Epigenetic profiling of human alveolar epithelial cells in health and disease

项目摘要：特发性肺纤维化(Ipf)是一种病因不明的致命疾病。 由进行性肺纤维化引起。除非患者接受肺移植，否则它会在5年内导致死亡。至.为止 美国每年诊断出3.4万例IPF。IPF肺的特征是增加 (Myo)成纤维细胞的积累，即具有收缩表型的激活的成纤维细胞，被认为是关键 导致基质沉积的效应细胞。尽管(Myo)成纤维细胞的来源尚未确定 最近已确定的研究表明，这些细胞中有很大一部分来自肺泡上皮细胞 (AEC)，通过上皮向间充质转化(EMT)的过程。大量的观察表明， AEC在IPF的发起和发展中的关键作用。肺泡上皮由立方的2型组成 (AEC2)细胞和长细1型(AEC1)细胞，每个细胞具有不同的形态、功能和模式 基因表达。AEC2是肺表面活性物质的主要来源。它们被认为既能自我更新，又能 作为AEC1的前体细胞，促进损伤后上皮完整性的恢复。相比之下，AEC1起到了调节作用 气体交换，并被认为是终末分化的，不能增殖。已向AEC展示 在体内进行内胚层移植，以回应转化生长因子β(TGF-β)，产生(肌)成纤维细胞。 AEC分化可以在体外概括；在没有添加生长因子的情况下，纯化的AEC2 分化为AEC1样细胞。相反，当用转化生长因子-β处理时，AEC2经历EMT，产生 (Myo)成纤维细胞。这一独特的模型系统允许对推动差异的事件进行时间剖析 在健康和疾病条件下的外周肺。这项提案旨在全基因组范围内识别 人AEC2细胞向AEC1-1转化不同时间点的转录和表观遗传学变化 如细胞或(肌)成纤维细胞。作为比较，来自正常肺的原代AEC2和AEC1细胞，以及AEC2 并对IPF患者肺成纤维细胞(MIO)进行检查。具体目标是：1)译本 人AEC2向AEC1样细胞或(肌)成纤维细胞转变过程中的特征。健康来源的原代细胞 肺(AEC2和AEC1)和IPF肺(AEC2和(MIO)成纤维细胞)将进行类似的分析。2)表观基因组学 使用全基因组方法评估DNA甲基化模式，组蛋白，来自AIM 1的细胞的概况 修饰、多梳基复合体占有率、染色体重塑复合体占有率以及 选定的转录因子占有率。3)整合AIMS 1和AIMS 2的数据以模拟时态和 不同表观遗传修饰和转录之间的空间关系的mRNAs和小非 编码RNA。4)确认新识别的表达模式的子集、表观遗传控制网络和 使用AEC和(MIO)成纤维细胞独立培养的调控途径。转录方面的知识 而推动AEC(Mis)分化的表观遗传学程序将为以下方面提供潜在的新途径 这将有助于制定治疗和最终预防肺间质纤维化等疾病的战略。

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.