The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis-and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5-aza-2-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.Opioids exert their pharmacological and physiological effects through binding to their endogenous receptors. Three types of opioid receptors, mu (), delta (␦), and kappa (), all belonging to the G-protein-coupled receptor superfamily, have been cloned. Upon agonist binding, these receptors couple to G proteins and affect several signal transduction pathways thought to mediate a broad range of functions and pharmacological effects of endogenous and exogenous opioids (51). Previous studies suggested that the opioid receptor (MOR) plays a key role in mediating the major clinical effects of analgesics, such as morphine, as well as the development of tolerance and physical dependence upon prolonged administration (39). MOR is mainly expressed in the central nervous system, with densities varying greatly in different regions, which can display different functional roles (55). During mouse embryonic development, the MOR message was specifically observed as early as embryonic day 8.5 (E8.5) using the reverse transcription (RT)-PCR method (44). In contrast, MOR transcripts were detected only beginning at E12 using the radioligand binding method (70) and at E10.5 by in situ hybridization (85). Transcript levels gradually incr...

Poly(C)-binding proteins (PCBPs) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). They can be divided into two groups: hnRNP K and PCBP1-4. These proteins are involved mainly in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). In this review, we summarize and discuss how PCBPs act as transcriptional regulators by binding to specific elements in gene promoters that interact with the RNA polymerase II transcription machinery. Transcriptional regulation of PCBPs might itself be regulated by their localization within the cell. For example, activation by p21-activated kinase 1 induces increased nuclear retention of PCBP1, as well as increased promoter activity. PCBPs can function as a signal-dependent and coordinated regulator of transcription in eukaryotic cells. We address the molecular mechanisms by which PCBPs binding to single-and double-stranded DNA mediates gene expression. KeywordsPoly(C)-binding proteins; p21-activated kinase 1; DNA-binding proteins; Transcriptional regulationThe poly(C)-binding proteins (PCBPs) are characterized by high affinity for, and sequencespecific interaction with polycytosine, poly(C). In mammalian cells, these PCBPs belong to one of two subsets: hnRNP K/J, or the alpha-complex proteins (e.g., PCBP1-4) [1]. hnRNP K, PCBP1, and PCBP2 have been studied in the greatest detail. The latter two proteins are also known as αCP1 and αCP2, or hnRNPE1 and hnRNPE2 [2,3]. Recently, two other members of the αCP family were discovered: PCBP3 (αCP3) and PCBP4 (αCP4) [4].PCBPs are expressed broadly in human and mouse tissues and demonstrate poly(C)-binding specificity [2,4,5]. All members of the PCBP family are related evolutionarily. The common feature of all PCBPs is the presence of three hnRNP K homology (KH) domains [1]; these are RNA-binding modules of about 70 amino acids in length. PCBP1 and PCBP2 share the highest level of amino acid sequence similarity (89%) [6]. PCBP3 is more divergent, and PCBP4 is the most distantly related (52% divergence from PCBP2 [4,7]. Members of this family perform multiple functions through their poly(C)-binding ability, including mRNA stabilization [8- Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Structure of PCBPsThe PCBPs contain three KH domains, two consecutive KH domains at the amino terminus and a third KH domain at the carboxyl terminus, separated by an intervening sequence of variable length (Fig. 1A). The structure of each KH domain consists of three α-helices...

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The pharmacological action of morphine as a pain medication is mediated primarily through the μ-opioid receptor (MOR). With few exceptions, MOR is expressed in brain regions where opioid actions take place. The basis for this unique spatial expression of MOR remains undetermined. Recently, we reported that DNA methylation of the MOR promoter plays an important role in regulating MOR in P19 cells. In this study, we show that the differential expression of MOR in microdissected mouse brain regions coincides with DNA methylation and histone modifications. MOR expression could be induced by a demethylating agent or a histone deacetylase inhibitor in MOR-negative cells, suggesting that the MOR gene can be silenced under epigenetic control. Increases in the in vivo interaction of methyl-CpG-binding protein 2 (MeCP2) were observed in the cerebellum, in which the MOR promoter was hypermethylated and MOR expression was the lowest among all brain regions tested. MeCP2 is associated closely with Rett syndrome, a neurodevelopmental disorder. We also established novel evidence for a functional role for MeCP2’s association with the chromatin-remodelling factor Brg1 and DNA methyltransferase Dnmt1, suggesting a possible role for MeCP2 in chromatin remodelling during MOR gene regulation. We conclude that MOR gene expression is epigenetically programmed in various brain regions and that MeCP2 assists the epigenetic program during DNA methylation and chromatin remodelling of the MOR promoter.

Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5′-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions.