DNA Methylation Profiling of Chronic Pain

描述（由申请人提供）：DNA胞嘧啶甲基化是“细胞记忆”的守护者，稳定且能够（共同）决定基因表达。新生胞嘧啶甲基化发生在发育过程中，如果在成人中不适当地激活，可引起疾病。慢性疼痛通常在没有明显损伤的情况下出现，或在组织损伤愈合后持续存在。在啮齿类动物单神经结扎（SNL）疼痛模型中，L4和L5背根神经节（DRG）神经元和胶质细胞随着电生理、基因表达和细胞可塑性的改变而功能失调。这些变化背后的基本分子力是未知的。在这里，我们提出慢性疼痛反映在背根神经节细胞的DNA胞嘧啶甲基化变化中，并可能由其驱动。我们暗示胞嘧啶甲基化可能构成一个新的概念层，以添加到已知的疼痛机制。该假说认为，成人DRG神经元和/或胶质细胞中的胞嘧啶甲基化是可塑的；它会因实验性神经损伤而改变；它（共同）决定了疼痛状态特征的基因表达模式；这也是慢性疼痛患者难以康复的原因之一。我们的建议有两个目的。目的1：检测神经性疼痛大鼠DRGs中胞嘧啶甲基化是否发生全局改变。HELP检测（=HpaII微小片段通过连接介导PCR富集）将用于对100万个HpaII微小片段（HTF）进行全基因组分析，这些片段反映了SNL或假手术后14天收获的L4和L5 DRGs中约140万个独特的HpaII/MspI位点的甲基化状态。分析将确定胞嘧啶甲基化改变的基因组区域，以及甲基化变化最显著的个体位点和/或大基因调控区域。目的2：检测胞嘧啶甲基化是否在预测的调控区域发生局部改变。将从文献和HELP结果中选择70个疼痛基因，使用MassArray（一种结合碱基特异性切割和质谱的甲基化分析方法）对每个转录起始位点周围的每个CpG进行深入的甲基化分析。目标1和目标2的研究成果将在一组独立的动物中进行验证。如果现在进行，这项提议的研究将是第一个研究疼痛中的DNA胞嘧啶甲基化的研究。

DESCRIPTION (provided by applicant): DNA cytosine methylation is a keeper of "cellular memory", stable and able to (co-)determine gene expression. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause disease. Chronic pain often arises without visible insult or persists after tissue injury has healed. In the rodent single nerve ligation (SNL) pain model, L4 and L5 dorsal root ganglion (DRG) neurons and glia become dysfunctional with altered electrophysiology, gene expression, and cellular plasticity. The basic molecular force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to known pain mechanisms. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic; that it is altered by experimental nerve injury; that it (co-)determines gene expression patterns characteristic of the pain state; and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. The HELP assay (=HpaII tiny fragment Enrichment by Ligation-mediated PCR) will be used to perform a genome-wide analysis of 1 million HpaII tiny fragments (HTF) reflecting the methylation status of ~1.4 million unique HpaII/MspI sites in the L4 and L5 DRGs harvested 14 days after SNL or sham operation. Analyses will identify genomic regions where cytosine methylation is altered and sets of individual loci and/or of large gene regulatory regions where methylation changes are most marked. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using MassArray, a method of methylation analysis combining base-specific cleavage and mass-spectrometry. Research outcomes of Aims 1 and 2 will be validated in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain. PUBLIC HEALTH RELEVANCE: Cytosine is one out of the four building blocks that make up DNA. Unlike the other three building blocks, cytosine can be permanently chemically modified through a process called DNA cytosine methylation. This process leads to "methyl-cytosine", which is often referred to as the "fifth base" of the genome. Methyl cytosine is a keeper of "cellular memory", stable and able to (co-)determine how genes function. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause diseases like cancer. Chronic pain often arises without a clear cause and can persist after tissue injury has healed. In the rodent model, two small structures of the nervous system located near the spinal cord, the L4 and L5 dorsal root ganglion (DRG) become dysfunctional. The basic force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to our current knowledge about how chronic pain arises. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic; that it is altered by experimental nerve injury; that it (co- )determines gene expression patterns characteristic of the pain state; and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. We will answer this question using a new technology, which we have developed, the HELP assay. This method combines digestion of DNA, amplification, and binding to 2.1 million probes on a glass slide. It allows us to obtain a picture of cytosine methylation for the entire genome in a single experiment. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using another method termed MassArray, which has specific advanteages when results with high resolution are desired. Research outcomes of Aims 1 and 2 will be confirmed in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain.