ChIP-seq Profiling Identifies Histone Deacetylase 2 Targeting Genes Involved in Immune and Inflammatory Regulation Induced by Calcitonin Gene-Related Peptide in Microglial Cells

Abstract

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.

Epigenetic Connection of the Calcitonin Gene-Related Peptide and Its Potential in Migraine

“…Further works confirmed the role of CGRP in microglial activation and showed that it might mediate nociceptive signaling via the interaction with its receptors and ATP release in the dorsal horn [ 15 , 16 , 17 ]. Consistently, several studies suggest that CGRP plays an important role in the regulation of glial gene expression [ 18 ].…”

“…Therefore, CGRP is involved in neurogenic pain transmission through the methylation of H3 histone mediated by EZH2 in the spinal dorsal horn. Earlier, it was shown that mouse microglial cells (BV2) expressed CGRP receptors and the treatment of microglia with CGRP altered HDAC2 enrichment in the promoters of over 1200 genes in microglial cells; most of the HDAC2-enriched genes were linked to immune- and inflammation-related pathways [ 18 ]. Therefore, CGRP may induce an inflammatory response through the induction of differential HDAC2 enrichment in microglia cells.…”

Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain

“…The CGRP (1 μM, Tocris Bioscience), GSK126 (EZH2 inhibitor, 5 nM, MEC), CGRP8-37 (CGRP antagonist, 2 μM, MCE) or vehicle in a volume of 10 μL was injected into the spinal lumbar enlargement region through the intrathecal catheter, followed by 20 μL of saline to flush. Previous studies have demonstrated that these dosages of CGRP, GSK126, CGRP8-37, and other reagents in experiments proved to be effective in vivo and in vitro [8,9,13,18,19]. When the drug administration fell on the same day as the behavior analysis, behavior tests were completed prior to the drug administration.…”

“…It has been reported that CGRP induced the activation of microglia at the transcriptional level through expression of the immediate-early genes cfos in the spinal cord [10,12], suggesting that CGRP may play a physiological role as a regulator of microglial gene expression. Our previous research showed that CGRP is involved in the expression of immune and inflammation-related genes in microglia through epigenetic mechanism [13].…”

Calcitonin gene‐related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain

“… 7 , 8 There are a number of reports suggesting that H3K9ac enhances the transcription of cytokines and chemokines underlies the pathogenesis of chronic neuropathic pain after peripheral nerve injury. 9 , 10 Although some reports have shown that epigenetic mechanisms contribute to the development and maintenance of neuropathic pain, 11 , 12 further research is needed to clarify mechanisms regulating the expression of most key factors in this form of pain.…”