Targeted epigenetic activation of fibroblast genes in pulmonary fibrosis

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伤害，但该领域存在两个主要问题。首先，由于方法上的原因，对流行病学上确定的许多与乳腺癌相关的甲基化变化进行因果关系检验是不可能的。也就是说，没有办法特异性地将推定的表观遗传靶序列去甲基化，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的方法来满足这些需求。我们专注于一个特定的“测试案例”：肺纤维化，许多环境伤害的结果。我们试图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CXCL 11的表观遗传激活，CXCL 11是一种被甲基化抑制的潜在有益基因。为了特异性靶向和重新激活甲基化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靶向精度。 特定目标1将通过4种融合蛋白构建体优化3 T3成纤维细胞中CXCL 11启动子的靶向去甲基化，其中TDG通过聚甘氨酸接头与靶向鼠CXCL 11启动子的定制ZFA阵列结合。对照构建体将包括无催化活性的TDG（无脱甲基酶活性）和单独的ZFA。待测试的预测是，培养物将显示对刺激物（例如IFN？）并且将确认DNA去甲基化（通过焦磷酸测序CXCL 11启动子）。将通过全基因组转录谱分析评价预测的效应特异性。特异性目标2将测试靶向去甲基化在人肺成纤维细胞中的功效。由于对去甲基化的转录反应可能在不同的细胞类型中有所不同，为了增加研究的翻译潜力，我们将探索CXCL 11去甲基化在特发性肺纤维化（IPF）患者的人肺成纤维细胞中的作用。这些研究的成功完成将解决对特定方法的需求，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，并将为表观遗传疗法的开发提供平台。

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.