Up-Regulation of the μ-Opioid Receptor Gene Is Mediated through Chromatin Remodeling and Transcriptional Factors in Differentiated Neuronal Cells

Abstract

The effects of morphine are mediated mainly through the opioid receptor (MOR). Expression of the MOR is upregulated during neuronal differentiation in P19 embryonal carcinoma cells and epigenetic changes play an important role in MOR up-regulation. This study investigates the basis for differentiation-dependent alterations of MOR chromatin by studying the recruitment or dissociation of several factors to the remodeled chromatin locus. Chromatin immunoprecipitation assays were used to demonstrate the recruitment of the transcriptional activator Sp1 and the chromatin remodeling factors Brg1 and BAF155 to this promoter, as well as the dissociation of repressors [histone deacetylases, mSin3A, Brm, and methyl-CpG-binding protein 2 (MeCP2)]. Histone modifications (acetylation, induction of histone H3-lys4 methylation, and reduction of H3-lys9 methylation) were consistently detected on this promoter. Overexpression of Sp1 strongly enhanced MOR promoter activity, and the histone deacetylase inhibitor trichostatin A also increased promoter activity. In vitro DNA CpG-methylation of the promoter partially blocked binding of the Sp1 factor but induced MeCP2 binding. Coimmunoprecipitation studies also found novel evidence of an endogenous MeCP2 interaction with Sp3 but a weaker interaction with Sp1. Overall, the results suggest that during neuronal differentiation, MeCP2 and DNA methylation mediate remodeling of the MOR promoter by chromatin remodeling factors (Brg1 and BAF155) from a compacted state to a conformation allowing access for transcriptional factors. Subsequent recruitment of the activating transcription factor Sp1 to the remodeled promoter results in MOR up-regulation.Opioid drugs, including endogenous opioid peptides and the analgesic drug morphine, exert their pharmacological and physiological effects through binding to opioid receptors. The three major types of opioid receptors (, ␦, and ) all belong to the G protein-coupled receptor superfamily. On agonist binding, these receptors couple to G proteins and affect several signal transduction pathways, including inhibition of adenylyl cyclases and Ca 2ϩ channels, activation of inward-rectifying K ϩ channels, transient increases of intracellular Ca 2ϩ levels, and activation of phospholipase C and the mitogen-activated protein kinases extracellular signalregulated kinases 1 and 2 (Law et al., 2000).The opioid receptor (MOR) is expressed mainly in the central nervous system with densities varying greatly in different brain regions displaying different functional roles (Mansour et al., 1995). The MOR-expressing brain regions are involved in motivating and rewarding behaviors mediated by opiates and other drugs of abuse. Furthermore, response to morphine varies in different neuronal cells and brain regions depending on receptor levels. Several studies suggest that the MOR plays a key role in mediating the major

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1

“…As reported earlier from our laboratory (Hwang et al, 2010), the CG residues of the endogenous MOR promoter in P19 cells are heavily methylated and are recognized by methyl-CpG-binding protein 2, which recruits repressive Brm proteins under unstimulated conditions. However, upon neuronal differentiation of P19 cells or stimulation with TSA, these repressive chromatin marks are removed, which allows the binding of activating transcription factors such as SP1.…”

“…P19 cells can be induced to differentiate into neuronal cells by retinoic acid, a process that closely resembles mammalian neurogenesis in vivo (Monzo et al, 2012). As reported earlier, MOR gene expression starts to increase within 2 days after the induction of differentiation, and continues further as the cells fully differentiate into neuronal subtypes (Hwang et al, 2010). Interestingly, phosphorylation of all three major MAPKs (p38 MAPK, ERK 1/2, and JNK) also increases during neuronal differentiation of P19 cells, and follows MOR expression kinetics, among which pharmacological inhibition of p38 MAPK abrogates MOR gene expression (Wagley et al, 2013).…”

“…Similar reactions were performed using b-actin as an internal control (Hwang et al, 2007). qRT-PCR was performed as previously described (Hwang et al, 2010) using the Quantitect SYBR Green RT-PCR kit (Valencia, CA). To calculate relative mRNA gene expression, amplification curves of test sample and standard samples that contained 10 1 to 10 6 molecules of the MOR gene (using pcDNA3.1-mMOR, constructed in our laboratory) were monitored, and the number of target molecules in the test sample was analyzed using qCalculator version 1.0 software (http://www.genequantification.de/download.html#qcalculator) on the basis of the mathematical model of Pfaffl (Pfaffl, 2001).…”

“…Nevertheless, a number of recent studies have shown that MOR gene transcription is activated in response to various extracellular stimuli and follows multiple coordinated intracellular signals that converge at the MOR promoter (Kim et al, 2011;Wagley et al, 2013). Quite interestingly, MOR gene expression follows a unique spatial and epigenetic regulation in different mouse brain regions where opioid drugs act (Hwang et al, 2009 such, stimulation of P19 embryonic carcinoma cells with epigenetic modifiers such as a histone deacetylase inhibitor or a demethylating agent has been shown to increase de novo transcription of MOR gene in a tightly regulated event that repositions nucleosomes to mount the transcription machinery (Hwang et al, 2007(Hwang et al, , 2010). Yet, the precise role of signal transduction events during this critical chromatin-directed event remains largely elusive.…”

IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus

“…Brg1 interacts with several transcription factors, including Sp1, to activate target genes (9,22,23,41,47,51). Because Brg1 does not contain a sequence-specific DNA-binding domain, the selective recruitment of the SWI/SNF complex to target genes depends on protein-protein interactions through Brg1 and other transcription factors or transcription regulators (28,29,51).…”