Epigenetic profiling of human alveolar epithelial cells in health and disease

描述（由申请方提供）：特发性肺纤维化（IPF）是一种病因不明的致死性疾病，其特征为进行性肺纤维化。它会导致患者在5年内死亡，除非患者接受肺移植。美国每年诊断出多达34，000例IPF病例。IPF肺的特征在于（肌）成纤维细胞（具有收缩表型的活化成纤维细胞）的蓄积增加，这些细胞被认为是导致基质沉积的关键效应细胞。虽然（肌）成纤维细胞的来源尚未明确确定，但最近的研究表明，这些细胞中有很大一部分来自肺泡上皮细胞（AEC），通过上皮-间充质转化（EMT）的过程。大量观察结果表明AEC在IPF的发生和发展中起关键作用。肺泡上皮由立方2型（AEC 2）细胞和细长1型（AEC 1）细胞组成，每种细胞具有不同的形态、功能和基因表达模式。AEC 2是肺表面活性剂的主要来源。它们被认为既能自我更新又能作为AEC 1的祖细胞，用于在损伤后恢复上皮完整性。相反，AEC 1介导气体交换，被认为是终末分化的，不能增殖。已显示AEC响应于转化生长因子β（TGF-2）在体内经历EMT，产生（肌）成纤维细胞。AEC分化可以在体外重演;在没有添加生长因子的情况下，纯化的AEC 2分化成AEC 1样细胞。相反，当用TGF-2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在健康和疾病条件下驱动外周肺分化的事件进行时间解剖。该提案旨在确定人类AEC 2在不同时间点的全基因组转录和表观遗传变化，因为细胞转变为AEC 1样细胞或（肌）成纤维细胞。为了比较，将检查来自正常肺的原代AEC 2和AEC 1细胞以及来自IPF患者肺的AEC 2和（肌）成纤维细胞。具体目的是：1）在向AEC 1样细胞或（肌）成纤维细胞转变期间，人AEC 2的转录组谱分析。将类似地分析来自健康肺（AEC 2和AEC 1）和IPF肺（AEC 2和（肌）成纤维细胞）的原代细胞。2)来自目标1的细胞的表观基因组分析，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和选定的转录因子占据。3)整合来自目标1和2的数据，以模拟mRNA和小的非编码RNA的各种表观遗传修饰和转录之间的时间和空间关系。4)使用AEC和（肌）成纤维细胞的独立培养物验证新鉴定的表达模式、表观遗传控制网络和调控途径的子集。对驱动AEC（mis）分化的转录和表观遗传程序的了解将为肺恢复的潜在新途径提供见解，并将有助于制定治疗和最终预防IPF等疾病的策略。 公共卫生相关性：没有人知道为什么每年有多达34，000名美国人患上特发性肺纤维化（IPF），这是一种致命的疾病，其中肺部被瘢痕组织缓慢破坏，最终导致窒息。有证据表明，在IPF患者中，某些肺细胞分化为瘢痕组织，而不是正常生长。使用一个独特的人类肺细胞培养系统，我们将研究基因上的信息（“表观遗传”信息）如何使肺细胞以正常和异常的方式生长，目的是为IPF和其他肺部疾病开发新的治疗和预防策略。

DESCRIPTION (provided by applicant): 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-2), 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-2, 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. PUBLIC HEALTH RELEVANCE: Nobody knows why each year up to 34,000 Americans develop idiopathic pulmonary fibrosis, "IPF", a lethal disease in which the lungs are slowly destroyed by scar tissue, ultimately resulting in suffocation. Evidence suggests that in patients with IPF, certain lung cells differentiate into scar tissue instead of growing normally. Using a unique system of cultured human lung cells, we will examine how the information that is layered on top of the genes ("epigenetic" information) makes lung cells grow in normal as well as abnormal ways, with the objective to develop new treatment and prevention strategies for IPF and other lung diseases.