Epigenetic profiling of human alveolar epithelial cells in health and disease

项目总结：特发性肺纤维化（IPF）是一种病因不明的致死性疾病， 进行性肺纤维化它会导致患者在5年内死亡，除非患者接受肺移植。高达 美国每年诊断出34，000例IPF病例。IPF肺部的特征是增加 （肌）成纤维细胞的积累，具有收缩表型的活化成纤维细胞，被认为是关键的 效应细胞导致基质沉积。虽然（肌）成纤维细胞的来源还没有明确的 最近的研究表明，这些细胞中有很大一部分来自肺泡上皮细胞 (AEC)通过上皮-间质转化（EMT）过程。许多观察表明， AEC在IPF的启动和发展中的关键作用。肺泡上皮由立方2型组成 （AEC 2）细胞和长瘦型1（AEC 1）细胞，每种细胞具有不同的形态，功能和模式， 基因表达。AEC 2是肺表面活性剂的主要来源。它们被认为可以自我更新， 作为AEC 1的祖细胞用于损伤后上皮完整性的恢复。相反，AEC 1介导 气体交换，并且被认为是终末分化的并且不能增殖。AEC已被证明 转化生长因子β（TGF-β）在体内进行EMT，产生（肌）成纤维细胞。 AEC分化可以在体外重现;在没有添加生长因子的情况下，纯化的AEC 2 分化为AEC 1样细胞。相反，当用TGF-β处理时，AEC 2经历EMT，产生 （肌）成纤维细胞。这种独特的模型系统允许对驱动分化的事件进行时间解剖， 健康和疾病条件下的外周肺。该提案旨在确定全基因组范围内的 在不同的时间点，随着细胞向AEC 1 - 1转化，人AEC 2的转录和表观遗传变化。 如细胞或（肌）成纤维细胞。为了进行比较，将来自正常肺的原代AEC 2和AEC 1细胞以及来自正常肺的AEC 2和AEC 1细胞进行了比较。 和来自IPF患者肺的（肌）成纤维细胞。具体目标是：1）转录组 在向AEC 1样细胞或（肌）成纤维细胞转变期间人AEC 2的谱分析。健康人的原代细胞 将类似地分析肺（AEC 2和AEC 1）和IPF肺（AEC 2和（肌）成纤维细胞）。2)表观 对来自Aim 1的细胞进行分析，使用全基因组方法评估DNA甲基化模式，组蛋白 修饰、多梳组复合物占据、染色体重塑复合物占据，以及 选择性转录因子占有率。3)综合目标1和2的数据， 各种表观遗传修饰和mRNA转录之间的空间关系以及小的非遗传修饰之间的空间关系。 编码RNA。4)验证新鉴定的表达模式、表观遗传控制网络和 使用AEC和（肌）成纤维细胞的独立培养物的调节途径。转录的知识 和表观遗传程序驱动AEC（错误）分化将提供深入了解潜在的新途径， 肺恢复，并将有助于制定治疗和最终预防IPF等疾病的策略。

PROJECT SUMMARY: Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by progressive lung fibrosis. It leads to death within 5 years unless patients undergo a lung transplant. Up to 34,000 cases of IPF are diagnosed in the United States each year. IPF lungs are characterized by increased accumulation of (myo)fibroblasts, activated fibroblasts with a contractile phenotype that are believed to be key effector cells leading to matrix deposition. Although the source of (myo)fibroblasts has not been definitively established, recent studies suggest that a significant portion of these cells arise from alveolar epithelial cells (AEC), through the process of epithelial-to-mesenchymal transition (EMT). Numerous observations point to a key role for AEC in the initiation and development of IPF. Alveolar epithelium consists of cuboidal type 2 (AEC2) cells and long thin type 1 (AEC1) cells, each with distinct morphologies, functions, and patterns of gene expression. AEC2 are a major source of pulmonary surfactants. They are believed to both self-renew and serve as progenitors for AEC1 for restoration of epithelial integrity following injury. In contrast, AEC1 mediate gas exchange and are believed to be terminally differentiated and unable to proliferate. AEC have been shown to undergo EMT in vivo in response to transforming growth factor beta (TGF-�), giving rise to (myo)fibroblasts. AEC differentiation can be recapitulated in vitro; in the absence of added growth factors, purified AEC2 differentiate into AEC1-like cells. In contrast, when treated with TGF-�, AEC2 undergo EMT, producing (myo)fibroblasts. This unique model system allows the temporal dissection of events that drive differentiation in the peripheral lung under conditions of health and disease. This proposal aims to identify genome-wide transcriptional and epigenetic changes of human AEC2 at distinct time points as the cells transition to AEC1- like cells or to (myo)fibroblasts. For comparison, primary AEC2 and AEC1 cells from normal lungs, and AEC2 and (myo)fibroblasts from the lungs of IPF patients will be examined. The Specific Aims are: 1) Transcriptome profiling of human AEC2 during the transition to AEC1-like cells or (myo)fibroblasts. Primary cells from healthy lung (AEC2 and AEC1) and IPF lung (AEC2 and (myo)fibroblasts) will be similarly analyzed. 2) Epigenomic profiling of the cells from Aim 1, using genome-wide approaches to assess DNA methylation patterns, histone modifications, polycomb group complex occupancy, chromosome remodeling complex occupancy, and selected transcription factor occupancy. 3) Integration of the data from Aims 1 and 2 to model the temporal and spatial relationships between the various epigenetic modifications and transcription of mRNAs and small non- coding RNAs. 4) Validation of a subset of newly identified expression patterns, epigenetic control networks and regulatory pathways using independent cultures of AEC and (myo)fibroblasts. Knowledge of the transcriptional and epigenetic programs driving AEC (mis)differentiation will provide insights into potential new avenues for lung restoration and will help develop strategies to treat and ultimately prevent diseases such as IPF.