Kyu Young Song

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.

Poly(C)-binding proteins as transcriptional regulators of gene expression

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.

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.

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.

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)

Novel function of neuron-restrictive silencer factor (NRSF) for posttranscriptional regulation

The neuron-restrictive silencer factor (NRSF) functions as a transcriptional repressor of neuronal genes in nonneuronal cells. However, it is expressed in certain mature neurons in adults, suggesting that it might have complex and novel roles depending on its cellular and physiological context. Overexpression of NRSF led to both increased opioid ligand-binding activity of the endogenous MOR and MOR-GFP fusion protein expression. In RNA immunoprecipitation and gel-shift assays, NRSF specifically interacted with the NRSE sequence of MOR mRNA. When MOR and NRSF genes were coexpressed, the specific ligand-binding activity of MOR was increased in neuroblastoma NMB cells, but decreased in PC12 cells result from its localization. Indeed, after overexpressing NRSF in NMB cells, the target RNA moved to the translationally active polysomal fraction. Overexpression of NRSF also led to enhanced phosphorylation of eIF4G. In contrast, knockdown of NRSF by siRNA transfection significantly decreased eIF4G phosphorylation. These findings indicate that NRSF may deliver the target MOR transcripts to the polyribosomal complex and activate eIF4G phosphorylation, resulting in translational activation. We report here a novel function of NRSF that enhance the translation of the mu opioid receptor (MOR) gene through its RNA binding sequence, the neuron-restrictive silencer element (NRSE).

Differential use of an in-frame translation initiation codon regulates human mu opioid receptor (OPRM1)

The pharmacological effects of morphine and morphine-like drugs are mediated primarily through the µ opioid receptor. Here we show that differential use of an in-frame translational start codon in the 5′-untranslated region of the OPRM1 generates different translational products in vivo and in vitro. The 5′-end of the OPRM1 gene is necessary for initiating the alternate form and for subsequent degradation of the protein. Initiation of OPRM1 at the upstream site decreases the initiation at the main AUG site. However, alternative initiation of the long form of OPRM1 produces a protein with a short half-life, resulting from degradation mediated by the ubiquitin–proteasome pathway. Reporter and degradation assays showed that mutations of this long form at the second and third lysines reduce ubiquitin-dependent proteasome degradation, stabilizing the protein. The data suggest that MOP expression is controlled in part by initiation of the long form of MOP at the alternate site.

Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

The pharmacological actions of morphine and morphine‐like drugs such as heroin mediate primarily through the mu opioid receptor (MOR). It represents the target of the most valuable painkiller in contemporary medicine. Here we report that poly(ADP‐ribose) polymerase 1 (PARP‐1) binds to the double‐stranded poly(C) element essential for the MOR promoter and represses promoter activity at the transcriptional level. We identified PARP‐1 by affinity column chromatography using the double‐stranded poly(C) element, followed by two‐dimensional gel electrophoresis and MALDI‐TOF mass spectrometry. PARP‐1 binding to the poly(C) sequence of the MOR gene was sequence‐specific as confirmed by the supershift assay. In cotransfection studies, PARP‐1 repressed the MOR promoter only when the poly(C) sequence was intact. When PARP‐1 was disrupted in NS20Y cells using siRNA, transcription of the endogenous target MOR gene increased significantly. Chromatin immunoprecipitation assays showed specific binding of PARP‐1 to the double‐stranded poly(C) element essential for the MOR promoter. Inhibition of PARP‐1s catalytic domain with 3‐aminobenzamide increased endogenous MOR mRNA levels in cultured NS20Y cells, suggesting that automodification of PARP‐1 regulates MOR transcription. Our data suggest that PARP‐1 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of PARP‐1 as a transcriptional repressor in MOR gene regulation.