reprogramming; Cell division; Cells; ChIP-seq; Chronic Disease; DNA Methylation; Data; Development; Dose; Endoderm; Enzymes; Epigenetic Process; Functional Regeneration; Gases; Gene Expression; Genes; Genetic Transcription; Homeostasis; Hyperoxia; Infectious Agent; Injury; Innate Immune Response; Knockout Mice; Laboratories; Lead; Lung; Metabolic; Metabolism; Methylation; MicroRNAs; Modeling; Molecular; Mus; Natural regeneration; Organoids; Oxidative Phosphorylation; Pathway interactions; Play; Pluripotent Stem Cells; Population; Process; Proliferating; Pulmonary alveolar structure; Research; Respiratory System; Rest; Role; Sentinel; Signal Transduction; Somatotype; Stimulus; Tissues; Untranslated RNA; alveolar epithelium; cell behavior; conditional knockout; derepression; histone modification; improved; in vivo regeneration; inhibitor; lung development; lung injury; lung regeneration; mutant; pollutant; premature; programs; pulmonary function; regenerative; repaired; response; response to injury; single-cell RNA sequencing; small molecule; stem cells; surfactant production; tissue regeneration

PROJECT SUMMARY The multiple tissue compartments or niches in the respiratory system display varying abilities to repair and regenerate after acute injury or in chronic disease states. The alveolar niche is critical for gas exchange as well as acting as a sentinel for environmental stimuli including infectious organisms and pollutants. Much of the regenerative power of the alveoli rests within the alveolar type 2 (AT2) cell, which is not only critical for surfactant production and innate immune responses, but also harbors the resident progenitor cell population. A building body of research has shown that subsets of AT2 cells can proliferate and differentiate into alveolar type 1 (AT1) cells after acute injury, which is critical for regenerating functional alveoli. These AT2 cells behaviors are regulated by signaling, transcriptional, and epigenetic mechanisms that have only recently started to be elucidated. To further our understanding of the role that epigenetic pathways play in lung alveolar regeneration, we performed a small molecule screen using an alveolar organoid assay to identify pathways that promote alveolar repair and regeneration. This screen identified multiple inhibitors of the Disruptor of Telomeric Silencing- 1 like (Dot1L) that regulate alveolar organoid size. Dot1L is the sole enzyme which is known to methylate H3K79 (H3K79me1/2/3 marks), and Dot1L has been demonstrated to play critical roles in promoting pluripotent stem cell reprogramming, and cellular responses to injury and tissue regeneration. Our data show that Dot1L inhibition increases alveolar organoid size in a dose dependent manner. To better understand the role of Dot1L in lung development and regeneration in vivo, we generated a Dot1L conditional knockout mouse allele and inactivated Dot1L during lung development and in multiple models of lung injury and regeneration. Loss of Dot1L during lung endoderm development results in the loss of H3K79 methylation and premature or enhanced expression of AT1 and AT2 marker genes, suggesting acceleration of AT1 and AT2 cell differentiation. In two models of lung alveolar regeneration, loss of Dot1L in AT2 cells results in dramatic acceleration of AT2-AT1 differentiation after lung injury. Single cell RNA-seq (scRNA-seq) combined with ChIP-seq analysis reveals that loss of Dot1L leads to the emergence of a new AT2 cell state characterized by a dramatic increase in the expression of the important transcriptional regulators Id1 and Id2 as well as an overall increase in expression of metabolism genes related to oxidative phosphorylation (OxPhos). Taken together, our data lead to the hypothesis that Dot1L plays an important role in regulating lung alveolar responses to acute lung injury by regulating the lineage barrier between AT2 and AT1 cells via de-repression of the critical transcriptional regulators Id1/Id2 and a switch to OxPhos metabolism, resulting in acceleration of AT2-AT1 differentiation.

项目摘要 呼吸系统中的多个组织室或壁nir显示出不同的修复能力和 急性损伤或慢性病状态后再生。肺泡的生态位也对气体交换至关重要 充当环境刺激的前哨，包括传染性生物和污染物。大部分 肺泡的再生能力存在于2型肺泡2（AT2）细胞中，这不仅对表面活性剂至关重要 生产和先天免疫反应，但也含有居民祖细胞群。建筑物 研究机构表明，AT2细胞的子集可以增殖并分化为1型牙槽（AT1） 急性损伤后的细胞，这对于再生功能肺泡至关重要。这些AT2细胞行为是 受信号，转录和表观遗传机制的调节，直到最近才开始 阐明。为了进一步了解表观遗传途径在肺肺泡再生中所起的作用， 我们使用肺泡器官测定法进行了一个小分子筛选，以识别促进的途径 牙槽修复和再生。该屏幕确定了端粒沉默的破坏者的多个抑制剂 - 1喜欢（DOT1L）调节牙槽器官大小。 dot1l是唯一的酶，已知是甲基化H3K79 （H3K79ME1/2/3标记），并且已证明DOT1L可以在促进多能茎中起关键作用 细胞重编程以及对损伤和组织再生的细胞反应。我们的数据表明dot1l抑制作用 以剂量依赖的方式增加牙槽器官的大小。更好地了解DOT1L在肺中的作用 在体内开发和再生，我们生成了一个有条件的敲除鼠标等位基因并灭活的DOT1L 在肺发育期间以及多种肺损伤和再生模型中的点1L。丢失dot1l在 肺内胚层发育导致H3K79甲基化和过早或增强表达的丧失 AT1和AT2标记基因，表明AT1和AT2细胞分化的加速度。在两种肺模型中 牙槽再生，AT2细胞中DOT1L的丧失导致AT2-AT1分化后的急剧加速 肺部受伤。单细胞RNA-seq（SCRNA-SEQ）与芯片序列分析结合了DOT1L铅的损失 出现新的AT2细胞状态，其特征是重要的表达表达急剧增加 转录调节剂ID1和ID2以及代谢基因表达的总体增加 氧化磷酸化（OXPHOS）。综上所述，我们的数据导致了Dot1l扮演的假设 通过调节之间的谱系屏障来调节对急性肺损伤的肺肺泡反应的重要作用 通过临界转录调节剂ID1/ID2的抑制和AT2和AT1细胞的转换 代谢，导致AT2-AT1分化的加速。