REST-mediated epigenomic and transcriptomic signatures in neuropathic pain

项目概要 慢性神经性疼痛很难治疗，尽管有大量的临床和治疗经验，但它仍然是一个主要的临床问题。 临床前研究。我们的长期目标是确定治疗/预防慢性神经病的新目标 疼痛。越来越多的证据表明，表观遗传机制和通过 microRNA (miR) 的调控是 与从急性疼痛到慢性疼痛的转变有关。该项目的目标是识别 REST- 和 G9a- 背根神经节（DRG）中相关的转录组和表观基因组调控网络及其在 神经性疼痛的发展。转录抑制因子 REST 是主要的表观基因组调节因子。在 神经损伤引起的神经性疼痛，DRG 神经元中 REST 过度表达导致的表观遗传沉默 与 Oprm1、Kcnd3、Kcnq2 和 scn10a 基因等靶标的抑制有关。组蛋白 甲基转移酶 G9a 也是许多转录因子（包括 REST）的表观基因组辅阻遏物。我们 最近发现 G9a 在 DRG 神经元中几乎所有 K 通道基因的表观遗传沉默中至关重要 在急性向慢性疼痛转变期间，基因启动子上的组蛋白 H3K9me2 增加。据此，小鼠 从 DRG 神经元中去除 G9a 后，神经损伤后不会出现慢性疼痛。因此，REST 和 G9a 提出合适的分子探针来破译慢性疼痛的遗传和表观遗传基础。然而， 关于与 REST 和 G9a 相关的转录组和表观基因组网络的知识有限 神经性疼痛的发展。这项多 PI 资助是两个实验室之间的合作，具有互补性 专业知识。除了上述 G9a 小鼠模型外，两个实验室还开发了一种创新型 实验系统由互补的 Rest 条件敲除 (cKO) 和新的条件敲除组成 人类 REST 过度表达 (coE) 小鼠模型。初步结果表明，虽然 DRG 特异性 休息 cKO 小鼠在神经损伤后表现出减弱的疼痛超敏反应，DRG 特异性 REST coE 小鼠表现出 疼痛过敏。因此，两种对比小鼠模型概括了慢性疼痛转变，如 基于该领域的工作预期，并提供一个体内系统来研究转录机制 慢性疼痛。在这里，我们提出检验 REST 和 G9a 参与的中心假设 通过独特的转录组和表观基因组特征，DRG 神经损伤后慢性疼痛的发展。如果 成功后，我们提出的研究将识别 DRG 神经元中的所有 REST 和 G9a 靶标，包括 mRNA 和 miR，以及 REST 和 G9a 介导的表观基因组变化在慢性疼痛的发展中 神经损伤。我们还将在功能上鉴定那些 REST 和 G9a 调节的 miR，可用于 阻断 REST 和 G9a 介导的慢性疼痛。我们将把我们的结果与化疗中获得的结果进行比较- 诱发慢性疼痛。总之，这项工作的结果很可能会产生新的签名和 可用于预测患者对慢性疼痛的易感性或抵抗力的生物标志物 发展。研究结果还可用于确定慢性疼痛的新的可行药物靶点。

PROJECT SUMMARY Chronic neuropathic pain is difficult to treat and remains a major clinical problem despite numerous clinical and preclinical studies. Our long-term goal is to identify new targets for treatment/prevention of chronic neuropathic pain. Mounting evidence suggests that epigenetic mechanisms and regulation via microRNAs (miRs) are associated with the transition from acute to chronic pain. The goal of this project is to identify REST- and G9a- related transcriptomic and epigenomic regulatory networks in the dorsal root ganglion (DRG) and their roles in the development of neuropathic pain. The transcriptional repressor REST is a major epigenomic regulator. In nerve injury-induced neuropathic pain, epigenetic silencing by REST overexpression in DRG neurons has been implicated in the repression of targets such as Oprm1, Kcnd3, Kcnq2, and scn10a genes. Histone methyl-transferase G9a is also an epigenomic corepressor of many transcription factors including REST. We recently found that G9a is critical in the epigenetic silencing of almost all K+ channel genes in DRG neurons via increased histone H3K9me2 on the genes' promoters during acute-to-chronic pain transition. Accordingly, mice with G9a ablated from DRG neurons do not develop chronic pain after nerve injury. Thus, both REST and G9a present suitable molecular probes to decipher the genetic and epigenetic bases of chronic pain. However, knowledge is limited about the transcriptomic and epigenomic networks associated with REST and G9a in neuropathic pain development. This multiple-PI grant is a collaboration between two labs with complementary expertise. In addition to the G9a mouse model described above, the two labs have developed an innovative experimental system consisting of complementary Rest conditional knock-out (cKO) and new conditional human REST overexpression (cOE) mouse models. Preliminary results indicate that whereas DRG-specific Rest cKO mice show attenuated pain hypersensitivity after nerve injury, DRG-specific REST cOE mice exhibit pain hypersensitivity. Thus, the two contrasting mouse models recapitulate the chronic pain transition, as anticipated based on work in the field, and provide an in vivo system to study transcriptional mechanisms of chronic pain. Here we propose to the test the central hypothesis that REST and G9a are involved in the development of chronic pain after DRG nerve injury via unique transcriptomic and epigenomic signatures. If successful, our proposed research will identify all REST and G9a targets in DRG neurons including mRNAs and miRs, as well as REST- and G9a-mediated epigenomic changes in the development of chronic pain after nerve injury. We will also functionally identify those REST- and G9a-regulated miRs that could be utilized to block REST- and G9a-mediated chronic pain. We will compare our results to those obtained in chemotherapy- induced chronic pain. In summary, the results from this work are likely to generate new signatures and biomarkers that could potentially be utilized to predict patient susceptibility or resistance to chronic pain development. The results could also be used to identify new actionable drug targets in chronic pain.