Targeted epigenetic activation of fibroblast genes in pulmonary fibrosis

肺纤维化中成纤维细胞基因的靶向表观遗传激活

基本信息

批准号：
8994282
负责人：
ALEXEY V FEDULOV
金额：
$ 22.19万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-12 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8994282
关键词：
Address Binding CXCL11 gene Cell Line Cell physiology Cells Chimeric Proteins Collagen Complex Custom DNA DNA Binding Domain DNA Methylation Data Development Disease Environmental Exposure Epigenetic Process Etiology Fibroblasts Fibrosis Gene Expression Gene Silencing Genes Genetic Transcription Genome Glycine Hamman-Rich syndrome Health Human Hypermethylation Individual Injury Interferon Type II Lead Link Lung Methods Methylation Mus Nitric Oxide Synthase Organism Outcome Pathway interactions Patients Phenotype Physiological Play Production Protein Array Proteins Pulmonary Fibrosis Research Role Site Specificity Stimulus Structure Test Result Testing Therapeutic Thymine DNA Glycosylase Zinc Fingers cell type demethylation design efficacy testing epidemiologic data fibrogenesis gene function genome-wide human disease improved novel novel therapeutics promoter pyrosequencing response success tool translational study

项目摘要

DESCRIPTION (provided by applicant): Many environmental exposures cause long-term harm through epigenetic effects on DNA methylation, but there are two major problems in the field. First, testing causality for the many exposure-related methylation changes identified epidemiologically has been impossible for methodologic reasons. Namely, there has been no way to specifically demethylate a putative epigenetic target sequence and then test predicted effects on gene expression and phenotype. Second, there is a need for novel therapeutic strategies to specifically reverse the epigenetic changes linked to environmental disease. This project will develop a novel method to address these needs. We focus on a specific 'test case': pulmonary fibrosis, an outcome of many environmental injuries. We seek to accomplish epigenetic activation of CXCL11, a potentially beneficial gene dampened by methylation, in fibroblasts using a novel method of targeted DNA demethylation that we have developed. To specifically target and re-activate the expression of individual genes silenced by methylation, we have designed fusion complexes of a demethylase Thymine-DNA-glycosylase (TDG) with DNA-binding domains (DBD) of zinc-finger protein arrays (ZFA). The zinc finger arrays provide the targeting precision needed to advance this approach. Specific Aim 1 will optimize targeted demethylation of the CXCL11 promoter in 3T3 fibroblasts via 4 fusion protein constructs in which TDG is bound via a poly-glycine linker to arrays of custom-built ZFAs targeting the murine CXCL11 promoter. Control constructs will include catalytically inactive TDG (no demethylase activity) and ZFAs alone. The predictions to be tested are that the cultures will show increased transcriptional responsiveness to stimuli (e.g. IFNγ) and that DNA demethylation will be confirmed (by pyrosequencing the CXCL11 promoter). The predicted specificity of the effect will be evaluated by genome-wide transcriptional profiling. Specific Aim 2 will test efficacy of targeted demethylation in human lung fibroblasts. Because transcriptional responsiveness to demethylation may vary in different cell types, and to increase translational potential of the stud we will explore the effect of CXCL11 demethylation in human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF). Successful completion of these studies will address the need for a specific method to test the functional importance of methylated sites identified by environmental epigenetics, and will provide a platform for development of epigenetic therapeutics.

描述（由适用提供）：许多环境暴露通过对DNA甲基化的表观遗传作用造成长期危害，但该领域有两个主要问题。首先，出于方法论原因，无法在流行病学上确定的许多与暴露有关的甲基化变化测试偶然性。也就是说，没有办法专门脱甲基化推定的表观遗传靶序列，然后测试预测对基因表达和表型的影响。其次，需要新颖的理论策略来特别扭转与环境疾病有关的表观遗传变化。该项目将开发一种新颖的方法来满足这些需求。我们专注于特定的“测试案例”：肺纤维化，这是许多环境伤害的结果。我们试图使用我们开发的新型靶向DNA脱甲基化方法来实现成纤维细胞中CXCL11的表观遗传激活，这是一种潜在的有益基因。为了特异性地靶向并重新激活被甲基化沉默的单个基因的表达，我们设计了脱甲基胸腺胺-DNA-糖基化酶（TDG）与锌指蛋白阵列（ZFA）的DNA结合结构域（DBD）的融合复合物。锌指阵列提供了推进这种方法所需的目标精度。特定的目标1将通过4个融合蛋白构建体优化3T3成纤维细胞中CXCL11启动子的靶向脱甲基化，其中TDG通过多聚糖接头绑定到针对鼠类CXCL11启动子的定制ZFA阵列。控制构建体将包括催化无效的TDG（无脱甲基酶活性）和ZFA。要测试的预测是，培养物将显示对刺激（例如IFNγ）的转录反应性增加，并且将确认DNA脱甲基化（通过对CXCL11启动子进行pyrosequencencence）。该作用的预测特异性将通过全基因组的转录分析进行评估。特定的目标2将测试靶甲基化在人肺成纤维细胞中的有效性。由于对脱甲基化的转录反应性可能在不同的细胞类型中有所不同，并且为了增加Stud的翻译潜力，因此，我们将探索来自特发性肺纤维化（IPF）患者的人肺成纤维细胞中CXCL11脱甲基化的影响。这些研究的成功完成将解决一种特定方法，以测试由环境表观遗传学鉴定的甲基化位点的功能重要性，并将为发展表观遗传学疗法提供一个平台。