H3K4me1和H3K27ac标记的增强子调控外周神经损伤后脊髓小胶质细胞激活的机制研究

Gene enhancer are noncoding segments of DNA that play a central role in regulating transcriptional programs. Abnormal production and activation of enhancers have been associate with many human diseases. Under the control of histone methyltransferases/acetyltransferases and several transcription factors, the monomethylation of the 4th lysine residue of H3 histone (H3K4me1) and the acetylation of the 27th lysine residue of H3 histone (H3K27ac) based on H3K4me1 mark the de novo and activated enhancers, respectively. It has been showed that peripheral nerve injury led to remarkable production and activation of enhancers in spinal microglia. Besides, our previous works demonstrate that peripheral nerve injury-induced neuropathic pain is alleviated by interfering the histone methyltransferases/acetyltransferases responsible for H3K4me1 and H3K27ac. However, the mechanism of transcription factors in regulating spinal microglial enhancers has not been determined following peripheral nerve injury, and the role of de novo and activated enhancer is also not clear in microglial activation. In this study, microglia will be harvested and sorted from the spinal cord after peripheral nerve injury, and the relationship between de novo and activated enhancers and gene transcription in microglia will be explored, by using the technique of chromatin immunoprecipitation sequencing and bioinformatics analysis. Afterwards, the role will be investigated about potential transcription factors in regulating the H3K4me1- and H3K27ac-marked enhancers at different DNA loci. Finally, the effects of enhancer production and activation in regulating microglial activation will be determined in vitro and in vivo. The above investigations will unveil the role of de novo and activated enhancers in the pathology of spinal microglial activation, so as to explore new strategies for the prevention and treatment of the neuropathic pain following peripheral nerve injury.

增强子是调控基因转录的重要DNA元件，它的异常产生和激活与人类多种疾病相关。H3组蛋白4位点赖氨酸单甲基化修饰（H3K4me1）和在此基础上发生H3组蛋白27位点乙酰化修饰（H3K27ac）分别是增强子产生和激活的重要标志，这一过程受到转录因子和组蛋白酶等因子的调控。外周神经损伤可重塑脊髓小胶质细胞增强子，我们在前期研究中也发现干预参与H3K4me1和H3K27ac修饰的组蛋白酶可缓解外周神经损伤导致的神经病理性疼痛，但转录因子对增强子的调控机制及增强子重塑在小胶质细胞激活中的作用尚不明确。本研究采用染色质免疫共沉淀-测序和生物信息学分析等技术探索外周神经损伤后脊髓小胶质细胞增强子产生和激活与基因转录的联系，通过基因干预等技术明确转录因子对不同DNA位点增强子的调控作用，并在体外和体内验证增强子调控小胶质细胞激活的作用机制，为小胶质细胞激活所介导的神经病理性疼痛等重大疾病防治提供新的思路。

本研究主要探讨了标记增强子的组蛋白修饰在脊髓背角小胶质细胞激活和神经病理性疼痛发病中的作用。我们通过构建外周神经损伤动物模型，探索了脊髓背角小胶质细胞中标记增强子的组蛋白修饰在神经病理性疼痛发病过程中的变化规律，验证了H3组蛋白4位点赖氨酸单甲基化修饰（H3K4me1）介导下游基因转录与小胶质细胞激活的联系。通过组织细胞分选和动物实验验证，明确了组蛋白甲基转移酶SETD7是调控脊髓背角H3K4me1变化的关键上游因子，SETD7调控H3K4me1修饰并进而控制小胶质细胞中疼痛相关基因转录。体外和体内实验证实SETD7介导H3K4me1修饰是调控小胶质细胞形态和功能变化的关键通路。采用特异性的SETD7单甲基化活性抑制剂PFI-2可逆转脊髓背角小胶质细胞激活和缓解神经病理性疼痛。本课题研究证实了组蛋白甲基转移酶SETD7介导H3K4me1修饰在神经病理性疼痛发病中的作用，抑制SETD7单甲基化活性可能成为神经病理性疼痛治疗的潜在手段。