Control of lung alveolar regeneration by Dot1L/H3K79 methylation

项目摘要 呼吸系统中的多个组织隔室或小生境显示出不同的修复和修复能力。 在急性损伤后或慢性疾病状态下再生。肺泡生态位对气体交换也很重要 作为环境刺激物的哨兵，包括传染性生物和污染物。大部分 肺泡的再生能力取决于肺泡2型（AT 2）细胞，这不仅对表面活性剂至关重要， 它不仅是细胞生成和先天免疫应答的载体，而且也是常驻祖细胞群体的庇护所。建筑物 大量研究表明，AT 2细胞亚群可以增殖并分化为肺泡1型（AT 1）， 急性损伤后的细胞，这对于再生功能性肺泡至关重要。这些AT 2细胞的行为是 受信号传导、转录和表观遗传机制的调节，这些机制最近才开始被 阐明。为了进一步了解表观遗传途径在肺泡再生中的作用， 我们使用肺泡类器官分析进行了一项小分子筛选，以确定促进 肺泡修复和再生。该筛选鉴定了端粒沉默破坏因子的多种抑制剂- 1 like（Dot 1 L）调节肺泡类器官大小。Dot 1 L是已知使H3 K79甲基化的唯一酶 （H3 K79 me 1/2/3标记），Dot 1 L已被证明在促进多能干细胞中发挥关键作用。 细胞重编程以及细胞对损伤和组织再生的反应。我们的数据显示Dot 1 L抑制 以剂量依赖性方式增加肺泡类器官大小。为了更好地了解Dot 1 L在肺中的作用， 在体内发育和再生中，我们产生了Dot 1 L条件性敲除小鼠等位基因，并灭活了 Dot 1 L在肺发育和多种肺损伤和再生模型中的作用。Dot 1 L在 肺内胚层的发育导致H3 K79甲基化的丧失和H3 K79的过早或增强的表达。 AT 1和AT 2标记基因，表明加速了AT 1和AT 2细胞分化。在两个肺模型中， 肺泡再生，AT 2细胞中Dot 1 L的缺失导致AT 2-AT 1分化的急剧加速， 肺损伤单细胞RNA-seq（scRNA-seq）结合ChIP-seq分析显示Dot 1 L的缺失导致 一种新的AT 2细胞状态的出现，其特征在于重要的 转录调节因子Id 1和Id 2以及代谢相关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Dot 1 L扮演了一个 通过调节细胞间的谱系屏障在调节急性肺损伤肺泡反应中的重要作用 AT 2和AT 1细胞通过关键转录调节因子Id 1/Id 2的去抑制和OxPhos的转换 代谢，导致AT 2-AT 1分化加速。

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.