Chun Sung Kim

Evidence of Endogenous Mu Opioid Receptor Regulation by Epigenetic Control of the Promoters

ABSTRACT 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.

Epigenetic programming of μ-opioid receptor gene in mouse brain is regulated by MeCP2 and brg1 chromatin remodelling factor

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.

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene.

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator.

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

The effects of morphine are mediated mainly through the μ opioid receptor (MOR). Expression of the MOR is up-regulated 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.

Translational repression of mouse mu opioid receptor expression via leaky scanning.

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.

Mouse μ Opioid Receptor Distal Promoter Transcriptional Regulation by SOX Proteins

We have identified transcription factors that bind to specific sequences in 5′-distal promoter regulatory sequences of the mouse μ opioid receptor (mor) promoter using the yeast one-hybrid system. The sequence between −746 and −707 inmor distal promoter was used as the bait because it acts as a functional promoter element and binds several DNA-binding proteins. From an adult mouse brain cDNA library, five cDNA clones encoding three Sox gene family (Sry like high mobility group (HMG) box gene) transcriptional factors, mSOX18, mSOX21, and mSOX6, were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for SOX proteins in the −731/−725 region. Additionally, we have also established that the flanking regions outside the core Sox-binding site play an essential role in high affinity binding. DNase I footprint analysis indicates that proteins from mouse brain interact with the Sox-binding site within the mor distal promoter. Finally, we demonstrated that overexpression of mSOX18 and/or mSOX21 was able to up-regulate mouse mor distal promoter activity inmor-expressing neuronal cells (NMB). These data indicate that SOX proteins might contribute to the transcriptional activity of the mor gene and suggest that μ opioid receptor could mediate some of the developmental processes in which SOX proteins are included.

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

ABSTRACT Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells

MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA.

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Novel function of the poly(C)-binding protein αCP3 as a transcriptional repressor of the mu opioid receptor gene

The alpha‐complex proteins (αCP) are generally known as RNA‐binding proteins that interact in a sequence‐specific fashion with single‐stranded poly(C). These proteins are mainly involved in various post‐transcriptional regulations (e.g., mRNA stabilization or translational activation/silencing). Here we report a novel function of αCP3, a member of the αCP family. αCP3 bound to the double‐stranded poly(C) element essential for the mu opioid receptor (MOR) promoter and repressed the promoter activity at the transcriptional level. We identified αCP3 using affinity column chromatography containing the double‐stranded poly(C) element and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. αCP3 binding to the poly(C) sequence of the MOR gene was sequence specific, as confirmed by the supershift assay. In cotransfection studies, αCP3 repressed the MOR promoter only when the poly(C) sequence was intact. Ectopic expression of αCP3 led to repression of the endogenous MOR tran‐scripts in NS20Y cells. When αCP3 was disrupted using small interfering RNA (siRNA) in NS20Y cells, the transcription of the endogenous target MOR gene was increased significantly. Our data suggest that αCP3 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of αCP3 as a transcriptional repressor in MOR gene regulation.— Choi, H. S., Kim, C. S., Hwang, C. K., Song, K. Y., Law, P.‐Y., Wei, L.‐N., and Loh, H. H. Novel function of the poly(C)‐binding protein αCP3 as a transcriptional repressor of the mu opioid receptor gene. FASEB J. 21, 3963–3973 (2007)