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.

Figures

FIG. 1.

Differential opioid receptor expression during neuronal differentiation of P19 EC cells. To induce neuronal differentiation, P19 EC cells were plated on bacterial petri dishes and allowed to aggregate for 4 days in the presence of 0.5 μM RA and then replated on tissue culture dishes without RA. For each day after plating, the cells were harvested and used for RNA or protein isolations as described in Materials and Methods. (A) The sizes of MORs (mMOR-S1 and mMOR-AS1 [Table 1] spanning exons 1 and 2, respectively) and β-actin (9) PCR fragments are 350 bp and 230 bp, respectively. PCRs for opioid receptors (MOR, DOR, and KOR) and β-actin consisted of 32 and 20 cycles, respectively. RA, all-trans retinoic acid. The DNA sequences of PCR products were confirmed by sequencing. Lanes 1 and 9 (M) are 100-bp size markers from Invitrogen. Samples for lane 3 consisted of control P19 cells cultured identically to the differentiated culture but without RA treatment. Undifferentiated cells (normal) are shown as a control. P19 cells cultured from 1 to 5 days after plating are indicated in lanes 4 to 8. Expression profiles of other opioid receptor genes (δ as DOR and κ as KOR) in this P19 differentiation were also included and analyzed by RT-PCR. (B) Quantitative analyses were performed on the receptor PCR band signal. Data are presented with the receptor signal normalized to the β-actin signal and the relative band signal to the normal P19 signal. The data are shown as means ± standard errors of the mean from three independent experiments. (C) In order to determine if neuronal differentiation of P19 cells occurred, PCR primers (Table 1) for two neuronal markers (N-cadherin and βIII-tubulin) and GFAP as a glial cell marker were used for RT-PCR. The MOR PCR was included to monitor the integrity of the experiment, and β-actin PCR was used as a control. Adult mouse brain and NS20Y cells were used as positive and negative control samples, respectively. UD, undifferentiated P19 cells. (D) Expression of the MOR and βIII-tubulin genes in P19 cells analyzed by real-time qRT-PCR. Levels of MOR mRNA were determined by real-time PCR analysis using normal and differentiated P19 cells. Five micrograms of total RNA was treated with DNase I and reverse transcribed using reverse transcriptase (Roche) and primers combined with oligo(dT) and random hexamer. One-fortieth of this cDNA sample was used for real-time qRT-PCR analysis of gene expression, using the Quantitect SYBR Green PCR kit (QIAGEN) in an iCycler (Bio-Rad). The relative expression of mRNA species was calculated using the comparative threshold cycle method as described in Materials and Methods after normalization against β-actin as an internal control. Primer sequences are shown in Table 1. (E) Northern blot analysis of a neuronal marker, N-cadherin, during neuronal differentiation using a PCR probe generated from the primers described for panel C. N-cadherin levels increase early in neuronal differentiation and steadily throughout differentiation (lanes 2 to 5). (F) Induction of βIII-tubulin, as revealed by Western blot analysis, confirmed the proper neuronal differentiation of P19 EC cells.

FIG. 2.

TSA, VPA, and 5-aza-dC treatment induced MOR gene expression in P19 cells. The results of RT-PCR analyses on the MOR mRNA levels in P19 cells treated with different doses of 5-aza-dC (A), TSA (C), or VPA (D) using the procedure described in Materials and Methods are shown. The TSA and VPA samples were treated for 6 h and 24 h, and 5-aza-dC samples were treated for 72 h (3 days) to induce maximal effects on MOR levels in P19 cells. The neuronal markers N-cadherin and βIII-tubulin were included in RT-PCR (N-cadherin in panel A) and Western blotting (N-cadherin and βIII-tubulin in panel B) analyses to demonstrate that P19 cell differentiation was not induced by 5-aza-dC treatment. β-Actin was used as a control.

FIG. 3.

