TET1-mediated 5-hydroxymethylcytosine modification & airway hyperresponsiveness

 描述(申请人提供)：哮喘是一种以呼吸道高反应性(AHR)和呼吸道炎症为特征的复杂疾病，目前困扰着全球超过3亿人。尽管遗传因素无疑在哮喘中发挥了作用，但哮喘患病率的快速上升表明，环境因素可能也起着同样重要的作用。到目前为止，表观遗传调控被认为至少部分地介导了可能导致哮喘的复杂的基因与环境的相互作用。到目前为止，大多数研究都集中在阐明容易获得的细胞中的DNA甲基化模式，包括T细胞和B细胞 解释哮喘患者过敏原致敏的免疫学机制。很少有研究研究与慢性肺功能改变有关的环境诱因对肺组织的表观遗传学后果。我们利用下一代测序技术和适当的验证分析来鉴定一组新的基因，这些基因高度丰富Tgfb2信号分子，并与常见过敏原--屋尘螨(HDM)的暴露有关，并通过对呼吸道平滑肌(ASM)细胞表型的表观遗传调节而与AHR的发展相关。此外，我们还发现了一种新的5-MC双加氧酶TET1在体外调节ASM功能和体内过敏原驱动的AHR的发展中的作用。我们初步证实，Tet1缺乏降低了HDM诱导的小鼠急性AHR。引人注目的是，我们能够翻译人类哮喘ASM细胞的表观遗传学变化；表明Tet1介导的羟甲基化可能影响ASM细胞的增殖和收缩。我们的研究首次证明了Tet1蛋白在过敏原暴露的背景下受到调节，尽管调节过敏原诱导Tet1的机制尚不清楚。我们的初步数据支持，HDM暴露产生的氧化应激激活了Tet1及其介导的ASM基因上调。基于这些新的发现，我们提出了中心假说：HDM通过调节氧化还原循环激活ASM细胞中Tet1介导的羟甲基化。这种表观遗传的重新编程在ASM中持续很长一段时间，因此可能有助于ASM细胞的增殖、僵硬和收缩能力在人类哮喘中出现。为了解决这些新的假设，我们组建了一个多学科的研究团队，他们拥有广泛的专业知识，涵盖表观遗传学、ASM生物学、肺病学和病理生物学等领域。首先，我们将确定Tet1介导的DNA羟甲基化在慢性HDM诱导的AHR小鼠模型中AHR表型发展中的作用。其次，我们将在体外和体内的小鼠模型中，通过NADH的氧化还原循环来研究HDM如何改变Tet1的活性。最后，我们将确认TET1介导的羟甲基化是否调节人ASM细胞的表型。拟议的实验结果将为ASM及其在哮喘过度狭窄中的作用提供新的视角。这些见解可能会推动治疗这种衰弱疾病的新治疗方法的发展。

 DESCRIPTION (provided by applicant): Asthma is a complex disease characterized by airway hyperresponsiveness (AHR) and airway inflammation, which current afflicts over 300 million people worldwide. Although genetic factors unquestionably play a role in asthma, the rapid rise in asthma prevalence suggests that environmental factors likely play an equally important role. To date, epigenetic regulation is suggested to mediate, at least partly, the complex gene-by- environment interactions that can lead to asthma. Most studies to date have focused on elucidation of DNA methylation patterns in easily accessible cells including T cells and B cells to explain the immunological mechanisms driving allergen sensitization in asthmatics. Few studies have examined the epigenetic consequences of environmental triggers on lung tissues linked to chronic alterations in lung functions. We have utilized next generation sequencing techniques and proper validation assays to identify a novel set of genes which is highly enriched for Tgfb2 signaling molecules and associated with exposure to the common allergen, house dust mite (HDM), and the development of AHR through epigenetic modulation of the airway smooth muscle (ASM) cell phenotypes. In addition, we identified a novel role of the specific 5-mC dioxygenase, TET1, in the regulation of ASM function in vitro and in the development of allergen-driven AHR in vivo. Preliminarily, we demonstrated Tet1 deficiency reduced acute HDM-driven AHR in mice. Strikingly, we were able to translate the epigenetic changes in human asthmatic ASM cells; showing Tet1-mediated hydroxymethylation may influence ASM cell proliferation and contraction. Our study represents the first demonstration that Tet1 protein is regulated in the context of allergen exposure, although the mechanisms regulating Tet1 induction by allergens are unknown. Our preliminary data supported that oxidative stress generated by HDM exposure activates Tet1 and its mediated upregulation of ASM genes. Based on these novel findings, we propose the central hypothesis "HDM activates Tet1-mediated hydroxymethylation in ASM cells through regulation of redox cycling. This epigenetic reprogramming persists in ASM over long time spans, thus likely contributes to the increased ASM cell proliferation, stiffness and contractility seen in human asthma". To address these novel hypotheses, we have assembled a multi-disciplinary team of investigators with a breadth of expertise spanning the fields of epigenetics, ASM biology, pulmonology and pathobiology. First, we will determine the role of Tet1- mediated DNA hydroxymethylation in the development of the AHR phenotype in a chronic HDM-induced AHR mouse model. Second, we will investigate how HDM alters the Tet1 activity, through redox cycling of NADH in both in vitro and in vivo mouse model. Lastly, we will confirm if TET1-mediated hydroxymethylation regulates human ASM cell phenotypes. The results of the proposed experiments should provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma. These insights may fuel the development of new therapeutic approaches for the treatment of this debilitating disease.