Calcitonin gene-related peptide (CGRP) is a mediator of microglial activation at the transcriptional level. The involvement of the epigenetic mechanism in this process is largely undefined. Histone deacetylase (HDAC)1/2 are considered important epigenetic regulators of gene expression in activated microglia. In this study, we examined the effect of CGRP on HDAC2-mediated gene transcription in microglial cells through the chromatin immunoprecipitation sequencing (ChIP-seq) method. Immunofluorescence analysis showed that mouse microglial cells (BV2) expressed CGRP receptor components. Treatment of microglia with CGRP increased HDAC2 protein expression. ChIP-seq data indicated that CGRP remarkably altered promoter enrichments of HDAC2 in microglial cells. We identified 1271 gene promoters, whose HDAC2 enrichments are significantly altered in microglia after CGRP treatment, including 1181 upregulating genes and 90 downregulating genes. Bioinformatics analyses showed that HDAC2-enriched genes were mainly associated with immune- and inflammation-related pathways, such as nitric oxide synthase (NOS) biosynthetic process, retinoic acid-inducible gene- (RIG-) like receptor signaling pathway, and nuclear factor kappa B (NF-κB) signaling pathway. The expression of these key pathways (NOS, RIG-I, and NF-κB) were further verified by Western blot. Taken together, our findings suggest that genes with differential HDAC2 enrichments induced by CGRP function in diverse cellular pathways and many are involved in immune and inflammatory responses.

中文翻译：

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ChIP-seq分析鉴定了组蛋白脱乙酰基酶2靶向基因，涉及降钙素基因相关肽在小胶质细胞中诱导的免疫和炎症调节。

降钙素基因相关肽（CGRP）在转录水平上是小胶质细胞激活的介体。表观遗传机制在这个过程中的参与在很大程度上是不确定的。组蛋白脱乙酰基酶（HDAC）1/2被认为是激活的小胶质细胞中基因表达的重要表观遗传调控因子。在这项研究中，我们通过染色质免疫沉淀测序（ChIP-seq）方法检查了CGRP对小胶质细胞中HDAC2介导的基因转录的影响。免疫荧光分析表明，小鼠小胶质细胞（BV2）表达CGRP受体成分。CGRP治疗小胶质细胞可增加HDAC2蛋白的表达。ChIP-seq数据表明，CGRP显着改变了小胶质细胞中HDAC2的启动子富集。我们确定了1271个基因启动子，CGRP处理后，其小胶质细胞中HDAC2的富集度发生了显着变化，包括1181个上调基因和90个下调基因。生物信息学分析表明，富含HDAC2的基因主要与免疫和炎症相关的途径有关，例如一氧化氮合酶（NOS）的生物合成过程，视黄酸诱导型基因（RIG-）受体信号传导途径以及核因子kappa B（NF-κB）信号通路。这些关键途径（NOS，RIG-I，和NF-的表达κ B）通过Western印迹进一步证实。综上所述，我们的发现表明，由CGRP诱导的HDAC2差异富集的基因在多种细胞途径中起作用，许多基因参与免疫和炎症反应。