Methylation statuses of the promoter regions of the MOR gene in P19 cells. (A) The 5′-flanking region of the MOR gene contains 21 putative methyl CpG sites from −569 to +33 (with the ATG start codon designated +1). The numbers at the top of the figure (No. CpG) are arbitrary designations to indicate each methyl CpG site. ppTIS indicates the TISs of the major MOR PP containing four sites (61). The methylation statuses of the MOR gene in normal P19 (UD), AP2d (2 days after plating, i.e., intermediately differentiated P19 cells), and AP4d (4 days after plating, i.e., fully differentiated P19 cells) were determined by bisulfite genomic sequencing. Methylation-specific PCR was performed using primers MS-630 and MAS + 65 (Table 1), followed by TA cloning (Invitrogen). Each row of circles represents a single cloned allele, and each circle indicates a single CpG site at a specific location. The methylation statuses of 20 individual clones were analyzed for each cell type. The filled and open circles represent the methylated and unmethylated CpG sites, respectively. The percentages of methyl CpG versus unmethylated CpG are indicated for the first nine CpG sites. (B) Differentiation-dependent methylation changes within the MOR promoter. The percentages of methylation at CpG sites in the MOR promoter from the region of base pairs −233 to −569 (*, P n ≥ 3). For statistical analysis, the data are representative of three independent experiments with sequencing data of at least 10 clones for each sample, which were used to quantify the percentage of methylation in the above-mentioned CpG sites. (C) Methylation statuses of the DP and coding exon 1 region of the MOR gene in P19 cells. For the DP and its upstream region, methylation-specific PCR was performed using the primers MS-1754 and MAS-927 (Table 1), followed by TA cloning (Invitrogen) as described for panel A. Methylation-specific PCR primers MS + 19 and MAS + 352 (Table 1) were also used for the coding exon 1 and a part of intron 1. dpTIS indicates a TIS of the DP (46). Except as noted above, experiments were performed as described for panel A. The junction site of exon 1 and intron 1 is +285, as indicated. (D and E) Demethylation of the MOR gene promoters in P19 cells after 5-aza-dC treatment. The DNA methylation statuses of the basal proximal (D) and distal (E) MOR promoters in P19 cells after treatment with 5-aza-dC are shown. The results of 20 clones assayed by bisulfite-sequencing analyses are presented. The filled and open circles indicate the methylated and unmethylated CpG sites, respectively.

FIG. 4.

Differential expression from dual promoters of MOR. (A) Total RNAs from P19 and 5-aza-dC-treated P19 cells were used for DP- and PP-mediated transcription by RT-PCR using their specific PCR primers. Two primer sets were used for each transcript: D1 and D2 for DP transcript, P1 and P2 for PP transcript (Table 1). −RT and +RT indicate samples analyzed without or with reverse transcriptase, respectively. Quantitative analysis is shown below the figure as a graph representing the average of each transcript from two sets of primers. The error bars indicate standard errors of the mean. (B) Analysis of differentiated P19 cells and mouse brain tissue by RT-PCR, as described above. (C) Control RT-PCR performed using β-actin primers shows equal amounts of total RNA used. (D) Control PCR performed to show PCRs using DP- and PP-specific primers and MOR cDNA template.

FIG. 5.

Methylation statuses of the promoter regions of the MOR gene in mouse brain. (A) Methylation analysis of the PP and its downstream regions from adult mouse brain. (B) Similar methylation analysis of the DP and its upstream regions from adult mouse brain. The experiments were performed as described for Fig. 3.

FIG. 6.

Repression of MOR promoter-driven transcription by CpG methylation. Three different luciferase (LUC) constructs (pL450, pGL1.3k, and pLup) were mock methylated (mock) or in vitro methylated with HpaII (partial) or SssI (full) methylase and transfected into P19 cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The error bars indicate the range of standard errors.

FIG. 7.

