Up-regulation of the mu-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 mu 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.

Fig. 1.

MNase-Southern blot analyses. A, nuclei were isolated from UD, AP2d, or AP4d cells and treated with different concentrations of MNase. Top left, ethidium bromide-stained agarose gel of isolated genomic DNA. Southern blots using α-³²P-labeled probes as indicated are also shown. Asterisks (one or two) indicate mono- or dinucleosomal DNAs, respectively, in UD or AP4d cells. Arrows indicate smeared DNA in AP4d cells using probes 1 and 2. B, schematic of the MOR gene promoters (DP and PP) and the first exon. The minor DP (−794, relative to the translation start codon at +1) has a single transcription initiation site (Ko et al., 1997), whereas the major PP has four (Min et al., 1994). Locations of the probes used in A are indicated. The downward arrows represent potential MNase attack sites. Right, enhanced expression of the MOR gene in differentiating P19 cells. Levels of MOR mRNA were determined at different developmental stages induced by RA using RT-PCR and real time qRT-PCR. The identities of the PCR products were confirmed by sequencing. Lane 1 (M), 1-kb-plus size marker (Invitrogen); lane 2, UD cells (control); lane 3, P19 cells cultured in parallel without trans-RA treatment; lanes 4 to 8, P19 cells cultured 1 to 5 days after plating (AP).

Fig. 4.

Selective binding of Sp1 factor to unmethylated MOR promoter DNA confirmed by EMSA. A, γ-³²P-end-labeled probes for native (unmethyl Me-1) and methyl Me-1 (as shown below) were incubated with the nuclear extracts from P19 cells. Antibodies (MeCP2 and Sp1) were used to detect each factor's complex. Major protein-DNA complexes are indicated by arrows. Arrowheads indicate bands shifted by the addition of each antibody. Asterisks indicate specific protein-DNA bands. NS, nonspecific band. Lanes 1 and 6, probe alone (free probes); lanes 2 and 7, control reactions; lanes 3 and 8, 100-fold excess of unlabeled self-competitor; lanes 2 to 5, labeled unmethyl Me-1 probe + nuclear extracts; lanes 7 to 10, labeled methyl Me-1 probe + nuclear extracts. Bottom, schematic of probe location on the MOR promoter. Black ovals, GC boxes for Sp transcription factor binding (Ko et al., 1998, 2003); ▩, iGA site (Ko et al., 1998); asterisks, CpG sites for DNA methylation (Hwang et al., 2007). Numbers indicate the site location, relative to the translation start site (+1). B, interaction of MeCP2 with the Sp factors analyzed by coimmunoprecipitation. Five micrograms of MeCP2 antibody was used to immunoprecipitate and to visualize the interaction with Sp1 (lane 2) and Sp3 (lane 4) in P19 extracts. Lane 6, reverse immunoprecipitation using Sp3 antibody for immunoprecipitation and MeCP2 antibody for immunoblotting. IP, immunoprecipitation antibody; IB, immunoblotting antibody. Input lanes contain one tenth of each IP reaction. C, Western blot analysis of Sp1, Sp3, and MeCP2 stage-specific expression in the UD, AP2d, AP4d P19, and NS20Y cells. Anti-β-actin was used as a control. NS, nonspecific band.

Fig. 5.

ChIP analyses of chromatin modifications and nuclear factor interactions. A and B, primers specific for nucleosome position N1 of the MOR gene promoter (S-342 and AS-229; Table 1) were used to amplify genomic DNA sequences present in immunoprecipitates by ChIP-PCR. Amplification of soluble chromatin before precipitation was used as an input control. Antibodies for each ChIP reaction are indicated at the top (A) or the side (B) of each gel image. Parallel controls were performed without antibody or using a nonspecific antibody (anti-Gal4). Images are representative of at least two experiments. C, ChIP analyses to detect chromatin modification on the N1 and N2 positions of the MOR promoter. Primers specific for the N1 and N2 locations (S-497 and AS-182; Table 1) were used to amplify genomic DNA sequences that were present in each immunoprecipitate by ChIP-PCR. Conditions for the ChIP assays were similar to those used for the ChIP experiments (A and B). D, transcriptional effect of Sp1 factor on the MOR promoter of NMB cells. NMB cells were cotransfected with Sp1 expression plasmid along with the MOR promoter construct p189. NMB cells transfected with the MOR promoter construct also were exposed to TSA. Cells were harvested 48 h after transfection, lysed, and assayed for luciferase activity. Promoter activity was normalized by protein concentration. Graphs indicate the averages from at least two representative experiments. Asterisks indicate statistically significant findings (*, p < 0.05; **, p < 0.01) relative to the vector control. Error bars indicate the range of standard errors.

Fig. 2.

Mapping of the nucleosome sites in the MOR promoter. A, MNase-mediated LM-PCR analysis. Left, nuclei isolated from UD, AP2d, and AP4d cells were treated with MNase. Primer (Pr) set A (Pr 1, AS-210; Pr 2, AS-233) and the N1 location are illustrated (right); Pr 2 starts at position −233 relative to the translation site and is end-labeled with γ-³²P. The amplified fragments extend 142 bp to the 5′-border of N1 (position −345 relative to the translation site). Controls include purified chromosomal DNA (naked DNA) treated with 100, 25, 5, or 1 U MNase (lanes 2–5, respectively). Free probe was also present in all samples (below, indicating equal amounts of labeled primers). Right, genomic nuclear DNA analyzed by semiquantitative PCR show equal amounts of input DNA used for MNase-mediated LM-PCR analysis. Input DNA was diluted serially. PCR primers (Table 1) were chosen to detect either distal (DP-MOR: S-1318 and AS-1099) (Hwang et al., 2007) or proximal (PP-MOR, S-342 and AS-229) MOR promoter regions. B, LM-PCR analysis with primer set B (Pr 1, AS-260; Pr 2, AS-288 end-labeled with ³²P). Nuclei were treated with 60 U MNase. C, DNase I-mediated LM-PCR analysis with primer set C (Pr 1, AS-260; Pr 2, AS-285; Pr 3, ³²P-end-labeled AS-296). Genomic DNA from DNase I-treated nuclei was isolated and digested with EcoRI/PvuII restriction enzymes for LM-PCR analysis. D, LM-PCR analysis of nuclei isolated from P19 cells with (+) or without (−) 100 nM TSA for 24 h were treated with MNase. Primer set A was used for analysis. ▩, MOR core promoter region (positions −340 to −300). The positioning of the 5′ border of the N1 nucleosome at position −262 is induced by TSA (compared with its location at −345 in untreated controls). All LM-PCR experiments were performed at least twice independently.

Fig. 3.

PCR-based analyses of nucleosomes on the MOR promoter. A, purified monomer DNA (see B) was used as a template with several primer sets (below, and see Table 1). As a control, intact genomic DNA was used as a template with the same primer sets. Each primer set was designed according to the predicted nucleosome positions. The expected sizes of the amplified products for each primer set are indicated on the right of box. B, left, MNase-digested nucleosomes separated on an agarose gel before purification. Mono, mononucleosome; Di, dinucleosomes. Right, purified monomers from UD, AP2d, and AP4d cells were used as template DNA for real-time qPCR with a primer set designed to detect nucleosome N1's position (A and Table 1). Each experiment was performed at least twice independently. Genomic DNA (5 or 25 ng) was used as a positive control. Asterisks indicate statistically significant differences (*, p < 0.05; **, p < 0.01) relative to the UD sample.

Fig. 6.

Proposed molecular mechanism for MOR gene regulation through chromatin remodeling.