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：
8823321
负责人：
ALEXEY V FEDULOV
金额：
$ 26.57万
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 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8823321
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 Interferons 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 public health relevance pyrosequencing response success targeted sequencing 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. IFN?) 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（由申请人提供）：许多环境暴露通过对 DNA 甲基化的表观遗传效应造成长期危害，但该领域存在两个主要问题。首先，由于方法学原因，不可能测试流行病学上确定的许多与暴露相关的甲基化变化的因果关系。也就是说，还没有办法特异性地去甲基化假定的表观遗传目标序列，然后测试对基因表达和表型的预测影响。其次，需要新的治疗策略来特异性逆转与环境疾病相关的表观遗传变化。该项目将开发一种新方法来满足这些需求。我们关注一个特定的“测试案例”：肺纤维化，这是许多环境伤害的结果。我们寻求使用我们开发的靶向 DNA 去甲基化的新方法，在成纤维细胞中实现 CXCL11（一种受甲基化抑制的潜在有益基因）的表观遗传激活。为了特异性靶向并重新激活因甲基化而沉默的单个基因的表达，我们设计了脱甲基酶胸腺嘧啶-DNA-糖基化酶（TDG）与锌指蛋白阵列（ZFA）的DNA结合域（DBD）的融合复合物。锌指阵列提供了推进这种方法所需的瞄准精度。具体目标 1 将通过 4 个融合蛋白构建体优化 3T3 成纤维细胞中 CXCL11 启动子的靶向去甲基化，其中 TDG 通过聚甘氨酸接头与靶向小鼠 CXCL11 启动子的定制 ZFA 阵列结合。对照构建体将仅包括催化失活的 TDG（无脱甲基酶活性）和 ZFA。要测试的预测是，培养物将显示出对刺激（例如 IFN？）的转录反应性增加，并且 DNA 去甲基化将得到证实（通过对 CXCL11 启动子进行焦磷酸测序）。预测的效果特异性将通过全基因组转录谱进行评估。具体目标 2 将测试人肺成纤维细胞靶向去甲基化的功效。由于不同细胞类型对去甲基化的转录反应可能有所不同，为了增加研究的翻译潜力，我们将探讨 CXCL11 去甲基化对特发性肺纤维化 (IPF) 患者的人肺成纤维细胞的影响。这些研究的成功完成将解决对特定方法的需求，以测试环境表观遗传学鉴定的甲基化位点的功能重要性，并将为表观遗传学疗法的开发提供平台。