ChIP analysis of the release of MeCP2 from the MOR promoter induced by neuronal differentiation. (A) Primers specific for the MOR gene promoter (especially the PP region overlapped by primer set e), the β-actin gene, and the H19 promoter (16) were used to amplify genomic DNA sequences that were present in each immunoprecipitate with 32 cycles of PCR. Recruitment of the MeCP2 to the MOR gene promoter was reduced in a time-dependent manner during P19 cell differentiation (lanes 7 to 11). Two percent of each lysate was used as an input control. A “no antibody” control for ChIP was included in a separate parallel run. (B) The specificity of MeCP2 antibody was assessed by Western blot analysis in normal and differentiated (including AP1d to AP3d) P19 cells. MeCP2 protein levels in neuroblastoma NS20Y cells and mouse brain are also shown. Anti-β-actin was used as a control. (C) The locations (shaded boxes) of five different PCR primer sets (a to e) (Table 1) indicate their 5′-flanking regions of the MOR gene. Left- and right-direction arrows indicate sense and antisense PCR primers, respectively. (D) SChIP by real-time qPCR for MeCP2 interaction. Association of the MOR promoter with MeCP2 during P19 cell differentiation was reduced specifically in the PP region covered by primer sets c, d, and e relative to normal P19 cells. Amplification of soluble chromatin before precipitation was used as an input control. Amplification of each primer set was normalized against its input after calculating individual amounts of real-time qPCR product based on each standard curve (see details in Materials and Methods). A “no antibody” control for ChIP was performed separately in parallel.

FIG. 8.

Alleviation of repression from hypermethylated MOR promoters after targeted reduction of MeCP2 using siRNA. P19 cells were transfected with siRNA-targeting mRNA encoding MeCP2, scb control, and MBD2. (A) After siRNA treatment, MOR transcription was assessed by RT-PCR as described in Materials and Methods. (B and C) Reductions in the levels of the targeted mRNA (MeCP2 [B] and MBD2 [C]) were monitored by RT-PCR analysis using the corresponding gene-specific primers (Table 1). (D) β-Actin was included as a control. (E) Reduction of MeCP2 protein was monitored by Western blot analysis using MeCP2 antibody and β-actin antibody as a control. Quantitative analyses of the RT-PCR and Western blot experiments measured changes in mRNA and protein levels, shown as a graph below each result. The data were normalized against β-actin levels. The graph was generated by using Kodak molecular imaging software version 4.0 (Kodak) for RNA and ImageQuant TL (Amersham) for protein.

FIG. 9.

SChIP by real-time qPCR analysis for the statuses of histone modifications and corepressors associated with the MOR gene promoter. (A) Five PCR primer sets were used (as in Fig. 7). (B) The results shown in the graph were from normal P19 cells (UD) and differentiated P19 cells (AP4d) as described in the legend to Fig. 7, but using different antibodies. The chromatin modification status of the MOR promoter region was examined by SChIP assays with anti-AcH3, anti-AcH4, anti-H3dmK4, and anti-H3dmK9 antibodies. The DNAs precipitated by either NRS or the nonspecific antibody anti-gal4 were amplified with the same primers as negative controls. (C) The interaction of corepressors was also analyzed using anti-HDAC1 and mSin3A. RNA polymerase II (using anti-Pol II antibody) was bound more strongly to primer e locations (where the PP-driven TIS is localized) in differentiated P19 cells than in normal P19 cells. (D) A proposed regulation mechanism for the MOR gene. In P19 normal (EC) cells, hypermethylation of CpGs around the PP is coincident with densely interacted MeCP2 recruitment of corepressors. This might lead to compaction of the chromatin structure after histone modifications, followed by silencing of the MOR gene in these cells. In AP2d cells (i.e., intermediately differentiated P19 cells), demethylation of CpGs around the PP begins as MeCP2 and its corepressors start to dissociate, concurrent with histone modifications; this results in intermediate MOR expression. In fully differentiated P19 cells, nearly complete demethylation of the CpGs around the PP region is observed as MeCP2 and its corepressors dissociate. Hyperacetylation of histones also occurs in the promoter, suggesting active transcription of the MOR gene in the cells. All the components for active transcription shown in the figure, e.g., HAT (histone acetyltransferase), TF (transcription factors), and GTF (general transcription factors associated with Pol II), are putative factors for many genes, based on current knowledge from numerous studies.