Targeted epigenetic activation of fibroblast genes in pulmonary fibrosis

描述（由申请人提供）：许多环境暴露通过表观遗传效应对DNA甲基化造成长期危害，但该领域存在两个主要问题。首先，由于方法学上的原因，在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测试是不可能的。也就是说，目前还没有办法特异性地去甲基化一个假定的表观遗传靶序列，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专门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，这是许多环境伤害的结果。我们试图利用我们开发的一种靶向DNA去甲基化的新方法，在成纤维细胞中实现CXCL11的表观遗传激活，CXCL11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被甲基化沉默的单个基因的表达，我们设计了去甲基化酶胸腺嘧啶- 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瞄准精度。Specific Aim 1将通过4种融合蛋白构建优化3T3成纤维细胞中CXCL11启动子的靶向去甲基化，其中TDG通过聚甘氨酸连接物与针对小鼠CXCL11启动子的定制ZFAs阵列结合。对照结构将包括催化无活性的TDG（无去甲基酶活性）和单独的ZFAs。待测试的预测是，培养物将显示出对刺激（例如IFN?）的转录反应性增加，DNA去甲基化将被证实（通过对CXCL11启动子进行焦磷酸测序）。该效应的预测特异性将通过全基因组转录谱进行评估。特异性Aim 2将测试靶向去甲基化在人肺成纤维细胞中的效果。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，并且为了增加该研究的翻译潜力，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一种特定方法的需求，以测试由环境表观遗传学确定的甲基化位点的功能重要性，并将为表观遗传学治疗的发展提供一个平台。

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.