Control of lung alveolar regeneration by Dot1L/H3K79 methylation

项目总结 呼吸系统中的多个组织间隔或壁龛显示出不同的修复和修复能力 在急性损伤或慢性疾病状态下再生。肺泡龛也是气体交换的关键。 作为环境刺激的哨兵，包括传染性生物和污染物。世界上大部分的 肺泡的再生力在于肺泡2型(AT2)细胞，这不仅对表面活性物质至关重要 产生和先天免疫反应，但也窝藏着常驻的祖细胞群体。一栋建筑 大量研究表明，AT2细胞亚群可以增殖并分化为肺泡1型(AT1)。 急性损伤后的细胞，这是再生有功能的肺泡的关键。这些AT2细胞的行为是 受信号、转录和表观遗传机制的调控，这些机制只是最近才开始 已澄清。为了进一步了解表观遗传途径在肺泡再生中所起的作用， 我们使用肺泡器官化验进行了小分子筛查，以确定促进 牙槽骨修复和再生。这一筛选确定了端粒沉默干扰物的多个抑制物- 1类(DOT1L)，调节肺泡器质大小。DOT1L是已知的唯一甲基化H3K79的酶 (H3K79me1/2/3标记)，DOT1L已被证明在促进多能性干细胞中发挥关键作用 细胞重新编程，以及细胞对损伤和组织再生的反应。我们的数据显示DOT1L抑制 以剂量依赖的方式增加肺泡器质的大小。为了更好地了解DOT1L在肺中的作用 在体内的发育和再生，我们产生了一个DOT1L条件基因敲除小鼠等位基因并灭活 DOT1L在肺发育以及多种肺损伤和再生模型中的表达。过程中DOT1L丢失 肺内胚层发育导致H3K79甲基化缺失和H3K79过早或增强表达 AT1和AT2标记基因，提示AT1和AT2细胞分化加快。在两种肺模型中 肺泡再生，AT2细胞DOT1L缺失导致AT2-AT1分化显著加速 肺损伤。单细胞RNA-SEQ(scRNA-SEQ)结合CHIP-SEQ分析揭示DOT1L导联丢失 以新的AT2细胞状态的出现为特征的重要基因表达的急剧增加 转录调控基因Id1和Id2以及相关新陈代谢基因表达的全面增加 氧化磷酸化(OxPhos)。综上所述，我们的数据导致假设DOT1L扮演着一个 在调节肺泡对急性肺损伤反应中的重要作用 通过下调关键转录调控因子Id1/Id2并转换为OxPhos来抑制AT2和AT1细胞 代谢，导致AT2-AT1分化加快。

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.